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Two methodologies in pursuit of the elucidation of copper (II) - centered bioinorganic chemistry


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Two methodologies in pursuit of the elucidation of copper (II) - centered bioinorganic chemistry
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Wagner, William J ( William John )
University of South Florida
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Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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ABSTRACT: Copper is a widely distributed transition metal in the earth's crust and has been adopted in a variety of biological systems. In many ways the biochemical usefulness of copper stems from its positive redox potential. This positive redox potential allows copper to assist in the movement of electrons. Copper ions can be found in natural systems as either Cusuperscript I, Cusuperscript II or Cusuperscript III in part due to this redox potential. WhileCusuperscript II -centered biochemistry has been studied for years, mechanistic details in certain Cusuperscript II -centered redox reactions remain unresolved. This study presents two methodologies for studying natural systems with known Cusuperscript II -centered redox capabilities in order to better elucidate the mechanistic intricacies of Copper ion chemistry.The first method explored involves the promiscuous enzyme Streptomyces griseus aminopeptidase (SgAP) which although known primarily as a peptidase has been shown to oxidize catechol under near physiological conditions in vitro when its native Znsuperscript II ions are replaced by Cusuperscript II ions. Protein engineering techniques were utilized toward expression a functional recombinant enzyme in wild type and mutant forms. The goal was to utilize Site directed mutagenesis of residues in the active site to determine which residues are involved in both the hydrolysis and the oxidative activities of SgAP. The second methodology explored was the use of the N-terminus of Histatin-5, a naturally occurring peptide that is known to form complexes with Cusuperscript II, as a model system to study Cusuperscript II-centered oxidation chemistry.Metal-Peptide complexes are much more simplified model systems which use the same building blocks as proteins, but reduce the structure to the minimal functional unit necessary for activity. This in turn, simplifies the study of their catalytic chemistry as influences outside of the active region are greatly reduced. Furthermore, chemical synthesis of short peptides is easily performed and inexpensive in comparison to protein engineering, thus enabling further exploration, if deemed necessary, to be a feasible and economically viable possibility.
Thesis (M.S.)--University of South Florida, 2009.
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Two Methodologies in Pursuit of the Elucidation of Copper (II)Centered Bioinorganic Chemistry by William John Wagner A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Co-Major Professor: Li-June Ming, Ph.D. Co-Major Professor: Brian T. Livingston, Ph.D. Steven H. Grossman, Ph.D. Date of Approval: March 30, 2009 Keywords: aminopeptidase, streptomyces, copper, moonlighting, histatin Copyright 2009 William John Wagner


i Table of Contents LIST OF TABLES iv LIST OF FIGURES v ABSTRACT ix CHAPTER I. INTRODUCTION TO STREPTOMYCES GRISEUS AMINOPEPTIDASE 1 1.1 Hydrolysis 1 1.2 Hydrolysis of Esters 2 1.3 Hydrolysis of Peptides 2 1.4 Serine Peptidases 6 1.5 Cysteine Peptidases 6 1.6 Metallopeptidases 8 1.7 Angiotensin Converting Enzyme (ACE): a Mononuclear zinc containing peptidase 8 1.8 Aeromonoas proteolytica aminopeptidase (AAP): a di-zinc aminopeptidase 14 1.9 Streptomyces griseus aminopeptidase (SgAP) 19 1.10 Catalytic Promiscuity and its relationship To evolution 22 1.11 Copper Chemistry 29


ii CHAPTER II: SGAP MATERIALS AND METHODS 42 2.1 SgAP in Escherichia coli expression system 42 2.2 SgAP in Streptomyces lividans expression system 46 CHAPTER III: SGAP RESULTS AND DISCUSSION 49 CHAPTER IV: CONCLUSION TO SGAP 72 CHAPTER V: INTRODUCTION TO N-TERMINUS OF HISTATIN-5 73 5.1 Antimicrobial Peptides (AMPs) 73 5.2 Histatin-5 75 CHAPTER VI: HISTATIN-5 N-TERMINUS MATERIALS AND METHODS 82 6.1 Metal Binding Studies 82 6.2 Kinetics Studies 83 6.3 DCC Binding Studies 83 CHAPTER VII: HISTATIN-5 N-TERMINUS RESULTS AND DISCUSSION 85 CHAPTER VIII: HISTATIN-5 N-TERMINUS CONCLUSION 101 CHAPTER IX: REFERENCES 102 APPENDICES 117 Appendix A: Pertinent information and Protocols regarding SgAP project using E. coli expression system 118


iii Appendix B: Pertinent information and Protocols regarding SgAP project using S. lividans expression system 145


iv List of Tables Table 7-1: Comparison of Hs5N to full length Hs-5 100


v List of Figures Figure 1-1: Mechanism of Ester Hydrolysis 3 Figure 1-2: The mechanism of peptide hydrolysis 5 Figure 1-3: Catalytic mechanism of the serine peptidase Trypsin 7 Figure 1-4: The catalytic mechanism of the cysteine peptidase Papain 9 Figure 1-5: Comparison of the crystal structures of the active sites of thermolysin and tACE 12 Figure 1-6: The active site structure of Aeromonas proteolytica aminopeptidase 16 Figure 1-7: The mechanism of peptide hydrolysis of the di-zinc Metallopeptidase Aeromonas proteolytica aminopeptidase 17 Figure 1-8: Comparison of di-zinc metallopeptidase metal centers 21 Figure 1-9: Relative activity of SgAP 25 Figure 1-10: Mechanism of CuZn-SOD 34 Figure 1-11: Mechanism of tyrosinase 37 Figure 1-12: Alternate mechanism of tyrosinase 38


vi Figure 1-13: Comparison of metal centers of SgAP and tyrosinase 39 Figure 3-1: 0.7% agarose gel of the product of PCR Reaction 50 Figure 3-2: 0.7% agarose gel of the product of the crude mini prep 52 Figure 3-3: EcoRI and PstI digest of crude mini prep product and pQE-30 Xa vector 53 Figure 3-4: Sequencing results from Macrogen 54 Figure 3-5: Translation of sequence results from Macrogen 54 Figure 3-6: ClustalW2 alignment of colony 16 translation result aligned with ScAP 55 Figure 3-7: Successful purification 56 Figure 3-8: Failed refolding protocol due to precipitation 58 Figure 3-9: Purification from soluble fraction 60 Figure 3-10: Western blot results from soluble purification and Factor Xa 61 Figure 3-11: Construct sequence for recombinant ScAP (rScAP) to be expressed in S. lividans 64 Figure 3-12: rScAP digest out of pUC57 vector as shown on 0.7% Agarose Gel 65 Figure 3-13: Partial digest of pD730 on a 0.3% Agarose gel 67 Figure 3-14: Crude Mini prep digested with SacI 69


vii Figure 3-15: Sequencing DNA from Colony 5 using internal and external sequencing primers 70 Figure 3-16: Translation of sequence DNA from colony 5 70 Figure 3-17: Translation of sequence DNA from colony 5 with extraneous G deleted 70 Figure 3-18: Alignment results of modified colony 5 sequencing DNA to the construct rScAP 71 Figure 7-1: Optical titration of CuII binding to Hs5N 86 Figure 7-2: CuII binding to Hs5N 87 Figure 7-3: Activity profile for catechol oxidation with CuII titration 89 Figure 7-4: Graph of increasing catalytic rate with increasing substrate concentration until saturation 92 Figure 7-5: DCC Binding by Optical Spectroscopy 93 Figure 7-6: Optical Job Plot 94 Figure 7-7: Influence of hydrogen peroxide on catechol Oxidation by CuII 2-Hs-5N 95 Figure 7-8: Catechol oxidatio n by various concentrations of catechol and various concentrations of hydrogen peroxide 97 Figure 7-9: Hanes-Woolf Analysis 98 Figure A-1: Streptomyces griseus aminopeptidase (SgAP) nucleotide sequence 118


viii Figure A-2: Translated active SgAP aligned with translated active ScAP 119 Figure A-3: Streptomyces coelicolor aminopeptidase (ScAP) nucleotide sequence 119 Figure A-4: pQE-30 Xa Vector Map from The Qiaexpressionist 122 Figure B-1: Modified ScAP nucleotide sequence as synthesized by Genscript 145 Figure B-2: pUC57 Vector Map (contains Insert) 146 Figure B-3: pD730 Vector Map 147


ix Two Methodologies in Pursuit of the Elucidation of Copper (II)Centered Bioinorganic Chemistry William John Wagner ABSTRACT Copper is a widely distributed transition metal in the earths crust and has been adopted in a variety of biol ogical systems. In many ways the biochemical usefulness of copper stems fr om its positive redox potential. This positive redox potential allows copper to assist in the movement of electrons. Copper ions can be found in natural systems as either CuI, CuII, or CuIII in part due to this redox potential. While CuII -centered biochemistry has been studied for years, mechanistic details in certain CuII -centered redox reactions remain unresolved. This thesis presents two methodologies for studying natural systems with known CuII -centered redox capabilities in order to better elucidate the mechanistic intricacies of copper ion chemistry. The first method explored involves the promiscuous enzyme Streptomyces griseus aminopeptidase (SgAP) which al though known primarily as a peptidase has been shown to oxidize catechol under near physiological conditions in vitro when its native ZnII ions are replaced by CuII ions. Protein


x engineering techniques were utilized toward expression a functional recombinant enzyme in wild type and mutant forms. The goal was to utilize site directed mutagenesis of residues in the active site to determine which residues are involved in both the hydrolys is and/or the oxidative activities of SgAP. The second methodology explored was the use of the N-terminus of Histatin-5, a naturally occurring peptide that is known to form complexes with CuII, as a model system to study CuII -centered oxidation chemistry. Metal-Peptide complexes are much more simplified systems which use the same building blocks as proteins, but reduce the structure to the minimal functional unit necessary fo r activity. This in turn, simplifies the study of their catalytic chemistry as influences ou tside of the active region are greatly reduced. Furthermore, chemical synt hesis of short peptides is easily performed and inexpensive in comparison to protein engineering, thus enabling further exploration, if deemed necessary, to be a feasible and economically viable possibility.


1 Chapter I: INTRODUCTION TO STREPTOMYCES GRISEUS AMINOPEPTIDASE 1.1 Hydrolysis Hydrolysis is a type of chemical reac tion that breaks a bond by adding water through the activation of water to generate a powerful hydroxide nucleophile. This reaction is the prim ary mechanism for catabolism in cells and is able to break down all classes of macromolecules in the cell. Although hydrolysis can occur spontaneously, it is not particularly favorable, resulting in macromolecules being rather stable at physiological pH. Thus, the degradation of macromolecules requires enzymatic catalysis to proceed at a rate suitable for the metabolic functions of the cell. Generally, the breakdown of macromolecules is highly specific, with different classes of enzymes carrying out the degradation of different classes of macromolecules. The three-dimensional structure of the macromolecules and the geometry of the bonds that hold them together determine the structure and amino acid composition of the catalytic site of the enzyme responsible for degrading each macromolecule. Although variations occur within each class of enzyme, the structural constraints placed upon these enzymes by their substrates lead to similarities within each class. Herein, the hydrolysis of two types of macromolecules, peptides and ester-linked molecules, is discussed, which is


2 followed by a description of an enzyme that, although known primarily as a peptidase, also has the ability to hydrolyze ester linkages. 1.2 Hydrolysis of Esters Esters are an important target for hy drolysis in biological systems. Ester bonds can be found in energy storage uses (i.e. ester linkages of triglycerides in fats). Their presence in carbohydrates means they also contribute to structural integrity of plant cell walls1 and also the exoskeleton of insects.2 Esters are also found as the backbone of nucleic acids in the form of phosphodiesters as well as the short term energy carrier ATP. While the reaction mechanisms of peptide and ester hydrolysis are similar, there are key differences in phosphoester hy drolysis. In traditional esters the nucleophilic water attacks from the top or bottom of the carbonyl plane. In phosphoester hydrolysis, the hydroxide attacks in an in-line SN2-like manner attacking the carbonyl carbon from the side directly opposite the leaving group. This SN2-like attack leads to a tetrahedral transition state in carbonyl esters (RCOOR) but a trigonal bipyramidal transition state in phosphoesters. 1.3 Hydrolysis of Peptides Peptide bond cleavage in biologic al systems is a function performed by enzymes called peptidases. Peptidas es come in three general classes: metal-dependent, serine, and cysteine proteases. The significant role of peptide bond hydrolysis in biology cannot be overstated due to its involvement in digestion of peptides, regulation of hormones, and the


Figure 1-1: Mechanism of Ester Hydrolysis 3


4 maturation process of some proteins.3 In the absence of enzymatic assistance, the amide bond is particularly stable to all but the most extreme pH and temperature conditions.6 The mechanism for the hydrolysis of peptide bonds at alkaline pH is well esta blished. The hydroxide nucleophile attacks the carbonyl carbon from the top (or the bottom) of the plane of the peptide bond to form a tetrahedral transition state typical of peptide hydrolysis.7 This transition state is followe d by the breaking of the peptide bond into an amine and a carboxylate io n. Acid-catalyzed hydrolysis of the amide bond is slightly more complicated.8 The reaction begins with the protonation of the carbonyl oxygen followed by the attack of a water molecules to form the tetrahedral transition state. The nitrogen is then protonated by another hydronium ion. The acid is reformed when one oxygen atom is stripped of its proton to initiate lose of the transition state that ends with the amine being kicked off the carbon, resulting in a carboxylic acid and an amine. In addition to the three major types of peptidases, there are various minor ones as well (namely Threonine, Aspartic acid and glutamic acid). The classifications for peptidas es are based on what is most responsible for its catalytic activity. For instance, serine peptidases use the deprotonated side chain of a serine residue to act as the nucleophile for amide bond cleavage (as opposed to the hydroxide anion discussed in the preceding section). There are at least two essential parts to any peptidase. First, it must have the substrate binding/recognition site. Secondly, it must have a catalytic site. Furthermore, peptidase may be classified as either exoor


5 2: The mechanis m of peptide hydrolysis. Figure 1


6 endopeptidases, depending on whether the enzyme cleaves the protein in the middle (endo) or at the end (exo) of the polypeptide. 1.4 Serine Peptidases One of the most well studied serine peptidases is trypsin. Trypsin is one of the many enzymes used in the digestive system of animals to breakdown peptide bonds. Furthermor e, it gained widespread use in the biological fields in the determination of amino acid sequences in proteins (i.e. the Sanger Method) due to its specif icity for Lys and Arg residues. The general mechanism for serine proteases starts with a His in the active site acting as a general base to form an hydrogen bond with the serine. This activates the serine to attack the amide bond in a similar fashion described for peptide hydrolysis under basic co nditions. The protonation of the nitrogen in the transition state is carried out by the same His, which now is acting as a general acid. After the hydrolysis of the peptide bond, the amine is free to disassociate, but the carboxylic acid terminus of the amino terminal fragment of the substrate is covalently bound to Ser-195. Water is then activated by His to form hydroxide anion, which attacks the covalent intermediate, leading to another tetrahedral transition state. This results in the release of the amino terminal fragment and the regeneration of the active site of the enzyme for another catalysis.9 1.5 Cysteine Peptidases Cysteine peptidases function in a very similar way to serine peptidases. The main exception is that cysteine rather than serine acts as


Figure 1-3: Catalytic mechanism of the serine peptidase Trypsin. 7


8 the initial nucleophile by attacking the carbonyl carbon. Various other amino acids within the active site play supporting roles, depending on the particular enzyme. For example, papain uses Asn175 to activate His-159 (rather than Asp as in Trypsin) which can then dona te a proton to the nitrogen of the peptide bond. This, of course, is a necessary step in peptide bond hydrolysis. Papain also utilizes Gln-19 as well as the nitrogen of Cys-25 to stabilize the carbonyl of the target peptide.10 1.6 Metallopeptidases Metallopeptidases are the most diverse group of peptidases due to two important factors. First, a variety of metal components can be present. For instance, the peptidase may contain zinc, iron, or manganese in the active site. Second, a varying number of metal components present can complicate the catalytic mechanism. Generally speaking, the most common metallopeptidases have either one or two metals in their catalytic centers; however, trinuclear metal centers do exist. Moreover, the metal components need not be homogenous, but heterogeneous metal centers do exist as well. 1.7 Angiotensin Converting Enzyme (ACE): a mononuc lear zinc containing peptidase ACE is one of the most interesting mononuclear zinc peptidases. In mammals this enzyme is responsible for at least two extremely important functions: the conversion of angioten sin I to angiotensin II (a potent vasopressor) and the breakdown as well as inactivation of the important vasodilator bradykinin.11 The overall result of the action of this peptidase is


Figure 1-4: The catalytic mechan ism of the cysteine peptidase Papain. 9


10 to raise blood pressure. Due to the potent hypertensive effect of ACE, it has become the main target in anti-hypertension treatment. The drug class known as ACE inhibitors has become the first line of treatment not only for hypertension but also congestive heart failure and diabetes related and nondiabetes related nephropathy. A noteworthy feature of ACE homologues is their ubiquitous nature. Homologues of ACE can be found in organisms that lack both angiotensin and a cardiovascular system.12 ACE was first discovered in equine plasma samples; however, it is now known to be membrane bound, with highest concentrations in the epithelial cells of highly perfused tissues such as the kidney and lungs.12 In mammals, there are two forms of ACE. The somatic form, sACE, is ubiquitously distributed around several types of tissue and consists of two transmembrane, a large cytosolic, and an Nand C-terminal extracellular domains. The extracellular domains are similar, but each contains a different active site configuration and different substrate preferences12. The other form found in mammals is the germinal or testicular ACE (gACE or tACE). gACE is 701 amino acids long and is identical to the C-terminal of sACE aside from the first 36 amino acids.11 Whereas the function of sACE is well established, the function of gACE is st ill unknown. However, knockout mice studies have shown that deletion of gACE gene leads to greatly decreased fertility.12 Unfortunately, there have been insurmountable difficulties with purification and subsequent crystallizatio n of sACE. One of the main reasons


11 is that ACE is heavily glycosylated, wh ich leads to microheterogeneity of the oligosaccharides on the surface, pr eventing the ordered packing necessary for crystallization.11 Despite these difficulties, gACE was finally crystallized as well as the Drosophilia homologue, AnCE. The similarities both in sequence and crystal structure between the active sites of another peptidase, thermolysin, and ACE suggest a similarity in mechanism.11 The accepted mechanism for thermolysin was proposed by Brian Matthews in 1988.13 The key feature is the zinc binding motif (HEXXH) in which the two active site His residues coordinate to the zinc ion. There is also a Glu and a water molecule which complete the tetrahedral ligand field for the zinc.12 According to Matthews,13 the zinc cation fulfills an important role in the catalysis of peptide hydrolysis First, it binds the carbonyl of the target amide bond; this attack on the carbonyl carbon makes the resulting product more energetically favorable by balancing the oxygen anion. The zinc also helps to activate the coordi nated water molecule to form hydroxide and initiates the attack. The active site then donates the proton to the amine group of the peptide bond.13 This stabilizes the amine group and by giving it a positive charge encourages the electron cascade from the carbonyl oxygen. The crystalographic study by Gordon et al.14 revealed other interesting aspects of this ACE. The study show s why ACE preferentially digests small unfolded peptides over larger folded pr oteins. The crystal structure contains a deep cleft that extends 30 into the in terior of the globular protein, which


Figure 1-5: Comparison of the crystal structures of the active sites of thermolysin and tACE. 12


13 is guarded by several charged residues at the clefts opening. This cleft restricts the types of substrates that can possibly come in contact with the active site. The study also revealed significant structural homology between tACE, AnCE, and neurolysin despite their low sequence homology.11 This interesting situation where there is significant structural homology without sequence homology reveals a phenomenon of biology. Nature follows the rules set forth by physical la ws. In order to facilitate the breaking of the surprisingly strong peptide bond, an enzyme must have both the correct three-dimensional structure to interact with the peptide bond and the appropriate amino acid side chains in the active site positioned in such a way that they facilitate the movement of atoms and/or electrons required for catalysis. As such, it frequently occurs that enzymes that perform similar tasks have similar three dimensional structures. There are two possible theories as for how these enzymes come to resemble each other so strikingly. First, proteins encoded by genes that all arose from the same ancestral gene, which have undergone only conservative mutations, will result in a structure that remains largely unchanged, even though the actual sequence can differ to varying degrees. This is termed divergent evolution. Alternatively, the particular structure could have evolved multiple times from different gene lineages. This convergent evolution postulates that earlier structures that did not resemble each other eventually evolved to form a three dimensional shape that bears a strong resemblance to each other. The evolutionary history of genes that enco de proteins with similar functions can easily be determined using bioinformatics tools for phylogenetic analysis.


14 The important thing, however, is that it is through these relationships a study about one enzyme in a class can be inferred to another enzyme within its class. It is known that certain sequences, such as the HEXXH motif,13 occur often and perform similar functions in different enzymes, and through this a starting point for future research on new enzymes can be found. This inference system is often used to prop ose mechanisms of enzymes that have yet to be thoroughly studied by comparing their structural homology (or sequence homology) to well-studied enzymes 1.8 Aeromonas proteolytica aminopeptidase (AAP): a di-zinc aminopeptidase Di-zinc aminopeptidases similar to AAP have been implicated in many disease states. Many drugs used in the treatment of cancer and HIV can be linked to their effect on dinuclear metallopeptidases1520. The similarities among peptidases of this class enable the inference of mechanisms relevant to all members from studies on particul ar model proteins. As such, studying a similar di-zinc aminopeptidase from Aeromonas proteolytica and how inhibitors interact with it can serve as a model and lead to the design of new drugs with greater efficacy. Sequence and structural studies of AAP have revealed that there is a small hydrophobic pocket which is ju st large enough to support the Nterminal amino acid. This explains wh y AAP is an aminopeptidase and not an endopeptidase. Inhibitor studies suppo rt the preference for hydrophobic residues in that alcohols with longer carbon chains and more steric hindrance


15 decreased the Ki from 860 mM with methanol to 0.98 mM with 3-methyl-1butanol.21 As is usual for peptidases, the tetrahedral transition state analogs are known inhibitors of AAP. These i nhibitors mimic the transition state of peptide hydrolysis and so are thought to compete for the active site. Metal chelating agents are also inhibitors of AAP by sequestering the metal ions necessary for catalysis21. Through the inhibition as well as st ructural studies, the mechanism for AAP has been elucidated. The zinc ions are in similar coordination spheres, both having one His residue directly coordinated to the zinc ion, a bridging water, and a bridging Asp. Zn1 is coordinated to a Glu, while Zn2 is coordinated to an Asp. This gives a distorted tetrahedral coordination sphere to each Zn ion.21 Upon substrate binding to the hydrophobic pocket, the bridging Asp coordination is replaced (on Zn1) by the carbonyl oxygen of the amide bond. Water is then deprotonated by Glu-151 to form hydroxide, which attacks the carbonyl carbon. The Zn1-carbonyl oxygen interaction stabilizes the tetrahedral transition st ate that came about by the hydroxide attack on the carbonyl carbon. The proton gained by the Glu from the activation of water to hydroxide is transferred to the nitrogen of the amide bond along with the breaking of the amide bond and the release of the second amino acid. The carbon to oxygen double bond then reforms as the oxygen dissociates from Zn1 and is replaced by the original bridging Asp. Other interesting studies from AAP involved the manipulation of the metal centers. By removing the metals from the enzyme and then


Figure 1-6: The active site structure of Aeromonas proteolytica aminopeptidase. 16


Figure 1-7: The mechanism of pe ptide hydrolysis of the di-zinc metallopeptidase Aeromonas proteolytica aminopeptidase.21 17


18 reconstituting with two equivalents of another transition metal (copper, cobalt, or nickel) the activity is altere d (some greatly increased activity while others reduced it).22 Addition of one equivalent of a different transition metal to the apo-AAP followed by an equivalent of ZnII dramatically increases the activity as well as changes the binding affinity ( KM).22 Although not medically relevant in any direct manner, the fact that nature has evolved only a few ways to perform peptide hydrolysis with a dinuclear center provides ample reason to study AAP. The study of AAP over the medicinally relevant enzymes is preferred due to the simplicity of purification from natural sources. Furthermore, the growth of bacteria is generally easier, and purification from bacteria is simpler. As such, using bacteria as expression systems is pref erable over eukaryotic cells. Further complicating matters, acquiring reco mbinant proteins using DNA from a eukaryotic source contai ns many more hurdles than using a prokaryotic source. Controlling of the expression of eukaryotic genes is more complex than bacterial genes, since the eukaryotic DNA is more complicated than bacterial DNA with the presence of in trons and exons, necessitating posttranscriptional modification (a problem overcome by using reverse transcriptase and processed mRNA). Another complication in trying to express a eukaryotic gene in bacteria, ev en after removing the exons, is the great differences in codon usage frequencies between species that diverged so long ago. This results in some codons calling for tRNAs that are available in abundance in the original eukaryote that simply do not exist in bacteria.


19 Thus, the translation is interrupted, resulting in a truncated and useless protein. 1.9 Streptomyces griseus aminopeptidase (SgAP) The aminopeptidase from the culture medium of Streptomyces griseus is a single peptide chain with 284 residues (from the protein data bank) and a molecular weight of approximately 30kDa.23 Native SgAP contains two ZnII ions and requires both for peptidase activity.24 There is also a calcium cation that enhances activity of native SgAP, but due to the great distance between the calcium binding site and the catalytic center (25 ) the mechanism of this activation remains unknown. The substr ate preference for SgAP is for large hydrophobic amino terminus residues24 preferably leucine25 and is maximally active at pH 8.0.26 The ligand environment for the first ZnII, based on x-ray crystallography studies, is proposed to be His-85, Asp-160, and a bridging Asp-97 and bridging dibasic phosphate. The ligand environment for the second Zn2+ is His-247 and Glu-132 as well as the bridging Asp and dibasic phosphate.27 More recently, however, NMR studies suggest that the bridging phosphate is a product of the crystalliz ation medium and is not bound to the active site in solution. Instead, a wa ter molecule or a hydroxide is proposed to replace the phosphate in the active site.26 This active site is quite similar to that of other aminopeptidases such as Aeromonas AP which was described previously. The incredibly similar ac tive site structures between AAP and SgAP suggest a similar mechanism for peptide hydrolysis, however key differences have been discovered (most notably those revealed by metal


20 titration and inhibitor studies). The Active site of AAP and SgAP are compared in Figure 1-1. Metal titration studies to SgAP have demonstrated that the two metal sites are not identical and that binding happens sequentially. This is particularly noticeable when CoII is used as the titrant in the absence of calcium at pH 6. Furthermore, these studies have shown that SgAP requires both metals for activity as detectable activity is shown only when greater than one equivalent of CoII has been added. There is a sharp rise in activity between 1 and 2 equivalents with an activity plateau forming shortly after 2 equivalents have been added to solution.25 Although cobalt is not the native metal for SgAP, it is used because of its sequential binding, in the absence of calcium. It should be noted, however, that sequential binding takes place with zinc as well. It is, however, not as well pronounced.25 Given the similarities between AAP an d SgAP it is difficult to see why one would want to study SgAP. There is, however, something peculiar about SgAP that sets it apart from most aminopeptidases. The unique aspect of SgAP is that it is able to take a tran sition state analogue inhibitor like bispnitrophenylphosphate (BNP P) and catalytically hydrolyze it. BNPP has become the standard for colorimetric assays of phosphoesterase activity.28 That SgAP is able to hydrolyze both peptide bonds ane phosphoesters is a property entitled catalytic promiscuity. Thus SgAP falls into an exclusive class of enzymes that seem to defy the fundamental dogma of enzymology.


Figure 1-8: Comparison of di-z inc aminopeptidase metal centers. Side by side comparison of the active sites of AAP (Right) and SgAP (Left). Clearly the ligand environments are very comparable. One difference that is noteworthy is the inclusion of phosphate in the picture of the SgAP active site. However, this is not seen in solution phase, but rather an artifact of the crystallization medium used to determine the active site composition. In solution, a bridging hydroxyl or water is proposed (as is seen in AAP). 21


22 1.10 Catalytic Promiscuity and its relationship to evolution Central to the dogma of enzymology is that enzymes have evolved to catalyze a particular reaction, generally showing a strong preference for an exceptionally small range of substrates. This was termed specificity Over the years, researchers have stumbled across many examples where not only did an enzyme catalyze a different type of substrate in the same class of reaction (hydrolysis of peptide bonds and hydrolysis of ester bonds, for example) but although considerably mo re rare, can also catalyze different types of reactions (hydrolysis and oxidation). There are various classes of catalytic promiscuity. The most common class is functional group anal ogues where the bonds broken differ but the mechanism is likely the same. This is common, for example, in a peptidase that also hydrolyzes esters.29 Another class involves the change in mechanism. A mutation in the enzyme that decreases reaction rate but does not eliminate function must follow a less efficient path and therefore a different mechanism.29 An example of this is observed in a protein-tyrosine phosphatase,30 in which the catalytically important Cys was converted to Asp. This mutant retains some activity, and since the catalytic functional group was changed, the observed ac tivity must be resulted via a different mechanism.29 Another class involves a chan ge in substrate specificity. Changing the substrate specificity leads to slight changes in the transition state and can be considered as a promiscuous function.29


23 Merely being catalytically promiscuous is not uncommon. The rate at which SgAP hydrolyzes BNPP is exceptionally high: 4x 1010 greater than the auto-hydrolysis of BNPP31 and this is very unusual. Whereas most promiscuous activities show a rate that is a fraction of the native activity, SgAP activity towards 1 mM BNPP is 33.7 nmol min mg versus SgAPs activity to 1 mM Gly-pNA of 19.3 nmol min mg.32 Furthermore, the activity of SgAP on BNPP surpasses that of several natural alkaline phosphatases: those from chicken intestine and Escherichia coli are 16.5 and 11.1 nmol min mg, respectively.28 While there is a great deal of difference between the hydrolysis of peptides and the hydrolysis of phosphoesters, most notably in transition state geometry, in essence they are both hydrolysis reactions. The promiscuity of SgAP extends beyond mere hydrolysis of phosphoesters. More interesting is the catechol oxidase activity. Da Silva and Ming33 showed that SgAP was able to catalyze the oxidation of 3,5-di-tert -butylcatechol (DTC). This oxidation was accomplished with the di-substituted Cu,Cu-SgAP, by contrast the natural state of SgAP is Zn,Zn-SgAP. The enzymatic parameters given were kcat= 1.45 s and KM= 0.44 mM and kcat/ KM 3295 M s. Which is comparable to a natural catechol oxidase from gypsywort ( kcat/KM 32 mM s).33 There is a large body of evidence that supports the claim that these three activities are catalyzed in the same active site.32 First, dizinc aminopeptidase inhibitors bestatin, 1-aminobutylphosphonate and Leuhydroxamate inhibit both hydrolytic ac tivities and the oxidative activity.32,33 Second, BNPP is shown to be a competitive inhibitor toward Leu-pNA


24 hydrolysis.32 Finally, the activity of BNPP hydrolysis follows that of Leu-pNA hydrolysis on cobalt titration studies.32 Relative activity versus copper ion titration revealed very similar patterns for both Leu-pNA hydrolysis and DTC oxidation (Figure 1-2). This indicates that as the active site that hydrolyses peptide bonds is completed by sequential addition of metals, so is the active site that hydrolyses BNPP, as is the ac tive site for the oxidation of DTC and as such must be the same site. There are two groups of scientists that have an interest in catalytic promiscuity: organic chemists and evol utionary biologists. The reasons for their interest are usually quite differen t, and as such what they qualify as being significant is different. Orga nic chemists are most interested in catalytic promiscuity because they have recently discovered the ability of enzymes to produce stereo-selective products using a broad range of synthetically useful molecules. Furthermore, the idea that small changes can be introduced into the enzymes primary sequence, which can enable a promiscuous reaction, can lead to even greater synthetic usefulness for the chemists. This alternate catalysis of phosphoesters is of interest to chemists in that the phosphoester is particula rly stable. Furthermore, SgAP can


Figure 1-9: Relative activity of SgAP DTC oxidation compared to Leu-pNA hydrolysis by CuII titration to apo-SgAP. Reprinted from da Silva and Ming.33 25


26 handle high temperatures, withstanding 69 C for 7 hours and maintaining approximately 80% activity,32 and so, is an enzyme that has potential for industrial applications. Evolutionary bi ologists are more concerned with what this catalytic promiscuity means for the natural history of this enzyme, or its family, which can provide a better understanding on how evolution can happen at a molecular level. Since the two groups have very different concerns or uses for catalytic promiscu ity, their definition of relevance and focus of study are significantly differen t. The more discerning of the two groups is undoubtedly the biologists. They will not be concerned with a promiscuous reaction that occurs in vitro if it has no biological relevance in vivo. Biologists have a more pressing reason to be interested with catalytic promiscuity. Understanding evolution on a molecular level may become extremely valuable for the agricultural and medical fields. This will aid in better elucidating the mechanism by which bacteria rapidly evolved to the pesitices and antibiotics introduced in the 20th century. There are two informal but important features that are crucial for this promiscuity. The first is an enzymes plasticity, which is its ability to change the mechanism, binding efficiency, or substrate preference with minimal changes in the primary sequence. A good example is the muconate lactonizing enzyme II (MLE). A single amino acid mutation enables MLE to perform the o-succinylbenzoate synthase activi ty with high efficiency while retaining its muconate lactonizing activity.34 Plasticity is a common feature among enzymes with promiscuous activity. The importance of plasticity in evolution is evident in the fact that if an enzyme requires a complete


27 modification of its primary sequence to change function, random mutation(s) in the sequence would be insufficient for adapting to new conditions. The other important feature for an enzymes promiscuity is its robustness. This is a way of stating that an enzyme has the ability to endure mutations with little impact on its native function and structure. With promiscuous enzymes, the mutation(s) can result in a drastic increase in the rate of the promiscuous function, with little change on the rate of the original activity.35 Studies on robustness have defined an x factor as the probability that a particular protein will be inactivated by a single, random amino acid substitution.36 Low percentages define the enzyme as being highly robust. Some reported examples are 3-methyladenine DNA glycolase with x factor value of 34 6%,37 T4 lysozyme ~16%,38 and barnase ~5%.39 These examples show that robustness can vary widely, but certain proteins can be particularly robust and thus are stable against random mutations. An enzyme can display both robustness and plasticity. The reason for this is simple. Not every amino acid is equivalent; the location in the primary sequence as well as the nature of the amino acid is important. The nature of the protein as a whole is also important. If it is mostly hydrophobic residues, the probability that a mutation will occur at a hydrophobic residue is high. Since the bulk of the naturally occurring 20 amino acids occurring in proteins are hydrophobic, a random mutation will probably produce another hydrophobic residue. Thus, the impact of mutation is minimal. The mutation that gives rise to plasticity typically occurs at the amino acid(s) near the active site, and the effect of the change can vary from little to extreme. In a more extreme


28 case, a simple change in the primary sequence such as a single amino acid alteration could result in a drastic change in substrate affinity and specificity and thus can be labeled promiscuous. If the mutated amino acid is involved in electron transfer or shuffling, the overall mechanism may be impaired, and the enzyme may become less efficient or completely lose its function. An interesting piece of knowledge that has been discovered from the laboratory studies on directed evolution and promiscuity is that the mutations that increase promiscuous function with little effect on native function predominately occur on the outski rts of the active site. In this way the mutation does not affect the catalytic machinery nor the scaffolding that holds the catalytic machinery in its correct orientation. These mutations often occur on surface loops that are part of the substrate binding pocket, and exhibit high conformational flexibility.35 Evolutionary biologists should be particularly interested in SgAP in that this appears to be a snapshot of enzyme evolution. It was discussed earlier that many enzymes have a plasticity th at allows for a very small number of mutations to result in a drastic change in promiscuous activity.35 While this has been able to be performed in the laboratory, SgAP appears to be rare in that it is a naturally occurring acquisit ion of promiscuous activity. Moreover, it shows an example of a mutation that occurred without damage to the native function (i.e. robustness). An important target for research has been to find what amino acid residue(s) acts as the molecular switch. That is what residue(s) was


29 changed that contributed to the promiscuous function or what may be changed to further favor one or the other. For the biologist, the molecular switch in SgAP is important in that it allows one to gain a better idea of how evolution is working on a molecular level in a natural system. The organic chemist can learn from the molecular switch by gaining a better understanding of how one can change other enzymes to enhance their promiscuous functions for stereoselective synthesis. Perhaps the most interesting of SgAPs promiscuity is its catechol oxidase activity. This is because the hydrolysis of peptides and the hydrolysis of phosphoesters are essentially very similar (they are both hydrolytic reactions). That SgAP has evolved, naturally, the ability to perform redox chemistry, as well as main tain its hydrolytic functionality, is exceedingly rare and thus warrants specia l attention. In all fairness, this redox chemistry is only present when the proper metal substitution has occurred. It is, however, also fair to point out that copper ions are ubiquitous in biological systems. Thus, a greater understanding of the mechanics of copper ions in protein systems is vital. 1.11 Copper Chemistry The composition of copper in the earth crust is low (approximately 0.7ppm). The rarity of copper combined with its extensive use emphasizes the importance of its unique properties for biological systems. On the other hand, a high concentration of copper ions is toxic. This is the result of its inherent ability to activate molecular oxygen and create destructive reactive


30 oxygen species. The biological useful ness of copper ions stems from its high redox potential, giving copper ions the ability to function as a means of electron transfer (i.e. plastocyanin an d hemocyanin) as well as participating in many oxidation reactions (i.e. tyrosinase and catechol oxidase). Therefore, the regulation of copper ions, both in utilizing their redox properties as well as preventing high concentrations of free ions, is a vital endeavor of evolutionary processes. In biological systems, copper-containing enzymes or proteins have been divided into three classes based upon differences in structural and spectroscopic characteristics.40 The first class, aptly named Type-1 copper centers (i.e. in blue copper proteins) ar e found in electron transfer proteins. All blue copper proteins have a similar deep blue color which is the result of Cys S-Cu(II) charge transfer transition with ( max >2000 M cm).41. Furthermore, the ligand environment for type-1 copper centers is consistent in that they position the copper into a distorted tetrahedral which is thought to be the transition state between the favored geometries of CuII (square planar or tetragonally di storted octahedral) and CuI (tetrahedral).41 One of the prototypical proteins of this clas s is plastocyanin. Plastocyanin was initially discovered by Katoh.42 Early on, its involvement with photosynthesis was postulated in part due to previous findings by Neish43 and Green,44 which showed that copper was present in ch loroplasts by the inhibition of photosynthesis when copper ion antagonists were present. Gorman and Levine46 later showed that the electron transport mechanism proceeded in a series. The chlorophyll of photosys tem II first transfers electrons to


31 cytochrome f which then transfers them to plastocyanin and finally the electrons are transferred to photosystem I. Plastocyanin, as a type-1 copper center, possesses certain spectroscopic properties, most notably is the intense absorption band at 600nm ( max ~5000 M cm) giving plastocyanin its characteristic blue color.46 It is likely that th e polypeptide enforces this ligand environment rather than the metal ion as the metal binding site of apo-plastocyanin, as well as the HgII-plastocyanin isomorph retains essentially the same shape as the native CuII-plastocyanin metal binding site.46 Interestingly, while it is nece ssary for blue copper proteins to maintain the distorted tetrahedral shape, slight changes in the axial ligands are able to fine tune the reduction potentials as well as the reorganization energy to suit the particular function of their proteins.47 The plastocyanin from Populus nigra (black poplar), for example, has a redox potential of 370 mV and is thus well suited for transferring between cytochrome f (redox potential of 340 mV) and P700+( 490 mV). Whereas the blue copper protein rusticyanin from Thiobacillus ferrooxidans has a redox potential of 680 mV and would therefore be incapable of s huttling electrons between cytochrome f and P700+.47 This provides some mechanism in which evolution might change the polypeptide primary sequence slightly and thus affect its function either by changing the ligands or ev en by adjusting the ligands in three dimensional space to change the degree of distortion in the distorted tetrahedral. Furthermore, it is likely that this method of fine tuning is likely to be applied to a greater or lesser degree in the other copper center classes.


32 The Type-2 copper centers (i.e. the non-blue copper centers) are generally oxidases or oxygenases.41 The copper centers in type-2 usually have three to four ligands and at least one of which is the imidazole of a histidine49. Cysteine, an amino acid required for type-1 copper centers, is absent in type-2 copper centers and thus they lack the characteristic blue color. In contrast to type-1 copper centers which restrict the copper to a distorted tetrahedral geometry, type-2 co pper centers restrict the copper to a distorted square pyramidal or octahedral geometry46. This, however, is more of a generalization than a rule in that the geometry of a copper center is rarely perfectly octahedral, but rather it is often distorted to varying degrees due to the Jahn-Teller effect. This is the case in CuIIZnII-superoxide dismutase (CuZn-SOD). CuZn-SOD is one of the well studied type-2 copper centers. This enzyme catalyzes the di sproportionation reaction of superoxide to hydrogen peroxide and dioxygen: 2O2 + 2H+ O2+ H2O2 Thus, CuZn-SOD enables an organism to deal with the reactive oxygen species (O2 ) that form during cellular respiration and as a result of living in an oxygen-rich atmosphere. CuZn-SOD catalyzes this reaction in a two step ping-pong mechanism as such: (1) O2 + CuIIZnSOD O2+ CuIZnSOD (2) O2 + CuIZnSOD + 2H+ H2O2 + CuIIZnSOD In human CuZn-SOD, the metal-binding environment of copper consists of four histidines (His 46, His48, His63, and His120) and a water which restrict


33 the coordination geometry to a distorted square pyramid.48 The zinc ion is coordinated by His63 (bridged to the Cu center), His71, His80 and Asp83 which fill its tetrahedral coordination sphere.48 A superoxide anion, which has previously been nonspecifically protonated, binds the CuII center. The proton, during the first transition state is exchanged for the CuII by His63. The superoxide then transfers a single electron to the copper, facilitating the CuII to CuI transition. At this point the superoxide is oxidized to molecular oxygen and disassociates. A second nonspecifically protonated superoxide binds to the CuI center. This time, the CuI transfers an electron to the superoxide creating a hydroperoxyl anion bound to a CuII. During the second transition state the proton from His63 is exchanged for CuII while the bond between CuII and hydrogen peroxide is broken. This is a classic example of a ping-pong (double displacement) mechanism because one product (O2) is leaves before the second substrate (HOO) has bound. Most important is the substantial change the metal center has undertaken between the first substrate binding and the second substrate binding, which is accepting an electron and becoming CuI, providing the driving force for the second reaction. The mechanism is shown in Figure 1-3. Not all ligands are merely structural; for example the ligand that bridges the two metal centers (His63) plays an important role in being a likely source for one of the protons involved in the superoxide dismutation. Furthermore, the zinc ion likely makes the N 2 proton of histidine more acidic, further enhancing the overall activity.


Figure 1-10 : Mechanism of CuZn-SOD Adapted from Pelmenschikov and Siegbahn but printed in Macpherson.48 34


35 The last class of copper centers, type-3, is characterized by a homonuclear di-copper center. The type-3 copper-containing proteins generally function as oxidases or oxy genases such as catechol oxidase and tyrosinase, respectively and in some cases as molecular oxygen carrier proteins such as hemocyanin.41 Among them, tyrosinase has received considerable scholarly interests for a variety of reasons including its role in fruit browning after damage, involvement in disease states (albinism), wound healing and immune response, and cosmetics (inhibition of tyrosinase in order to lighten the skin).49 Tyrosinase has both the monooxygenase and catechol oxidase activity.41 It can accept a substrate with a monophenol moiety such as the amino acid tyrosine and add a hydroxyl group in the ortho position to afford a diphenolic compound, catechol. Then, it can further catalyze the oxidization of catechol to the corresponding ortho -quinone.41 It is these quinones that are able to spontaneously polymerize to form melanins as the mechanism to fruit browning.41 Catechol oxidase, by comparison, lacks the monooxygenase activity and can only catalyze the conversion of catechol into ortho-quinone.41 The copper atoms in the type-3 center are antiferromagnetically coupled with a ground state of S = 0 in the presence of molecular oxygen (in the reduced form) or hydrogen peroxide (in the oxidized form).41 Upon binding, the di-copper center adopts the oxy -state with absorbance maxima at 340 nm and 580 nm caused by O2 to CuII charge transfer transition. The deoxy state which contains CuI:CuI is diamagnetic.43 Both copper sites in the tyrosinase from Streptomyces castaneoglobisporus crystallized by Matoba et al.51 are each coordinated to


36 three histidine residues at the N atom. This histidine rich coordination sphere is a similar feature for all type-3 copper centers including catechol oxidase and hemocyanin.49 The mechanism by which tyrosinase oxidizes a monophenol through the catechol-moiety to ortho-quinone is complex, and a definitive mechanism is still under debate. In one example, the monophenol binds the oxy-state and is monooxygenated. The catechol-moiety is then free to disassociate. A catechol-moiety can bind the met-state copper center and is oxidized to o-quinone, leaving the copper center in the deoxystate. The deoxy -state binds molecular oxygen to reestablish the initial oxystate.41 This mechanism is shown in Figure 111. Another proposed mechanism is similar except that once the monophenol binds the copper center it does not disassociate until it is o-quinone.49 This mechanism is shown in Figure 1-12. Given the ability of SgAP to perform catecholase activity it is clear that it shares more common features with a type-III CuII center. Thus a comparison of the active sites of tyro sinase and SgAP could provide useful information in looking for the molecular switch to enhance catecholase activity (Figure 1-13). An obvious diff erence is the composition of the active site. In tyrosinase the ligands are all His residues, while in SgAP a blend of acidic residues and His residues is present. Thus, potential targets for enhancing catecholase activity in SgAP would be Asp-97, Asp-160 and Glu132. In searching for the molecular switch for switching between the hydrolysis reactions, the most obvious place to start looking was where


Figure 1-11 : Mechanism for tyrosinase Starting from mono-phenol moiety through quinone.41 This displays the theory that the intermediate catechol is released and then subsequently rebound. 37


Figure 1-5 : Alternative mechanism for tyrosinase This mechanism does not allow the relase of the catechol moiety but rather continues through to the final quinine as championed by Decker et al.49 38


Figure 1-13: Comparison of metal centers of SgAP and tyrosinase. Side by side comparison of SgAP27 (Left) and tyrosinase from Streptomyces castaneoglobisporus (Right).49 39


40 Harris et al.26 showed a likely phosphate binding moiety. This is because the alternate substrate, BNPP, has a phosphat e and it is the phosphoester that is cleaved in the promiscuous function. As such, places where phosphate could bind would make ideal candidates. The NMR studies, as well as phosphate inhibition studies (showing that phos phate was a noncompetitive inhibitor), indicated that the most likely candidate would be the Arg residue near the active site, Arg202.26 Harris was able to use chemical methods to modify the Arg and found that kcat decreased and the Km increased significantly toward Leu-pNA. Also the Ki for phosphate inhibition increased as well. These results indicate that the phosphate an d the Leu-pNA interact with the Arg202.26 The chemical modification can be manipulated in such a way to protect certain Arg residues. By incubating SgAP in a buffer with phosphate before and during the modi fication step those Arg that are binding phosphate should be protected. Following modification the sample with phosphate protection showed little change in activity, thus Arg-202 when unprotected was changed and a decrease in activity was seen. Furthermore, protection with 1-aminobutylphosphonate, which binds at the active site, indicates that the important Arg is also close to the active site, and the best candidate is Arg202. The problem with using chemic al methods to modify an amino acid is that it is difficult to be selective. It is usually impossible to modify a single amino acid, especially in a protein with as many residues as SgAP. As such, the possibility that an amino acid, similar but separate, to the intended target was modified and it is this change that is responsible for observed results (in SgAP there are 8 arginine residues that could be changed). For


41 this reason molecular biology has been employed in this research dilemma. Through site-directed mutagenesis biolog ists are able to change one amino acid to another, delete an amino acid or add an amino acid at the DNA level. This, unlike chemical modifications, is absolutely specific leading to a change only as specified. The goal of cu rrent research is to understand the molecular mechanisms of this hydrolysis reaction. This will be accomplished through the exploitation of molecular biology techniques to determine what the functions of the active site residues are.


42 Chapter II: SGAP MATERIALS AND METHODS 2.1 SgAP in Escherichia coli expression system Isolating insert DNA from Streptomyces coelicolor The Streptomyces coelicolor genome was purchased from ATCC. The primers used were 5-AAT CCC GGG TAG GCG ACA GTT CCC AGA C-3 and 5/5PHOS/AAC GTC AAG GCC CAT CTG AGG-3 and purchased from Integrated DNA Technology (IDT). The reagents for use in the polymerase chain reaction (PCR) were purchased from Promega. The PCR was performed on an Eppendorf Mastercycler Gradient. The PCR products were purified using Amicon Spin Columns from Millipore. Agarose gel electrophoresis was utilized to confirm the PCR was successful. For a detailed protocol please see Appendix A. Cloning The vector, pQE30Xa, was purchased from Qiagen. This vector contains the gene for ampicilin resistance as well as a C-terminal 6x-His tag and a peptide sequence recognized by Factor Xa protease for removing the His-tag and has an approximate size of 3.5 kb. The pQE30Xa was digested with XmaI and StuI (both purchased from New England Biolabs). The purified PCR product insert was digested with only XmaI (as the PCR product


43 was designed with a blunt end that woul d anneal with the blunt end created by StuI). Following digestion both reactions were quenched by denaturing the restriction endonucleases at 65 C for 10 minutes. The DNA products were then purified using Amicon Spin Columns. The digested vector was then treated with shrimp alkaline phosphatase, purchased from Promega. The use of shrimp alkaline phosphatase removes phosphates on the vectors digested ends that could provide the necessary energy to self-ligate. Self-ligation is unlikely because the ends were incompatible; however, by removing the phosphates with shrimp alkaline phosphatase the reaction is effectively impossible. Again, the DNA product from shrimp alkaline phosphatase was purified using Amicon Spin Columns. Ligation materials were purchased from Promega. The ligation reactions were carried out at various ratios of vector to insert (1, 2, 3, 5, 10, 15, 20, 50, 100, and 150). Of which, success was achieved with a 20:1 vector to insert ratio. The ligation reaction product was used to transform XL1-blue E. coli This bacterial cell line is transformed using heat shock technique and was then plated on LB-Amp (100 ug/mL ampicilin) plates and grown overnight at 37 C. Successful colonies were picked and transferred to a 3 mL liquid LBBroth and shaken overnight at 300 rpm at 37 C. This liquid culture was then tested for possessing a vector with approximate size of 4.5 kb (vector and insert) using a crude mini-prep (reagents from Qiagen) and then agarose


44 gel electrophoresis. Those that appeared to have said band were then digested with PstI and EcoRI to separate the insert from the vector (XmaI was not used because it is unnecessarily expensive and the EcoRI and PstI cut sites are close enough to not be noticed on agarose gel electrophoresis. Successful ligations following diges tion would have two bands, one representing the vector with an approximate size of 3.5 kb and one representing the insert with an approximate size of 1 kb. More liquid cultures for colonies that passed the digestion test were produced, their vector DNA purified using a perfect prep (kit purchased from Qiagen) and then sent to Macrogen, USA for sequencing. The sequencing primers used were: 5-AAG CGC AGG TGC TTC GTC GG-3 and 5-ATC TGA GGC AGC TGG AGT CGA TCG-3. For detailed protocols see Appendix A. Expression of Recombinant Protein Expression was induced using Isopropyl -D-1-thiogalactopyranoside (IPTG) purchased from Fisher BioTech. A quick expression test was us ed to determine which of the successfully ligated colonies was producing more protein than the others. The colony that appeared to be producing the most recombinant protein (with an approximate MW of 30 kDa) was then selected for future use, while glycerol stocks of it as well as the other colonies were stored in 0 C storage. Expression was further tested to determine if temperature, IPTG concentration, and time would affect whether or not the protein was in the


45 soluble or insoluble fractions after th e cells were lysed and the fractions separated. Expression could be observ ed via SDS-PAGE or Western Transfer. See Appendix A for detailed protocols. Purification of Recombinant Protein Purification of recombinant proteins was performed using an affinity column utilizing the His-tag engineered into the protein by the pQE30Xa vector. The His-tag strongly binds Ni-NTA Agarose (purchased from Qiagen). The insoluble fraction was made solubl e using a urea buffer or a guanidium HCl buffer (which proved cost prohibitive and not effective enough to justify its continued use). The Ni-NTA Agarose was added to this solution (urea buffer and insoluble fraction) and subsequently passed through a column which was then washed and eluted using a pH gradient. Purification could be observed on via SDS-PAGE or Western Transfer. See Appendix A for detailed protocols. Refolding of Recombinant Protein Several attempts were made to refold the recombinant protein and gain activity. In most cases dialys is using Spectra/Por dialysis tubing purchased from Spectrum having a molecular weight cut-off of 6000-8000 daltons was utilized. Various methods of stepping down the urea concentration to induce folding spontaneously, as well as greatly diluting the concentration of protein to assist with refolding, were attempted. Following refolding protocols, concentrating the product was accomplished using an affinity column and Ni-NTA agarose or using YM-10 membrane and Amicon


46 Concentrator. Experiments were perform ed using reducing agents to reduce the disulfide bonds. Detergents usin g Tween-20 or Triton X-100 were used in different experiments to see if this could dissolve the inclusion bodies and then removing the detergent with Bio-Beads SM-2 Adsorbents from Bio-Rad. Including acetone in the refolding buffers was attempted. Refolding experiments were attempted with ZnII, CaII, both, or neither. All relevant protocols can be found in Appendix A. Following refolding protocols a sample was tested for peptidase functionality using Leucineparanitroaniline (L-pNA) in pH 8.0 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulf onate (HEPES ) buffer with 2 mM Ca(NO3)2. Activity was monitored at 405 nm. Another sample from the refolding product was treated with Factor Xa protease to remove the His-tag and then purified using Amicon Spin Columns. Following purification the sample was again tested for peptidase activity. This was performed to ensure that the His-tag was not affecting peptidase activity. Protocols may be found in Appendix A. 2.2 ScAP in Streptomyces lividans system Isolating Insert DNA from pUC57 Following the example set by Ni et al.52 the gene was designed to include the pre-pro sequence from Streptomyces limosus -amylase and Streptomyces griseus protease B. This designed gene was custom made by Genescript and delivered in the pUC57 vector. The pUC57 vector was transformed into XL1-Blue E. coli and grown on LB-Amp plates. One


47 successfully transformed colony was then picked and used to inoculate a 500 mL LB-Amp broth in order to be used with a Maxi-Prep (Qiagen) to isolate large quantities of the pUC57 vector (with the gene of interest). The pUC57 vector was then digested using NcoI and SacI. This separated the vector DNA from the insert (gene of interest) DNA. The vector DNA and insert DNA were then separated using agarose gel electrophoresis. The agarose was then separated from the DNA using MO-BIOs Ultraclean Gel Spin Kit. All relevant protocols can be found in Appendix B. Cloning The vector used for expression in S. lividans, pD730, as well as a sample of S. lividans was graciously received from Dr. David Wilson, Ph.D and Dr. Diana Irwin, Ph.D from Cornell University. This vector required a total digest with SacI and a partial digest with NcoI. Following digestion the vector was separated using agarose gel electrophoresis and MO-BIOs Ultraclean Gel Spin Kit. The pD730 vector was treated with shrimp alkaline phosphatase and then purified using Amicon spin columns. A ligation reaction was then carried out to ligate the gene of interest to the new vector. The ligation reaction product was then used to transform XL1-Blue E. coli which were grown overnight at 37 C on LB-Amp plates. Successfully transformed colonies were then used to inoculate 3 mL cultures to be used in a crude mini-prep. Samples from the crude mini-prep that displayed bands on an agarose gel corresponding to approxim ate size of 8 kb were considered


48 candidates for the digestion. Upon di gesting with SacI a successfully ligated vector and insert construct would have a band 1 kb larger than the vector alone. These were then selected and sent to MWG sequencing. Four sequencing primers were ut ilized. Two of the primer s would bind outside the gene of interest, on the vector, which would sequence into the gene. These primers were 5-TCG AAT CCT GCG GAA GGA G-3 and 5-GGT ACC GCA TGC CAT ATG GA-3. The other two primers would bind inside the gene of interest and sequence out towards the vector DNA. These primers were 5-ACA CGA GTC GTG AAG CC-3 and 5-GGG TTA CTT CGT CTA GGA CG-3. After sequencing it was apparent that the gene would be sequenced out of frame. As such site directed mutagenesis primers were designed to delete a single G nucleotide, thus putting the entire pept ide back into frame. These primers were 5-CCT GCG GAA GGA GCC CCA CCA TGA TGC GCA TCA AGC GGA CC-3 and 5-GGT CCG CTT GAT GCG CAT CAT GGT GGG GCT CCT TCC GCA GG-3. Site directed mutagenesis was performed using reagents from QuikChange II from Stratagene. All relevant pr otocols can be found in Appendix B.


49 Chapter III: SGAP RESULTS AND DISCUSSION At the time when this project was started the exact genomic sequence of Streptomyces griseus aminopeptidase (SgAP) was unknown. As such designing primers in order to use PC R to extract the insert from the S. griseus genome was not possible. In retrospect, there is the potential to use degenerative primers for PCR. Essentially, a primer for every possible combination of codons is created and the mix used for PCR. Whichever of the combinations is the successful will bind and produce the desired inserts while the incorrect ones will not bind an d not produce insert fragments. That said, there are obvious cost issues, as well as further complications from having competing primers. The genome of Streptomyces coelicolor was known, however, and it had previous ly been shown to have a putative aminopeptidase similar to SgAP. Moreover, the active sites in both enzymes were identical, and as such the promiscuous activity seen in SgAP is likely to also occur in ScAP. Thus, ScAP became the target because the DNA sequence was known and primers for PCR could be designed to isolate the ScAP gene while they could not currently be designed to isolate the SgAP gene. Running the PCR product on a 0. 7% agarose gel (Figure 3-1) clearly shows that the PCR produced a gene with an approximate size of 900 bp, the appropriate size for the target insert.


Figure 3-1 : 0.7% agarose gel of the product of PCR Reaction The bright bands are situated well between the 750bp and 1kb bands on the molecular weight marker 50


51 Following ligation 28 of the success fully grown colonies were picked and grown in liquid cultures and their vector DNA isolated using a crude mini prep protocol. This vector DNA was ru n on an agarose gel to determine the size of the recombinant vector and compare that size with the pQE-30 Xa vector. The insert DNA having a size of 900 bp and the pQE-30 Xa vector having a size of 3.5 kb it would be po ssible to see a difference between the 4.5 kb construct (vector and insert) from the vector itself (Figure 3-2). Crude mini prep products that exhi bited an upward shift and the pQE30 Xa were digested with EcoRI (located just before the His-tag of pQE-30 Xa) and PstI (part of the multiple cloning site of pQE-30 Xa). EcoRI and PstI were chosen instead of XmaI and StuI for fiscally responsible reasons (XmaI) and because the cut site is not present after the blunt end ligation (StuI). If the insert were in the construct a band of approximately 900 bp would be seen. If, however, there were no insert no DNA would appear below the vector band because the lost DNA would be too small to appear on an Agarose Gel (Figure 3-3). Colonies 13, 16, 17 and 22 were processed through a perfect prep and the product DNA sent to Macrogen for sequencing. Of which colony 16 proved the most successful (Figure 3-4 through Figure 3-6).


Figure 3-2 : 0.7% agarose gel of the product of the crude mini prep. The last lane in both levels of the gel represents the vector by itself (pQE-30 Xa) in order to be compared to the products of the crude mini prep. Lanes with the construct wo uld exhibit an upward shift. Lanes labeled 3, 9, 10, 11, 15, 16, 17, 22 and 25 exhibit potential and so were selected for further processing. 52


Figure 3-3 : EcoRI and PstI digest of crude mini prep product and pQE-30 Xa vector Clearly samples 9, 10, 11, 13, 15, 16, 17, and 22 have the appropriate band at 1kb. The band is not at 900bp as was said before, but this likely is the result of slight increased from cutting at EcoRI and PstI rather than XmaI and StuI. 53






Figure 3-7 : Successful purification MW= molecular weight marker, C1, C2, D, and E coorespond to washes and elutions as discussed in protocols in Appendix A. FT= Flow through. 56


57 Clearly, based upon the sequencing data, the translation of it, and its alignment with ScAP the cloning was su ccessful. As such colony 16 became the focus for expression, purification and refolding attempts. Based upon the purification shown in Figure 3-7, it is evident that purification was successful, however, so much protein is being produced that all the Ni-NTA agarose used during the purification process is saturated with His-tagged protein and thus the excess His tagged protein is lost in the flow through and wash steps. Thus indicating that more Ni-NTA agarose should be used in future purifications. After an other successful purification, dialysis was performed on the E fraction to remove the denaturant so that the protein might refold spontaneously. After multiple changes of the dialysis buffer a precipitate formed and coated the dialysis bag but was easily dislodged into the dialysis buffer. Cent rifugation separated the dialysis buffer from the precipitate and samples of both supernatant and precipitate were taken, and mixed with 2x SDS-PAGE buffer. The results of which are shown in Figure 3-8.


Figure 3-8 : Failed refolding protocol due to precipitation. MW = molecular weight, E= fraction E before dialysis, S= dialysis supernatant, P= dialysis precipitate. 58


59 Unfortunately, the majority of the protein seemed to have been in the precipitate. Furthermore, the little pr otein that remained in the supernatant, upon concentrating through a YM-10 membrane showed no activity with LeupNA. Regarding the identity of the second band in the E fraction it seems possible that it is the result of some proteolytic cleavage of ScAP during purification possibly by some enzyme native to E. coli or another unknown phenomenon. After trying unsuccessfully many times to refold the protein and achieve activity it was found that expression at 25.7 C and 0.1 mM IPTG shifted some of the expressed protein to the soluble fraction (while the majority still was in the insoluble fraction). The purification proved challenging however, as shown in figure 3-9. Purification of the soluble fraction was never achieved to the level that was attained by the insoluble purification Nonetheless, the elution fractions were tested for aminopeptidase activity against Leu-pNA but activity was not detected. To ensure that this was not due to the His-tag Factor Xa was utilized to remove the His tag. To ensure the removal of the His tag a Western blot was used. The western blot clearly shows that the His tag was removed by Factor Xa (Figure 3-10). The Factor Xa protease reaction dilutes the sample by half, and given the sensitivity of the Western blotting technique it is impossible to have the quality bands we have in the untreated lanes and nothing in the


Figure 3-9 : Purification from soluble fraction From Left to right: MW, Wash 1, Elutions 1 though 4. The dark band on the bottom is lysozyme, a byproduct of the purification and lyses of the cellular wall 60


Figure 3-10 : Western blot results from soluble purification and Factor Xa. A side by side comparison of the SDS-PAGE Gel, and a western blot attained from an identical gel. Lanes in both (from right to left) correspond to: Molecular weight marker, Wash 1, Elutions 1 through 4, 4Units Factor Xa 2 hours, 4Units Factor Xa 3 hours, 10U Factor Xa 2 hours, 10U Factor Xa 3 hours, 20U Factor Xa 2 hours, 20U Factor Xa 3 hours. 61


62 treated bands based simply upon a 50% reduction. As for the smaller bands that show up in the last three elution samples it is likely that these are the result of proteolytic cleavage during the purification process by an unknown peptidase. The new bands shown in the treated lanes represent the Factor Xa. Factor Xa is 44kDa, however this is really two polypeptides connected by a disulfide bond. The SDS-PAGE loading dye reduce s the disulfide bond thereby separating the larger polypeptide from the smaller one, hence the large band at approximately 30 kDa and the sma ller band above the lysozyme band. Testing the Factor Xa treated samples with Leu-pNA revealed no activity. Following months of attempts to get the insoluble fraction to refold to an active enzyme and the lack of activity with the soluble fraction enzyme it became apparent that the possibility that the protein cannot fold correctly in E. coli should be investigated. An article by Henderson et al55 compared two putative enzymes from S. griseus that revealed that the propeptide is likely vital for secretion of proteases in S. griseus Given that SgAP is a putative enzyme it too must have a propeptide region, although neither DNA nor amino acid sequence has yet been published. Furthermore, a fully folded protein is not able to be secreted as it cannot diffuse through the plasma membrane. Therefore, in the case of S. griseus the protein does not fold in the cytosol of the bacteria but rather fold s as it is excreted in the medium in which the bacteria is growing. Moreover, the protein may not fold correctly as it is translated on the ribosome, that is, in S. griseus with the propeptide


63 sequence intact, chaperone proteins bind and prevent folding and so the first sequences to be translated might not fold until later parts have been able to fold. The active conformation may not be the lowest potential energy, but rather located in an energy trough the result of folding in a particular order. In the E. coli expression system used herein, the peptide lacks a propeptide sequence and furthermore given the very different physiology of S. griseus and E. coli the propeptide sequence from S. griseus may not have any corresponding chaperone proteins in E. coli. As such, expressing ScAP in an expression system more closely related to the native system may yield more positive results. One technique for doing this was published by Ni et al52 in which they expressed and purified recombinant Streptomyces griseus in S. lividans As the propeptide sequence was unknown Ni et al. designed their own using the amino terminus of the signal peptide from S. griseus protease B and the carboxy terminus of the signal peptide from S. limosus alpha-amylase. The exact sequence of the propeptide sequence of Ni et al. was not published, however, a propeptide sequence using hints in their article was put together (Figure 3-11). The construct sequence for rScAP was received in a plasmid and thus required digestion the use of agarose ge l separation and purification (Figure 3-12). The vector pD730 was generously supplied by Dr. David Wilson and Dr. Diana Irwin from Cornell University. pD730 is designed for multiplication in both E. coli and S. lividans but only for expression in S. lividans In




Figure 3-12: rScAP digest out of pUC57 vector as shown on 0.7% Agarose Gel Lane 1: Molecular Weight Marker (F rom bottom: 500 bp, 1 kb, 1.5 kb, 2.0 kb, 3.0 kb, 4.0 kb, 5.0 kb, 6.0 kb, 8.0 kb, 10.0 kb) Lane 2: pUC57 (superco iled and nicked circular) Lane 3: pUC57 Digested with SacI Lane 4: pUC57 Digested with SacI and NcoI. Insert is lower band with ~1000bp Lane 5: Purified product of Insert (faint but present and the concentration can be determined using A260 65


66 preparation for ligation it needed to be digested and the correct fragment separated by agarose gel electrophoresi s and purified from the gel (Figure 313). The vector pD730 had to be subjected to a partial digest protocol because it had various NcoI recognition sequences. The fragment required for ligation had a size of 7619 bp while the entire vector has a size of 7654 bp. As such, there would be no way to separate them, however, the incorrectly cut fragments would all be considerably smaller, the closest of which would be 6.3 kb. The highest band on the gels with the double cuts is a mixture of two populations, the first a single cut vector, and a double cut (correct two cuts). The ratio between these two populations depends on the amount of time exposed to NcoI and as such for purification out of the gel the 20 minute sample was chosen. It appears that 2 hours of exposure to the second restriction enzyme is enough to remove the resi dual uncut vector there should therefore be no difference in population between the 20 min NcoI/2 hour SacI and the 20 min NcoI /24 hour SacI top bands. As such purification was chosen on the 20 min NcoI/2 hour SacI sample and the concentration was determined by A260. Ligation with the vector should no t prove a difficult situation despite the vector being a mixed population of single cut vectors and correctly double cut vectors. The logic behind this is first that the insert can only ligate with a correctly cut vector due to compatibility issues between the ends. Secondly, the vector is trea ted with Calf intestinal alkaline phosphatase. This removes the phosph ates from the 5 ends of the DNA


Figure 3-13: Partial digest of pD730 on a 0.3% Agarose gel Lane 1: Molecular weight marker (F rom bottom 500 bp, 1.0 kb, 1.5 kb, 2.0 kb, 3.0 kb, 4.0 kb, 5.0 kb, 6.0 kb, 8.0 kb, 10.0 kb) Lane 2: uncut pD730. Lane 3: pD730 digested with Sac. Lanes 4-6: 10min exposure to NcoI with varied exposure to SacI. Lanes 7-9: 15min Exposure to NcoI with varied exposure to SacI. Lanes 10-12: 20min Exposure to NcoI with varied exposure to SacI. 67


68 which prevents easy self ligation. This prevents the vector from self-ligating and therefore becoming a working vect or that can transform bacteria but yields no value due to the lack of an insert. Successfully transformed bacteria that grew on LB-Amp agar plates were grown in liquid broth cultures and their DNA extracted and digested with SacI. The product of was run on an agarose gel and is displayed on Figure 3-14. While in no way is the Figure 3-14 gel perfect, especially when compared to above Figure 3-12 and Figure 3-13, it is not used by itself to determine which bacterial conlonies were successfully transformed. Sequencing is the only way to truly determine if ligation was successful as it will also show if the sequence is in frame and if expression will proceed correctly. The determined sequen ce is shown in Figure 3-15. The translation from colony five did not code for the rScAP as expected, Figure 3-16. Upon evaluation of the Sequencing DNA the reason for this becomes apparent in that the G as highlighted in Figure 3-15 is extra, a byproduct of engineering an NcoI cut site so the insert could be used with pD730. This extra G nucleotide throws the sequence out of frame. At the time of writing this our laboratory is currently working on eliminating this extra G nucleotide, the new translation of which is expected to push eliminate the frameshift. The translation of the expected sequence without the extraneous G is shown in Figure 3-17, and the alignment with the designed construct (figure 3-11) is shown in Figure 3-18.


Figure 3-14 : Crude Mini prep digested with SacI. Dark horizontal line indicates furthest region the Vector lane reached. Samples with band above this line may have insert ligated and ar e selected for sequencing. Therefore, samples 2, 5 and 15 were chosen. 69






72 Chapter IV: CONCLUSION TO SGAP Like countless biochemists and bi ologists before, the complex and still unknown mechanisms of protein folding have caused infinite frustration and near psychosis for all involved. Gi ven the previous success of Ni et al.52 in expressing the recombinant SgAP in a similar expression system to the native SgAP there is hope that using S. lividans as an expression system for ScAP will prove fruitful. At present a plasmid that can easily be modified (deleting the superfluous G nucleotide ) is available. Furthermore, by following the roadmap set forth by Ni et al.52 acquiring the first unmutated recombinant ScAP should be merely a matter of time. Considering the simplicity of doing site directed mutagenesis the mutants should follow quickly. The only foreseeable difficulty could be the time to learn and adjust the laboratory accordingly to using a new bacterium ( S. lividans ) including learning the new techniques for transformation and expression. Isolating the protein after expression should prove simple as S. lividans will secrete rScAP into the growth medium and the presence of the engineered His-tag on rScAP will facilitate easy purification.


73 Chapter V: INTRODUCTION TO HISTATIN-5 N-TERMINUS 5.1 Antimicrobial Peptides (AMPs) One of the most interesting class of peptides are the antimicrobial peptides (AMP), owing to their broad spectr a of microbicidal functionality. Alternatively, they are known as cationic host defense peptides .55 In addition to the antimicrobial activity, AMPs have various essential functions in the hosts innate immunity. Thus, the la tter name better describes its overall function. AMPs can be ubiquitously found in nature, ranging from plants to humans.55 The exact classification for AMPs is broad; in general, the length of the peptide ranges anywhere from 12 to 50 amino acids, with 29 positvely charged residues and at most 50% hydrophobic residues.55 Furthermore, they are commonly thought to have similar three dimensional structures due to the similarities of their compositions. In hydrophobic environments, such as that of the plasma membrane, they generally have sheet or -helix conformations. Both secondary structures are well suited for membrane spanning roles, which is a common antimicrobial mechanism among AMPs and is necessary for many of them to function successfully as antimicrobials.56-58 The amphipathic nature of these peptides enables insertion into the membrane, which leads to pore formation. This, in turn,


74 disrupts the integrity of the cell membrane, leading to cell lysis and loss of metabolites to the extracellular environment.59-62 It is interesting that AMPs are selectively active toward the foreign but not host cell; this feat is accomplished by the slight difference in the composition of celluar membranes. The bacterial membranes are more negatively charged on the surface, thus enabling the positively charged AMPs to interact quite strongly. However, this is not how all AMPs work. They have also been proposed to play major roles in the hosts innate and adaptive immunity,63-66 including lysing of tumor cells,67 preventing excitotoxicity caused by lipopolysaccharide(LPS),68 and acting as chemical markers during antiinflammatory responses and angiogenesis.68 Furthermore, some AMPs function in a fashion similar to normal antibiotics; they bind to the microbial enzyme or protein and inhibit its functi on. Examples include attacins, which block the transcription of omp gene in E. coli ,69 Buforin II, which binds DNA and RNA,70 or pyrrhocoricin, which binds and inactivates the chaperone DnaK.71 It seems only natural that AMPs have generated a storm of commercial interests. The potential for possible therapy, combined with the alarming decrease in efficacy for current generat ion of antibiotics have brought money and enthusiasm to this search. Unfortunately, only few AMPs have made significant progress, in part to the need for high doses and the low therapeutic index, making them unacceptably dangerous for medicinal use.72 Nonetheless, a couple of successful AM Ps have been brought to the market. These have a common feature in that they are topical, enabling them to have


75 a high local but low systemic concentration.73 Further research may lead to a better understanding both its toxicity to humans and interaction with microbial targets, which could lead to more pharmaceutical and commercial interests. 5.2 Histatin-5 Histatins (Hs) are found in the saliva of higher primates and named due to their histidine rich character.74-75 The wide range of microbicidal activity of Hs includes one of the most prevalent fungi, Candida albicans,76-78 and several families of bacteria.79 Interestingly, only two separate genes are responsible for the expression of histatins. These genes, HTN1 and HTN2, encode the primary sequence for hist atin-1 and histatin-3, respectively.80 The remaining Hs variants (up to 50) found in the whole saliva are the products of post translational modifica tion, namely proteolytic cleavage, of either Hs-1 or Hs-3.81 Histatin-5, for example, consists of the first 24 amino acid residues of Hs-3 (DSHAKRHHGY10 KRKFHEKHHS20 HRGY).81 One interesting aspect of Histatin-5 is that while it is clearly antimicrobial, its exact mechanism remains under debate. There are three schools of thought currently on this su bject. The first being proposed by Edgerton et al.82-85 is that the histatin-5 induce s the non-lytic loss of ATP, which then binds to purinergic-like receptors which initiate apoptosis. In this mechanism, the peptide interacts with th e heat shock proteins (Ssa1/2) of C. albicans and is internalized. Once inside the cell it binds to the potassium transporter (TRK1) which leads to the lo ss of cellular integrity, and cellular


76 death.82-85 The second proposed mechanism by Oppenheim et al.64-65 involves the breakdown of the yeasts respiratory machinery. In this mechanism the peptide is internalized due to the electrochemical gradient (the inside of the cell bei ng negative and histatin-5 being very positive). Once inside, it migrates to the mitochondria and is again internalized via a similar mechanism. Histatin-5 is able to disrupt the electron transfer process of the mitochondria which leads to the accumulation of reactive oxygen species, oxidative stress and finally cellular death.86-87 A third mechanism has more recently been proposed by Mochon et al.88 which contradicts both Oppenheim64-65 and Edgerton82-85 by saying that Histatin-5 does indeed cause a lytic event, although is unconventional in how it does that. In this mechanism physio logical levels of Histatin-5 were shown to create a single breach in the membrane allowing the rapid loss of ATP and K+. This single breach site is the result of the fact that Histatin-5 acts this way only because of the membrane potential. Upon creation of one lytic site, the membrane potential is lost and th erefore a second lytic site is not possible.88 Amino acid residues such as histidine, serine, tyrosine, and acidic aspartate and glutamate are well known to participate in metal binding.89 The primary sequence of Hs 5 consists of seven histidines, two serines, two tyrosines, one glutamate, and one aspa rtate, creating multiple potential metal-binding sites.81 The presence of multiple metal-binding sites has been confirmed by a number of studies us ing calorimetric, and spectroscopic


77 techniques. According to previous studies, there are three common metalbinding motifs, which are the zinc-binding HEXXH13 the copper binding HXXXH ,90 and the copperand nickel-binding motif ATCUN (Amino Terminal CuII and NiII Binding) consisting of DSH.91 Gusman et al.92 used spectrophotometric competition assays to determine whether Hs-5 binds both CuII and/or ZnII. This study showed that free murexide, a metallochrome, in so lution absorbs maximally at 520 nm, and upon addition of CuII or ZnII, the peak shifts to 500 nm, indicating the metal-murexide complex formation. Addition of Hs-5 to this solution shifted the absorption maximum back to 520 nm, which clearly indicates the removal of metal ion from the original complex by the peptide to give free murexide. The absorption at 520 nm due to free murexide was further confirmed by using EDTA, a good metal chelator, in place of the Hs-5 peptide.92 Melino et al.93 used UV-vis spectroscopy to show the similarities between Histatin-5 N-terminus metal binding and that of the ATCUN region of human serum albumin. In this study, the disruption of the ATCUN site by substituting His-3 into Ala-3 drastically changed the UV-Vis spectrum.93 The major flaw of both these studies is that it did not go far enough into investigating to what stoichiometric ratios does metal bind. Given the bountiful nature of Hs-5s primary structure with regard to its possible metalbinding ligands, it seems obvious that this peptide can bind more than one metal ion. Nonetheless, these studies overlooked or discounted such


78 possibility. Brewer and Lajoie94 investigated the potentials of Hs-5 in complexing with more than one metal ion using electrospray ionization mass spectrometry (ESI-MS). The mass difference between one versus multiple metal ion bound complexes can be determined using ESI-MS. In addition, the study clearly showed that Hs-5 has di fferent affinities for different metal ions under different conditions. Fo r instance, with 5:1 metal-topeptide stoichiometric ratio in solution three CuII ions were bound to Histatin-5 (but no FeII).94 With a change in the pH from 6.5 to 7.0 another CuII ion is bound.94 The ratio of metal to peptide used in preparation greatly affects the number of bound metals.94 For instance when prepared with a 2:1 CuII to peptide ratio only one CuII ion is bound, whereas at a 10:1 ratio four CuII ions are bound.94 Again, this study is limited to only the metal-binding ability of Hs-5 and does not provide mechanistic information toward its in vivo antimicrobial activity. The above potential mechanisms, particularly the ones proposed by Mochon et al.88 and Edgerton et al.,82-85 provide an interesting situation that arises when one takes in to account the metal binding characteristics of Histatin-5. Both Mochon and Edgerton emphasize the importance of the positive charge on Histatin-5. For one, the positive charge is necessary for the peptide to interact with the negati vely charged outer leaflet of the plasma membrane. Second, the positive charge provides a necessary function in making entrance into the cell favorable based on the net negative charge inside the cell. Changing Lys-13 to Glu and Arg-22 to Gly decreased candidacidal activity significantly.95 It was later shown that these changes


79 effect the entrance into the cell and this could be the cause of the decreased candidacidal activity.88 The overall charge of the peptide is certainly important for its function as an AMP. Would metal binding affect this activity by neutralizing the apparent charges? If so, what metal binding species is present in the human salivary environment? Further, would this species lack sufficient charge to attack C. albicans as Edgerton and Mochon proposed? In consideration of the metal-binding ability of Hs-5 it is essential to determine the possible role of CuII in the in vivo antimicrobial activity of Hs-5. In fact, a previous study suggested a possible invo lvement of metal, particularly Zn, in the interaction of Hs-5 and cellular membranes. The study showed that Hs-5 can selectively fuse the negatively charged small unilamellar vesicles in the presence of Zn.96 In order to investigate how the binding of metals affects the catecholase properties of Hs-5, Tay97 used UV-vis spectroscopy to monitor catechol oxidation (using 3m ethyl-2b enzo t hiazolinone h ydrazone Hydrochloride as an indicator for o-quinone, the oxidative product of catechol) by Hs-5 upon addition of various ratios of CuII. It was evident that there was no activity until greater than 1 equivalent of CuII was added and a steady increase in activity was observed until approximately 8 equivalents of CuII were added.97 In context of the ESI-MS study,94 it seems unlikely that all 8 equivalents of CuII were bound to the peptide, but rather that maximal activity should fall somewhere between 3 and 4 CuII ions bound. This study was further refined using the 4,5-di-chloro-catechol (DCC) as the substrate. The benefit of using DCC is that cataly tic turnover of DCC is approximately


80 200-fold smaller than that of catechol. As such, any metal-ligand-substrate complex that forms should exist long enough to be observed using optical UV-Vis spectroscopy; whereas catechol would form complex and react too quickly to be well observed. The result of this experiment revealed a 3:1 DCC:CuII 4-Hs-5 ratio. This infers that three metal centers exist in the peptide each of which can bind DCC. Tay97 was further able to assign metal binding sites using CoII titration as monitored by NMR. However, in this experiment only two metal sites were assigned, the first being the HEXXH ZnII binding motif (or for that matter the HXXXH CuII-binding site) and the second being the DSH ATCUN site. The third site was not apparent using NMR with up to 6 equivalents of CoII. This of course, based on the ESI-MS study by Brewer and Lajoie,94 may be a result of the rather large differences that may arise with a variation in metals used. At a 4:1 metal-to-peptide ratio using CuII we may see three metals bind, but with CoII perhaps only two may bind. Further titration of CoII may have revealed the third binding site. These questions however are outside of the scope of this current study. The primary interest is not in its ability to kill C. albicans but rather in its ability to utilize metal to perform oxidative chemistry. In recent years, there has been a great deal of interest in the study of peptides that have the ability to catalyze enzymatic reactions. The idea is that these peptides can be utilized as model systems to study the mechanisms for the more complex proteins. The goal of my research is to reveal greater insight into copper ion chemistry using Histatin-5 as a natura l system. So, while there is obvious direct medical relevance in studying an y AMP, the exact nature of this study


81 deals not with the medical significance of the subject, but rather with the Histatin-5s other unique properties, namely oxidative chemistry under near physiological conditions in vitro In determining the oxidative properties of the full length Hs-5 three potential metal binding sites for CuII were observed. Theoretically, by synthesizing the peptide in two separate fragments one might be able to better isolate the proposed metal binding sites. From this better characterization of the metal binding sites, their affinities and oxidative properties may be determined.


82 Chapter VI: HISTATIN-5 N-TERMINUS MATERIALS AND METHODS Histatin-5 and the N-terminal histatin-5 fragment (Hs-5N) were synthesized and purified and characterized at the University of South Florida Peptide Synthesis and Mass Spectroscopy Center. The concentration was determined using A280 with =1440 M cm. Anhydrous copper sulfate and HEPES was purchased from Fisher Scientrific. pyrocatechol (~99%) and 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (98%), MBTH, were acquired from SigmaAldrich Inc. The o-quinone from catechol oxidation reacts with MBTH to form a colored adduct that with a maximum absorption at 500 nm ( = 32500 M cm). All spectroscopic studies were conducted on a Cary-50 UV/Vis spectrometer unless stated otherwise. 6.1 Metal Binding Studies Metal binding was monitored in two ways. The first method was CuII titration while monitoring catechol ox idation at 500 nm. In the second method the formation of CuII-Hs-5N complex was monitored directly over 200-800 nm range. Catechol oxidatio n experiments were conducted over a range of 0.0-5.0 equivalents of CuII with 1.0 mM catechol and equal molarity of the oquinone-specific indicator (MBTH) in 100 mM HEPES at pH 7.0 with


83 1.01 M Hs-5N. Optical CuII titration was performed using a CuII range of 0.0-20.0 equivalents with 100 M Hs-5N in 100 mM HEPES at pH 7.0. 6.2 Kinetics Studies Kinetics experiments were monitore d at 500 nm from the red MBTHo quinone adduct. Catechol and MBTH were varied from 0.0 to 6.4 mM were incubated with 1.0 M Cu2 II-Hs-5N in 100 mM HEPES at pH 7.0. The initial rate was determined from the slope of the change in absorbance versus time over a period of 0-5 minutes in the linear range. Kinetic constants were determined using Sigma Plot 11 by fittin g data to the appropriate equations. A mechanistic Job plot was constructed by continuously varying the mol fraction between the CuII-Hs5N complex and the substrate, while keeping the overall concentration constant. Kinetic studies were also performed in the presence of hydrogen peroxide, H2O2. In one experiment H2O2 was titrated over a range of 0.0 to 32.0 mM while holding constant the catechol, MBTH and CuII-Hs5N constant at 3.2 mM, 3.2 mM and 1.5 M, respectively. For another series of experiments the peroxide concentration was held constant while varying the catechol and MBTH concentrations (between 0.0 to 6.0 mM catechol/MBTH). The oxidation of catechol was determined in the presence of several concentrations of H2O2 ranging 0.0-32.0 mM. 6.3 DCC Binding Studies 4,5-di-chloro-catechol, DCC, was used to study the substrate binding due to its significantly smaller reactivi ty compared to catechol, in order to monitor the interaction between this substrate and the Cu2 II-Hs-5N complex.


84 DCC was dissolved in DMSO due to its low solubility in water or buffer. Up to 3 equivalents of DCC was titrated into Cu2 II-Hs-5N complex. The binding was monitored over the 200-800 nm. An optical Job plot was also attained as described above.


85 Chapter VII: HISTATIN-5 N-TERMINUS RESULTS AND DISCUSSION Metal binding to the full length Histatin-5 (Hs-5) was investigated previously and was able to concretely determine two metal binding sites, the ATCUN region as well as the copper ion binding domain (HXXXH).97 In the same study Tay determined that each hi statin-5 peptide can bind more than 2 metal ions.97 In analyzing the metal binding to Hs5N the goal was to determine the stoichiometry of metal binding to the Hs5N fragment using both optical studies as well as kinetics to determine the redox active species. Upon addition of CuII to the Hs5N solution a pea k at approximately 525 nm appears. Subsequent addition of CuII increased the intensity of the 525 nm peak until 2.0 equivalents of CuII. Further addition of CuII shifted the peak towards the longer wavelengths. This shift is caused by the CuII filling the second binding site (absorbance around 600nm). This data is displayed in Figure 7-1. The absorption at 52 5 (A525) versus concentration of CuII is shown in Figure 7-2. From Figures 7-1 and 7-2 two valuable pieces of information are acquired. First, that th e N-terminal region of Histatin-5 has two CuII binding sites. Second, the dissociation constant, Kd, for CuII binding Hn5N is 6.37 x 10 M. The absorbtion maximum at 525 nm is indicative of a copper center with square planar geometry.98


Cu Binding Optical TitrationWavelength (nm) 400500600700800 Abs 0.000 0.005 0.010 0.015 0.020 0.025 Figure 7-1: Optical titration of CuII binding to Hs5N Optical titration of CuII with 100 M Hn5N in 100 mM HEPES pH 7 and 25 C, which displays main peak at 525 nm, with shift to wards 800 nm after 2 equivalents of copper ion(relative to Hs-5N). The shif t towards 800 nm is likely the result of increased absorption by the free CuII ion in solution. Each spectra represents a 0.2 equivalents of CuII from 0-2 equivalents followed by 0.4 equivalents from 2-4.4 equivalents. 86


Molarity 0.00000.00010.00020.00030.00040.0005 Absorption 0.000 0.005 0.010 0.015 0.020 [Cu(II)]M vs A525 [Cu(II)]M vs A650 Figure 7-2: CuII binding to Hs5N. Titration of CuII showing saturation of the A525 binding site when ratio CuII to Hs-5N is 2:1. 100 M Hn5N, 100 mM HEPES pH7 and 25.0 C. The A525 was fit to a quadratic equation, thus 1:1 binding with a KD of 6.37 x 10 M. 87


While the ability of Hs-5N to bind copper is important, more important is how this copper activates Hs-5N to promote oxidative catalysis. There was no increase in activity over the uncatalyzed reaction until greater than one equivalent of copper was added. Considering the flexible stretch of peptide between the two potential metal binding sites it is possible that with one copper bound the peptide might fold over the copper ion in such a way as to prevent the substrate from binding. It is further possible that one CuII center is simply insufficient for catecholase activity; catecholase activity requires a two electron transfer and therefore a single CuII to CuI (single electron transfer) is not enough as seen with tyrosinase. This is a similar phenomenon that Tay observed.97 The rate at various concentrations of catechol and MBTH while keeping the Cu2 II-Hs-5N complex concentration constant (at 1.0 M) in 100 mM HEPES pH 7.0 at 25 C (Figure 7-4) was tested. This study showed increased catalytic rate until saturation at higher levels of substrate. The data displays Michaelis-Menten like steady state kinetics and thus can be well fitted to the Michaelis-Menten equation: CAK CAVNHs Max 5 0 Where Vo is the observed rate, Vmax is the maximum rate, KHs5N is the Michaelis-Menten constant, and [CA] is th e concentration of substrate, in this case catechol. This is based upon the assumption that substrate 88

PAGE 100

Catechol Oxidation by His5N with Cu EqEq. Cu 024681 01 2 v500 (M/s) 0.0 5.0e-9 1.0e-8 1.5e-8 2.0e-8 2.5e-8 3.0e-8 3.5e-8 Eq Cu vs Rate (M/s) Figure 7-3: Activity profile for ca techol oxidation with CuII titration. Equivalents of CuII titrated into 1 M Hn5N with 1 mM Catechol/MBTH in 100 mM HEPES pH 7.0 and 25 C. 89

PAGE 101

concentration is much greater than the complex concentration, i.e. [catechol]>>[Hs5NCuII]. Further assumed is that the reaction proceeds thusly: 90 [Cu(II)-Hn5-CA] [Cu(II)-Hn5] + [CA] [Cu(II)-Hn5] + o-quinone k1k-1kcat Based upon the data in Figure 7-4, it is possible to determine the KHs5N to be 3.2 x 10 M, the kcat to be 2.20 x 10 s, and the second order rate constant (kcat/KM) to be 6.88 M s. This represents a 4.6 x 104 rate enhancement over the autooxidation of catechol (4.74 x 10 s). In an effort to better understand the interaction between the metalpeptide complex and the substrate, the slow reacting substrate, DCC, was utilized in an optical study (Figure 7-5). During this study, DCC was titrated into the 1.0 mL cuvette that contained 100 M Complex (2:1) in 100 mM HEPES pH 7. Upon addition of DCC to the 2:1 complex two new absorption peaks appears at 306 nm and 426 nm both of which saturate at a 1:1 ratio DCC to complex. It appears that the Cu2 II-Hs-5N complex can bind a single equivalent of DCC. This finding can be further supported by Figure 7-6 in which a Job plot is shown. The Job plot in Figure 7-6 supports the one to one substrate binding first proposed by the optical DCC titration study shown in Figure 7-5. It makes sense that the Cu2 II-Hs-5N complex can bind just a single substrate makes sense considering the proposed mechanism of catechol oxidation.

PAGE 102

Since catechol oxidation to oquinone requires a twoelectron transfer, of which each CuII may contribute one (becoming CuI), and Hs-5N can bind two copper ions, it seems primed particularly for a single substrate reaction. Finally, the effect of hydrogen peroxide on the Cu2 II-Hs-5N system was explored. Previous studies have shown that hydrogen peroxide acts as a second substrate,97 and increases the rate of catechol to o-quinone. Similar studies were carried out to determine if this second substrate interacts with the metal center on the N-terminus of Hi statin-5. First, an experiment with increasing concentration of H2O2 (0-32 mM) while holding the concentration of the 2:1 complex and Catechol/MBTH constant at 1.5 M and 3.2 mM, respectively. This demonstrated that saturation of hydrogen peroxide was possible and thereby implying Michael is-Menten like steady-state kinetics (Figure 7-7). The data were fit to th e modified Michaelis-Menten equation to determine the relevant constants: 22 22 022OHK OHVOH Max Background The KH2O2 is 5.4 x 10 M, a value 1.7 times higher than the KHs5N for catechol, therefore displaying slightly weaker affinity for hydrogen peroxide (or a higher kcat). The kcat for H2O2 catalyzed reaction is 4.5 x 10 s, thus giving a second order rate constant (kcat/KM) of 8.3 s M, an enhancement of 2.0 and 1.2, respectively, compared to catechol oxidation by Hs-5N without H2O2. 91

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Michaelis Menten His5N[Catechol] M 0.000 0.002 0.004 0.006 v500 M/s 0.0 2.0e-9 4.0e-9 6.0e-9 8.0e-9 1.0e-8 1.2e-8 1.4e-8 1.6e-8 1.8e-8 [Catechol] M vs avg rate (M/s) Figure 7-4: Graph of increasing catalytic rate with increasing substrate concentration until saturation. 1.0 M Cu2 II-Hs-5N complex in 100 mM HEPES pH 8.0 25 C. The data is fitted to a the Michaelis-Menten equation in order to determine rate constants KM, kcat, and kcat/KM. 92

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DCC binding His5nWavelength (nm) 280300320340360380400 Abs 0.0 0.2 0.4 0.6 DCC Binding by His5N optical at 320nm[DCC] M 0.0000 0.0001 0.0002 0.0003 A320 0.0 0.1 0.2 0.3 0.4 0.5 Figure 7-5 : DCC Binding by Optical Spectroscopy Optical of DCC binding studied with 100 M Cu2 II-Hs-5N complex in mixture HEPES and DMSO (in order to keep DCC soluble). Saturation appears slightly over 1 equivalent DCC. DCC binding was fit to a quadratic equation giving a dissociation constant of 16 M. 93

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Mol Fraction Hn5N/( mol DCC+ mol Hn5N) Absorbance 0.45 0.50 0.55 0.60 0.65 0.70 Figure 7-6: Optical Job Plot Job plot based on change of absorbance at 306 nm as the mol fraction of His5N is varied. Experimental conditions: 0200 M Hs5N (0-400 M CuII, i.e. 2:1 complex), 0-200 M DCC, 100 mM HEPES pH 7.0 and 25 C. 94

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[Catechol] = 32 mM[H2O2] M 0.00 0.01 0.02 0.03 v 500 M/2 0 2e-8 4e-8 6e-8 8e-8 Col 1 vs jan 11 h202 titration x column 1 vs y column 1 Figure 7-7: Influence of hydrogen peroxide on catechol oxidation Catechol oxidation by Cu2 II-Hs-5N complex in the presence of varied amounts of hydrogen peroxide in 100 mM HEPES pH 7. Data display Michaelis-Menten like kinetics and so is fit to the modified Michaelis-Menten equation, from which relevant constants KM, kcat, and kcat/KM can be obtained. 95

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To better elucidate the manner in which the two substrates, hydrogen peroxide and catechol, influence the binding of the other to the active site, varying concentrations of catechol were incubated with 1.5 M Cu2 II-Hs-5N complex with varying concentrations of hydrogen peroxide. This study provided multiple graphs that showed Michaelis-Menten-like kinetics as a function of either [Catechol] or [H2O2] (as shown in Figure 7-8). Furthermore, a second graph can be attained from the same data, that is a The basic Michaelis-Menten equation, as stated above, can be replotted in a linear relationship in accordance with the Hanes-Woolf plot: 96 Catechol K V K OH V Catechol K OHInt Catechol App OH App Catechol1 1max 22 max 0 2222 From which two secondary plots may be attained, one which relates the yintersect of all the lines on Figure 7-9 part A to the inverse of the catechol concentration (Figure 7-9 part B) and an other which relates the slopes of the same lines to the inverse of the catechol concentration (Figure 7-9 part C). From combining data represented on these graphs the various affinity constants may be ascertained, which are depicted in Table 1, as well as a comparison to the affinity constants in previous studies.99 Catechol V KK V K yInt Catechol App OH App OH1 intmax max22 22

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[Catechol] M 0.000 0.002 0.004 0.006 V500 (M/s) 0.0 2.0e-8 4.0e-8 6.0e-8 8.0e-8 1.0e-7 1.2e-7 [Catechol] (mM) vs Rate M/s 1mM [Catechol] (mM) vs Rate M/s 2mM [Catechol] (mM) vs Rate M/s 4mM [Catechol] (mM) vs Rate M/s 8mM [Catechol] (mM) vs Rate M/s 16mM [Catechol] (mM) vs Rate M/s 32mM Figure 7-8 : Catechol oxidation by various concentrations of catechol and various concentrations of hydrogen peroxide 1.5 M complex (2:1) with varying amounts of catechol (0-6.0 mM) and hydrogen peroxide (0-32 mM) in 100 mM HEPES pH 8.0. The graph clearly shows saturation of hydrogen peroxide above 16mM. From this data the Hanes-Woolf plot shown in Figure 6-9 is accomplished. 97

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[Catechol] -1 M -1 050010001500200025003000 y-intercept 0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5 3.0e+5 3.5e+5 [Catechol]-1 M-1 050010001500200025003000 Slope 0.0 2.0e+7 4.0e+7 6.0e+7 8.0e+7 1.0e+8 1.2e+8 [H2O2] M 0.00 0.01 0.02 0.03 [H2O2]/v s 0 1e+6 2e+6 3e+6 4e+6 5e+6 6e+6 0.2mM Catechol 0.4mM Catechol 0.8mM Catechol 1,6mM Catechol 3.2mM Catechol 6.0mM Catechol Figure 7-9: Hanes-Woolf Analysis Hanes analysis of the oxidation of catechol by Cu2 II-Hs-5N complex in the presence of varied amounts of hydrogen peroxide based upon data presented in Figure 7-8. The smaller submissive plots below are a replot of the various data represented in the larger dominatrix plot above. From which the values in Table 1 were gathered. 98

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Catechol V K V slopeApp Catechol1 1max max There are a few interesting things to note based on the data presented in Table 1. First, the kcat for the catechol oxidation reaction with H2O2 shows a 1.97 x 105 fold enhancement versus the autooxidation reaction. Second, the apparent KH2O2 is 33 % higher than the intrinsic KH2O2 while the apparent Kcatechol is nearly unchanged than the intrinsic Kcatechol. This indicates that the binding of H2O2 has no effect on the binding of catechol, while the binding of catechol slightly inhibits or blocks the binding of H2O2. Third, in essence this is similar to the results previously shown99 for the full length peptide, the only difference is the degree to which the apparent KH2O2 is lowered relative to the intrinsic value. This indicates th at the catalytically relevant region of the peptide, in so much as the activity in which we are measuring, may largely take place in the N-terminus, and not in the C-terminus. 99

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100 Constant Histatin-5 N terminus Histatin-5 Full length97 kcat with H2O2 9.33 x 10 s Not reported Apparent KH2O2 4.21 x 10 M 5.23 10 M Intrinsic KH2O2 3.16 x 10 M 2.26 x 10 M Apparent KCatechol 3.42 x 10 M 3.63 x 10 M Intrinsic KCatechol 3.55 x 10 M 3.06 x 10 M Table 7-1: Comparison of Hs-5N to full length Hs-5. The valuable affinity constants and catalytic rate constants ascertained from the Hanes-Woolf Plot compared to the ones collected by Dr. William Tay for the full length peptide.97

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101 Chapter IIX: HISTATIN-5 N-TERMINUS CONCLUSION This study focused essentially on reducing the metal binding sites in the Histatin-5 peptide system which is well known for antimicrobial activity, candidacidal and bactericidal. The aim of this was to determine whether the amino terminus of Hs-5 showed the ox idative activity of the full length peptide. In many ways, it mirrored the study done by Dr. Tay,97 but the goal was to determine what truly is active in histatin-5. As was discussed earlier, the primary sequence of hi statin-5 shows many sites which might bind metal and therefore be vital for the oxidatio n activity displayed by histatin-5. Bisecting the sequence effe ctively halves the metal binding sites to two sites, the ATCUN region and the HHGY sequence (the most likely candidate knowing that Hs5N binds two metals, one of which we know is the ATCUN region). From the data presented, there can be no doubt that the Nterminus has the ability to bind CuII, a redox-active metal, and the substrate (catechol), but furthermore shows similar affinity constants, as well as kinetic parameters (kcat and kcat/KM) and peroxide influences (Table 7-1) of the full length peptide. With this in mind, it is reasonable to say that the N-terminus is responsible for the oxidative activity se en in the full length peptide. It is impressive to see such a small and simple system that is able to perform such complex chemistry attaining a 1.97 x 105-fold rate enhancement.

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113 [79] Murakami, Y.; Xu, T.; Helmerhorst, EJ.; Ori, G.; Troxler, RF.; Lally, ET.; Oppenheim, FG. (2002) Inhibitory effect of synthetic histatin 5 on leukotoxin from Actinobacillus actinomycetemcomitans. Oral Microbiol Immun 17:143-149 [80] Sabatini, L. M.; Ota, T.; Azen, E. A. (1993) Nuleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family. Mol. Biol. Evol. 10:497511. [81] Xu, L.; Lai, K.; Santarpia, R. P. ; Pollock, J. J. (1993) Salivary proteolysis of histidine-rich polypeptides and the antifungal activity of peptide degradation products. Arch. Oral Biol. 38: 277283 [82] Edgerton, M.; Koshlukova, S. E.; Lo, T. E.; Chrzan, B. G.; Straubinger, R. M.; Raj, P.A. (1 998) Candidacidal activity of salivary histatins. J. Biol. Chem. 273 : 2043820447 [83] Koshlukova, S. E.; Lloyd, T. L.; Araujo, M. W. B.; Edgerton, M. (1999) Salivary Histatin 5 induces non-lytic release of ATP from Candida albicans leading to cell death. J. Biol. Chem. 274: 1887218879 [84] Li, X. S.; Reddy, M. S.; Baev, D.; Edgerton, M. (2003) Candida albicans Ssa1/2p is the cell envelope binding protein for human salivary histatin 5. J. Biol. Chem. 278: 2855328561

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114 [85] Baev, D.; Rivetta, A.; Vylkova, S.; Sun. J. N.; Zeng, G.-F.; Slayman, C. L.; Edgerton, M. (2004) The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, histatin 5. J. Biol. Chem. 279: 5506055072. [86] Helmerhorst, E. J.; Troxler, R. F.; Oppenheim, F. G. (2001) The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species. Proc. Nat. Acad. Sci. 98: 1463714642. [87] Helmerhorst, E. J.; Breeuwer, P.; vant Hof, W.; Walgreen-Weterings, E.; Oomen, L. C. J. M.; Veerman, E. C. I.; Amerongen, A. V. N.; Abee, T. (1999) The cellular target of histatin 5 on Candida albicans is the energized mitochondrion. J. Biol. Chem. 274: 7286291. [88] Mochon, A.B.; Liu, H. (2008) The Antimicrobial peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm PloS Pathog 4(10) [89] Glusker, J.P. (1991) Structural aspects of metal liganding to functional groups in proteins. Adv. Protein Chem. 42: 1-76 [90] Todd, R.J.; Van Dam, M.E.; Ca simiro, D.; Haymore, B.L.; Arnold, F.H. (1991) Cu(II)-binding properties of a cytochrom c with a synthetic metal-binding site: His X3His in an alpha-helix. Proteins 10:156-161

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115 [91] Peters Jr., T. (1960) Interaction of one mole of copper with the alpha amino group of bovine serum albumin. Biochim. Biophys. Acta. 39 :54647. [92] Gusman, H.; Lendenmann, U.; Grogan, J.; Troxler, R.F.; Oppenheim, F.G. (2000) Is salivary Histatin-5 a metallopeptide? Biochimica et Biophysica Acta 1545:86-95 [93] Melino, S.; Gallo, M.; Trotta, E.; Mondello, F.; Paci, M.; Petruzzelli, R. (2006) Metal-binding and nuclease activity of an Antimicrobial peptide analogue of the Salivary Histatin-5. Biochem. 45:15373-15383 [94] Brewer, D.; Lajoie, G. (2000) Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ioniza tion mass spectrometry. Rapid Comm. In Mass Spec 14: 1736-1745 [95] Tsai, H.; Raj, PA.; Bobek, LA. (1996) Candidacidal activity of recombinant human salivary histatin-5 and variants. Infect Immun. 64: 5000-5007 [96] Melino, S.; Rufini, S.; Sette, M.; Morero, R.; Grottesi, A.; Paci, M.; Petruzzelli, R. (1999) Zn2+ Ions Selectively Induce Antimicrobial Salivary Peptide Histatin-5 Fuse Negatively Charged Vesicles. Identification and Characterization of a ZincBinding Motif Present in the Functional Domain. Biochem. 30: 9626-9633

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116 [97] Tay, W.M. (2008) Metallopept idases As Model Systems for the Study of Cu(II)-Dependent Oxidation Chemistry. University of South Florida [98] Peters Jr., T.; Blumenstock, F. (1967) Copper-binding Properties of Bovine Serum Albumin and Its Amino Terminal Fragment. J. of Biol. Chem. 242: 1574-1578

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117 Appendices

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PAGE 130


PAGE 131

120 Appendix A (Continued) PCR Protocol for obtaining insert from S. coelicolor genomic DNA Master Mix (for single sample) *Done in PCR hood 1) Nano pure Water 28.0 L 2) 5x Taq Master 10.0 L (must first be thawed 15min at 65C) 3) 10x Taq Buffer 5.00 L 4) 10.0mM dNTP 1.00 L 5) 5-primer 0.50 L (stock solution 55.1 M) 6) 3-primer 0.50 L (stock solution 40.4 M) 7) Taq Polymerase 0.50 L PCR Sample Tubes *Done in PCR Hood 1) Master Mix 46.5 L 2) Nano pure Water 1.50 L 3) Mg2+ 1.00 L 4) Genomic DNA 1.00 L *Not added in PCR Hood (Need to find concentration of this) PCR Cycles 1) Hold at 95C for 3.5 minutes 2) Heat to 95C for 30 seconds 3) Cool to 50C and hold for 30 seconds 4) Heat to 72C and hold for 1.5 minutes

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121 Appendix A (Continued) 5) Repeat steps 2-4 for a total of 35 cycles 6) Hold at 72C for 10minutes 7) Cool to 4C until removed and stored for future use Purifying DNA using Amicon Spin Columns 1) Place spin column on tube. 2) Add 300 L nano pure water into reservoir a. Adjust based on sample but no less than 300, so total volume is 400 L 3) Add no more than 100 L sample reaction to reservoir 4) Place assembled device into centrifuge with cap strap inward 5) Centrifuge at 1000xg for 15 minutes 6) Remove from centrifuge save filtrate until sample has been analyzed 7) Place reservoir upright into clean vial. Add 20 L TE. 8) Invert reservoir into the clean vial and centrifuge at 1000xg for 2 minutes. 9) The sample should now be pure and ready for downstream applications

PAGE 133

Appendix A (Continued) A-4: pQE-30 Xa Vector Map from The Qiaexpressionist 122

PAGE 134

123 Appendix A (Continued) Restriction Digest Protocol Vector Digest Add to 0.65mL Eppendorf Tube: 1) Nano Pure Water 67.0 L 2) pQE-30 Xa 20.0 L (Stock Solution at 189ng/ L) 3) Buffer B 10.0 L 4) BSA 2.00 L 5) XmaI 0.50 L Then: 1) Place in 37C Water bath for 6.0 hours 2) Add 0.50 L StuI 3) Return to 37C Water bath for 16.0 hours 4) Purify product using Amicon Spin Columns (see above) Insert Digest Add to 0.65mL Eppendorf Tube: 1) Nano Pure Water 33.0 L 2) Insert DNA 10.0 L 3) Buffer B 5.00 L

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Appendix A (Continued) 4) BSA 1.00 L 5) XmaI 1.0 L Then: 1) Incubate at 37C for 6 hours 2) Purify using Amicon Spin Columns (See Above) Ligation Protocol Ligation Buffer: 1) Nano Pure Water 22.5 L 2) 1.0M Tris pH 7.5 50.0 L 3) 1.0M DTT 10.0 L 4) 100mM ATP 10.0 L 5) MgCl2 5.00 L 6) BSA 2.50 L Reaction Tubes: 1) Nano Pure Water 24.9 L 2) Ligation Buffer 3.00 L 3) Ligase 1.00 L 124

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125 Appendix A (Continued) 4) Insert 2.57 L (Concentration 202.5ng/ L) 5) Vector 0.50 L (Concentration 202.5ng/ L) This creates a 20:1 ratio, 20 inserts per vector Then: 1) Hold at 17.0C for 14-18 hours 2) Proceed to Transformation without purification Transformation Protocols XL1-Blue Transformation 1) Thaw glycerol stocks of cell line (stored at -80C) on Ice 2) Place 1.5mL eppendorf tubes on ice to pre-chill 3) Add 80 L XL1-Blue to 1.5mL Eppendorf tube 4) Add 2.00 L ligation reaction product 5) Mix well by flicking or brief vortexing 6) Hold on ice for 30.0 minutes 7) Heat shock at 42C for 45 seconds 8) Replace in ice for 2.0 minutes 9) Pipette 41.0 L onto agar plate with antibiotics (Ampicilin at 100 g/mL) a. Two plates per transformation 10) Grow at 37C for approximately 24 hours

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126 Appendix A (Continued) Rosetta Blue Transformation and BL21 Transformation 1) Thaw glycerol stocks of cell line (stored at -80C) on Ice 2) Place 1.5mL eppendorf tubes on ice to pre-chill 3) Add 20.0 L Rosetta Blue thawed cells to 1.5mL Eppendorf tube 4) Add 1.00 L ligation reaction product 5) Mix well by flicking or brief vortexing 6) Heat shock at 42C for 45 seconds 7) Incubate on Ice for 2.0 minutes 8) Add 80.0 L room temperature SOC medium 9) Shake at 250rpm, 37C for 1 hour 10) Dilute transformation into more SOC medium a. 10 L transformation with 30 L SOC, total volume of 40 L b. 20 L transformation with 20 L SOC, total volume of 40 L 11) Spread onto LB-AMP agar plates and incubate at 37C for approximately 24 hours 3mL overnight culture protocol 1) Add 3mL LB-AMP (100 g/mL) broth to sterilized test tube 2) Pick single colony off agar plate using sterilized toothpick 3) Add toothpick to 3mL LB-AMP broth in test tube 4) Shake in incubator at 300rpm, 37C for 16-18 hours (overnight)

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127 Appendix A (Continued) Crude Mini Prep Protocol 1) Centrifuge 1.0-1.5mL of 3.0mL overnight culture at 14,000xg for 1.0 minute 2) Decant supernatant, remove residual supernatant with pipette 3) Add 150 L Buffer P1 (with RNase A). Resuspend by vortexing 4) Add 150 L Buffer P2. Invert to mix. Hold at room temperature 5.0 minutes 5) Add 150 L Buffer P3. Invert to mix. Hold on Ice 5.0 minutes 6) Centrifuge at 14,000xg, 4C for 10.0 minutes 7) Add supernatant to 1.0mL ethanol (100%). Incubate -80C for 1 hour 8) Centrifuge at 14,000xg, 4C for 15.0 minutes 9) Decant supernatant. Wash pellet with 200 L 70% ethanol 10) Centrifuge at 14,000xg for 5.0 minutes 11) Decant 70% ethanol supernatant and air dry pellet 12) Add 30 L TE and dissolve pellet Perfect Mini Prep Protocol 1) Centrifuge 1.0-1.5mL of 3.0mL overnight culture at 14,000xg for 1.0 minute 2) Decant supernatant, remove residual supernatant with pipette 3) Add 100 L Solution 1 to the cell pellet. Resuspend by vortexing

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128 Appendix A (Continued) 4) Add 100 L Solution 2. Invert to mix. 5) Add 100 L Solution 3. Invert to mix. 6) Centrifuge at 14,000xg for 30 seconds. Transfer supernatant to spin column. 7) Vigorously and thoroughly mix the DNA binding matrix suspension before pipetting. 8) Add 450 L DNA binding matrix to the supernatant in the spin column. Mix by capping the spin column and inverting vigorously 9) Centrifuge the spin column assembly at 14,000xg for 30 seconds. Decant filtrate and replace spin column into the collection tube. 10) Add 400 L DILUTED purification soluti on to same spin column. Cap the tube, shake briefly and centrifuge at 14,000xg for 60 seconds 11) Decant filtrate and place spin column back into collection tube. Centrifuge at 14,000xg for 60 seconds to remove residual Purification solution. 12) Transfer spin column to fresh collection tube. Add 50-70 L of Elution Buffer directly to the DNA binding matrix in the spin column. Cap the assembly and vortex briefly. 13) Centrifuge at 14,000xg for 60 seconds.

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Appendix A (Continued) 14) Discard spin column and cap the collection tube. Eluted plasma DNA is ready for downstream applications or can be stored at 20C. Agarose Gel Electrophoresis Preparing the Gel (0.7%) Add to microwave safe Erlenmeyer Flask 1) 1X TAE 50mL (Volume Varies by gel rig) 2) Agarose 0.35g (depends on gel percentage) a. 3) Swirl to Mix 4) Microwave on High for 15 second intervals, swirling between intervals, until all agarose has dissolved. 5) Let cool 1-2 minutes, add 20 L ethidium bromide 6) Pour into gel rig, with comb, and let rest until polymerization is complete Running Conditions (general, may be adjusted as ne eded by samples) 1) 150 Volts 2) 35 minutes 129

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130 Appendix A (Continued) Overexpression protocol 1) To sterilized Erlenmeyer flask (250mL for this example) add 99mL Fresh LB-AMP (100 g/mL) broth 2) Add 1.0mL of 3.0mL overnight culture to 100mL Fresh LB-AMP 3) Shake in incubator at 300rpm, 37C until OD=0.4-0.6, use 1.0mL Fresh LB-AMP to blank spectrophotometer. a. While growing make IPTG solution. b. Add 0.024g IPTG to 3.0mL LB-AMP broth, invert to dissolve i. Makes 3mL of 33.6mM solution 4) Take uninduced sample, 1.0mL. Centrifuge at 14,000xg for 1.0 minute, remove supernatant and re-suspend pellet in 50 L SDSPAGE loading buffer. a. Put sample in boiling water bath for 5-7 minutes 5) Add IPTG solution, so final concentration is 1mM a. Based on above, add 3mL IPTG solution to 98mL culture (99mL (Fresh LB-AMP)+1.0mL (Overnight Culture) -1.0mL (to test OD)-1.0mL (for uninduced sample) b. Optional to take time point samples at 1, 2, 3, 4 etc. hours 6) Centrifuge remainder at 10,000xg for 20min to pellet cells. 7) Store in -20C until ready for use for purification

PAGE 142

131 Appendix A (Continued) Purification of ScAP under dena turing (for insoluble fraction) conditions 1) Thaw cells on Ice for 15.0-20.0 minutes 2) Re-suspend pellet in 15mL bacterial cell wall lysis buffer (for a 100mL overexpression sample) by vortexing or pipetting. a. 2-5mL cell wall lysis buffer per gram wet pellet weight 3) Add 1.7 mg lysozyme per mL of cell wall lysis buffer a. Optional to add protease inhibitors: i. 250 L 10mM PMSF ii. 250 L 10mM Benzamidine 4) Shake on ice for 60.0 minutes 5) Sonicate 60 seconds, take sample for SDS-PAGE 6) Pellet cells at 10,000xg, 4C, 20 minutes. a. Save supernatant for SDS-PAGE 7) Add 15mL Urea buffer to cell pellet and resuspend. a. Take sample for SDS-PAGE 8) Incubate 1.0 hour at 37C shaking at 200rpm. Take sample for a. SDS-PAGE 9) Centrifuge at 10,000xg, 4C, 20 minutes 10) Add 1mL Ni-NTA Agarose, shake on ice 45.0 minutes

PAGE 143

132 Appendix A (Continued) 11) Add to column and collect flow through. a. Take sample for SDS-PAGE 12) Wash 2x 4mL with Buffer C (pH 6.3) a. Take sample for SDS-PAGE 13) Elute with 2mL Buffer D (pH 5.9) a. Take sample for SDS-PAGE 14) Elute with 2mL Buffer E (pH 4.5) a. Take sample for SDS-PAGE 15) Run samples from above on SDS-PAGE. Purpose: a. Find target protein and Determ ine which elution fraction has bulk of target protein b. Ensure purification is working properly c. Determine if any modifications need to be made. 16) Optional modifications include a. adjust the volumes for washing or eluting. b. More or less Ni-NTA Agarose c. Adjusting volume of Urea Buffer d. Replacing Urea Buffer with Guandine Buffer

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133 Appendix A (Continued) Purification of ScAP insoluble frac tion using Detergents to denature 1) Thaw cells on Ice for 15.0-20.0 minutes 2) Re-suspend pellet in 15mL bacterial cell wall lysis buffer (for a 100mL overexpression sample) by vortexing or pipetting. a. 2-5mL cell wall lysis buffer per gram wet pellet weight 3) Add 1.7 mg lysozyme per mL of cell wall lysis buffer a. Optional to add protease inhibitors: i. 250 L 10mM PMSF ii. 250 L 10mM Benzamidine 4) Shake on ice for 60.0 minutes 5) Sonicate 60 seconds, take sample for SDS-PAGE 6) Pellet cells at 10,000xg, 4C, 20 minutes. a. Save supernatant for SDS-PAGE 7) Resuspend pellet in 10mL Cell Wall Lysis Buffer (no lysozyme) with 30 L Tween-20 (0.3%) a. Also tried .1% Tween-20 8) Add Stir bar and stir vigorously in fridge for 4 hours 9) Add 5 grams SM-2 Bio-Beads and stir in fridge overnight 10) Centrifuge at 10,000xg, 4C for 20 minutes

PAGE 145

134 Appendix A (Continued) Another Purification protocol using detergents to denature 1) Thaw cells on Ice for 15.0-20.0 minutes 2) Re-suspend pellet in 15mL bacterial cell wall lysis buffer (for a 100mL overexpression sample) by vortexing or pipetting. a. 2-5mL cell wall lysis buffer per gram wet pellet weight 3) Add 1.7 mg lysozyme per mL of cell wall lysis buffer a. Optional to add protease inhibitors: i. 250 L 10mM PMSF ii. 250 L 10mM Benzamidine 4) Shake on ice for 60.0 minutes 5) Sonicate 60 seconds, take sample for SDS-PAGE 6) Pellet cells at 10,000xg, 4C, 20 minutes. a. Save supernatant for SDS-PAGE 7) Resuspend pellet in 10mL Cell Wall Lysis Buffer (no lysozyme) with 30 L Tween-20 (0.3%) a. Also tried .1% Tween-20 8) Add Stir bar and stir vigorously in fridge for 4 hours 9) Add 5 grams SM-2 Bio-Beads and stir in fridge overnight 10) Remove liquid with as few biobeads as possible and run through column to remove remaining biobeads

PAGE 146

135 Appendix A (Continued) 11) Add 1mL Ni-NTA Agarose to flow through and shake on ice for 45.0 minutes 12) Add to column collect flow through, wash and elute as for soluble fraction (see below) Purification of ScAP under native (soluble fraction) conditions 1) Thaw cells on Ice for 15.0-20.0 minutes 2) Re-suspend pellet in 15mL bacterial cell wall lysis buffer (for a 100mL overexpression sample) by vortexing or pipetting. a. 2-5mL cell wall lysis buffer per gram wet pellet weight 3) Add 1.7 mg lysozyme per mL of cell wall lysis buffer a. Optional to add protease inhibitors: i. 250 L 10mM PMSF ii. 250 L 10mM Benzamidine 4) Shake on ice for 60.0 minutes 5) Sonicate 60 seconds a. Take sample for SDS-PAGE 6) Pellet cells at 10,000xg, 4C, 20 minutes. 7) Supernatant is soluble protein fraction. a. Take sample for SDS-PAGE 8) Add 1.0 mL Ni-NTA agarose to supernatant in Erlenmeyer flask. 9) Shake on Ice for 45.0 minutes

PAGE 147

136 Appendix A (Continued) 10) Add to column and collect flow through a. Take sample for SDS-PAGE 11) Wash 2x4mL with Wash buffer a. Take sample for SDS-PAGE 12) Elute 4x0.5mL with Elute buffer a. Take Sample for SDS-PAGE 13) Run SDS-PAGE 14) Optional modifications include a. adjusting the volumes for washing or eluting. b. More or less Ni-NTA Agarose Cell Wall Lysis Buffer 1) 50.0mM NaH2PO4 2) 1.0M NaCl 3) 10.0mM imidazole 4) pH 8.0 Urea Buffer (insoluble fraction) 1) 8M Urea 2) 100mM NaH2PO4 3) Triton X-100 (500 L per 50mL total volume)

PAGE 148

137 Appendix A (Continued) 4) 10.0mM Tris pH 8. 5) Adjust pH to 8 using NaOH Guanidine Buffer (insoluble fraction) 1) 6M Guanidine 2) 100.0mM NaH2PO4 3) 10.0mM Tris 4) Adjust pH to 8.0 Buffers C, D, and E (insoluble fraction) 1) 100mM NaH2PO4 2) 10mM Tris-CL 3) 8M Urea (6M Guanidine can be substituted) a. Buffer C pH adjusted to 6.3 b. Buffer D pH adjusted to 5.9 c. Buffer E pH adjusted to 4.5 Wash Buffer (soluble fraction) 1) 50mM NaH2PO4 2) 300mM NaCl 3) 20mM imidazole 4) Adjust pH to 8.0 using NaOH

PAGE 149

138 Appendix A (Continued) Elution Buffer (soluble fraction) 1) 50mM NaH2PO4 2) 300mM NaCl 3) 250mM imidazole 4) Adjust pH to 8.0 using NaOH Protein Refolding for insoluble fraction after purification Protocol Basic Refolding protocol 1) Add appropriate fraction (based on SDS-PAGE) into dialysis bag 2) Place dialysis bag (A pproximately 2mL) in 150mL beaker with Dialysis Buffer a. 50.0mM Tris b. 100.0mM NaCl c. Adjust pH to 8.0 d. Possible modifications i. 50mM Glycine ii. 2mM Beta-mercaptoethanol iii. Also substituted 50.0mM Tris with 100mM HEPES 3) Change buffer 4 times with at le ast 4 hours in between changes 4) Potential modifications a. Adding 50 M ZnCl2

PAGE 150

139 Appendix A (Continued) b. Adding 1mM CaCl2 c. pH 7.5 d. Diluting the appropriate fraction i. Ex. 1mL Fraction E + 39mL fr esh Buffer E placed into dialysis bag and dialyzed against Dialysis buffer (note that this requires a larger beaker for dialysis and therefore more dialysis buffer ii. Also tried 2mL Fraction E +198mL Buffer E iii. The goal of this was to prevent the protein from precipitating with the fast removal of denaturant (Urea or Guanidine e. Use a stepwise removal of denaturant i. Dialysis buffer one contains 6M urea, ii. Dialysis buffer two contains 3M urea, iii. Dialysis buffer three contains 1M Urea iv. Dialysis buffer four contains no urea, etc. Renaturation Protocol 1) Dilute 2mL Buffer E into 98mL Renaturation Buffer a. 20mM Tris b. 100mM NaCl c. pH 8.0

PAGE 151

140 Appendix A (Continued) 2) Shake on Ice 30.0 min 3) Add 1mL Ni-NTA Agarose 4) Shake on Ice 30.0 min 5) Add to column and collect flow through 6) Elute with 2mL Elution buffer (Same as for soluble fraction purification Elution Buffer see above) 7) Dialyzed against 2x250mL Buffer a. 20mM Tris b. 100mM NaCl c. 50 M ZnCl2 d. 1mM CaCl2 e. pH 8 Modified Renaturation Protocol 1) Instead of eluting insoluble fraction from column with Elution Buffer, add 5-10mL acetone/Guanidine buffer but do not run through column. a. 1.4M Acetone b. 0.7M Guanidine c. 1mM CaCl2 d. 50 M ZnCl2 e. pH 8.5

PAGE 152

141 Appendix A (Continued) 2) Swirl or pipette up and down to suspend His-tagged protein-Ni-NTA Agarose complex into the acetone/Guanidine buffer and remove from column. 3) Add to 40-45mL acetone/Guanidine buffer (total volume approx. 50mL) 4) Shake at 150rpm, 35C for 1.0 hour 5) Add back to column and collect flow through 6) Elute from column with 2mL Elution Buffer (Same as for soluble fraction purification Elution Buffer see above) 7) Dialyze against 2x250mL Buffer a. 20mM Tris b. 100mM NaCl c. 50 M ZnCl2 d. 1mM CaCl2 e. pH 8 Another renaturation attempt 1) Elute from column with Buffer E as stated in insoluble fraction purification protocol 2) Refold in Acetone/Guanidine buffer, shake at 150rpm, 35C for 1.0 hour 3) Add 1mL Ni-NTA Agarose, shake on ice for 45.0 minutes

PAGE 153

142 Appendix A (Continued) 4) Add to column, collect flow through 5) Elute with 2mL Elution buffer (Same as for soluble fraction purification Elution Buffer see above) Western Blotting Protocol 1) Perform SDS-PAGE, 150 volts for 1.5 hours 2) Equilibrate Membrane a. 3 seconds Methanol, b. 1-2 minutes H2O, c. 5-10 minutes western transfer buffer 3) Equilibrate Gel a. 5 minutes H2O b. 10 minutes western transfer buffer 4) Soak pad and paper for transfer in western transfer buffer 5) Assemble western transfer apparatus: a. Black side b. Pad c. Paper d. Gel

PAGE 154

143 Appendix A (Continued) e. Membrane f. Paper g. Pad h. Clear 6) Run in apparatus with ice pack and stir bar, 1.0 hour and 100 volts 7) Soak in 200mL 1X-PBS blotto at room temp for 1.0 hour 8) Soak in 10mL blotto, .5% Tween-20 and 40000-fold dilution of primary antibody a. Primary antibody is RGS-His from Qiagen 9) Wash 3x 30 minutes in 600mL Blotto 10) Soak in 9mL Maleic Acid Buffer + 1mL 10X blocking reagent with 5 L secondary antibody a. Secondary antibody___ 11) Wash 3x 30 minutes with 600mL Maleic Acid buffer with 0.3% Tween-20 12) Add to bag with 1mL BM Purple in dark and soak for 10 minutes 13) Remove from bag and rinse with nano pure H2O

PAGE 155

144 Appendix A (Continued) SDS-PAGE Running Buffer 1) 3g Tris 2) 14.4 g Glycine 3) 10mL 10% SDS 4) Add H2O to 1L Western Transfer Buffer 1) 6.0g Tris 2) 14.4g Glycine 3) 200mL Methanol 4) Add H2O to 1L Maleic Acid Buffer 1) 100mM Maleic Acid 2) 150mM NaCl 3) pH to 7.5 4) Autoclave 15 minutes at 121C 1X-PBS Blotto (1 liter) 1) 100mL 10X phosphate buffer saline 2) 50 grams nonfat dry milk (5%) 3) Add H2O to 1L

PAGE 156


PAGE 157

Appendix B (Continued) B-2: pUC57 Vector Map (contains Insert) 146

PAGE 158

Appendix B (Continued) B-3: pD730 Vector Map 147

PAGE 159

148 Appendix B (Continued) Restriction Digest Protocol pD730 Digest For One Sample (ie volumes can be multiplied accordingly, time adjusted as needed but not a multiple of the ingredients) 1) Add to 0.65mL Eppendorf Tube a. Nano pure H2O 2.7 L b. Multicore Buffer 1.5 L c. BSA 0.5 L d. SacI 0.3 L e. pD730 Vector DNA 10.0 L 2) Place in 37C water bath for 4 hours or overnight 3) Briefly centrifuge to bring conden sation down off tube walls and lid 4) Add 0.3 L NcoI 5) Return to 37C water bath for 20 min 6) Heat inactivate NcoI by placing in 65C water bath for 15.0 min 7) Run on Agarose Gel (0.3%) for maximum separation of high weight bands pUC57 Digest 1) Add to 0.65mL Eppendorf Tube a. Nano pure H2O 2.7 L

PAGE 160

149 Appendix B (Continued) b. Multicore Buffer 1.5 L c. BSA 0.1 L d. SacI 0.3 L e. NcoI 0.3 L f. pUC57 vector DNA 10.0 L 2) Place in 37C water bath for 2-4 hours 3) Heat inactivate NcoI by placing in 65C water bath for 15.0 min 4) Run on Agarose Gel (0.7%) for separation of low weight bands pD730 requires a partial digest due to the multiple cut for NcoI in its sequence (see vector map). Thus the time exposure to NcoI was varied. pUC57, however, does not have this issue and so the NcoI exposure is longer to ensure a complete digest. The appropriate fragment of the vector must be separated from the other bands prior to ligation, hence the agarose gels were used. These bands are then cut out of the gel in the next protocol Isolating DNA from Agarose Gels Uses MO-BIOs Ultraclean Gel Spin Kit 1) Cut band from gel and determine weight, less than or equal to 200mg 2) Place in spin filter basket, add three volumes of Gel Bind a. I.e. if gel weighs 0.2g then add 0.6mL Gel Bind

PAGE 161

150 Appendix B (Continued) 3) Incubate 2 minutes at 55C water bath. Invert to mix, continue to incubate at 1 minute intervals until gel is completely melted. 4) Centrifuge 10 seconds at 10,000xg 5) Remove filter, Vortex flow through for 5 seconds 6) Reload vortexed flow through onto fliter 7) Centrifuge 10 seconds at 10,000xg 8) Discard flow through 9) Add 300 L Gel Wash Buffer 10) Centrifuge 10 seconds at 10,000xg 11) Discard flow through, centrifuge an additional 30 seconds at 10,000xg 12) Discard flow through, add 50 L Elution Buffer. 13) Centrifuge 30 seconds at 10,000xg. Flow through is desired DNA 14) Concentration can be determined using A260 method SAPd Vector (this time with calf intestinal alkaline phosphatase) 1) DNA 20 L 2) Nano pure H2O 65 L 3) 10x CiAP buffer 10 L 4) CiAP 5 L 5) Incubate at 37C 1.0 hour

PAGE 162

151 Appendix B (Continued) 6) Purify using Amicon Spin column (see Appendix A) 7) Check A260 to determine concentration

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Two methodologies in pursuit of the elucidation of copper (II) centered bioinorganic chemistry
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Includes bibliographical references.
Text (Electronic thesis) in PDF format.
ABSTRACT: Copper is a widely distributed transition metal in the earth's crust and has been adopted in a variety of biological systems. In many ways the biochemical usefulness of copper stems from its positive redox potential. This positive redox potential allows copper to assist in the movement of electrons. Copper ions can be found in natural systems as either Cu[superscript I], Cu[superscript II] or Cu[superscript III] in part due to this redox potential. WhileCu[superscript II] -centered biochemistry has been studied for years, mechanistic details in certain Cu[superscript II] -centered redox reactions remain unresolved. This study presents two methodologies for studying natural systems with known Cu[superscript II] -centered redox capabilities in order to better elucidate the mechanistic intricacies of Copper ion chemistry.The first method explored involves the promiscuous enzyme Streptomyces griseus aminopeptidase (SgAP) which although known primarily as a peptidase has been shown to oxidize catechol under near physiological conditions in vitro when its native Zn[superscript II] ions are replaced by Cu[superscript II] ions. Protein engineering techniques were utilized toward expression a functional recombinant enzyme in wild type and mutant forms. The goal was to utilize Site directed mutagenesis of residues in the active site to determine which residues are involved in both the hydrolysis and the oxidative activities of SgAP. The second methodology explored was the use of the N-terminus of Histatin-5, a naturally occurring peptide that is known to form complexes with Cu[superscript II], as a model system to study Cu[superscript II]-centered oxidation chemistry.Metal-Peptide complexes are much more simplified model systems which use the same building blocks as proteins, but reduce the structure to the minimal functional unit necessary for activity. This in turn, simplifies the study of their catalytic chemistry as influences outside of the active region are greatly reduced. Furthermore, chemical synthesis of short peptides is easily performed and inexpensive in comparison to protein engineering, thus enabling further exploration, if deemed necessary, to be a feasible and economically viable possibility.
Mode of access: World Wide Web.
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Co-advisor: Li-June Ming, Ph.D.
Co-advisor: Brian T. Livingston, Ph.D.
Dissertations, Academic
x Chemistry
t USF Electronic Theses and Dissertations.
4 856