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2000 NMR, spectroscopic, and biochemical studies of calmodulin variants

Brokx, Richard D.

Brokx, R. D. (2000). NMR, spectroscopic, and biochemical studies of calmodulin variants (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/12816 http://hdl.handle.net/1880/40536 doctoral thesis

University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca NMR, Spectroscopic, and Biochemical Studies of Calmodulin Variants

Richard D. Brokx

A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREEOFDOCTOROFPHILOSOPHY

DEPARTMENT OF BIOLOGICAL SCIENCES

CALGARY, ALBERTA m,2000

O Richard Brokx 2000 National Library Bibliotheque nationale ($1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. rue Wellington OttawaON K1AON4 OttawaON KIA ON4 Canada Canada Your 6h9 Vorm reference

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Calmodulin (CaM1, the ubiquitous eukaryotic Ca2+-bindingprotein, is well-known and well characterized and can be produced in good yields in a reliable bacterial expression system. In this thesis, the proline analogs 3.4-dehydroproline (Dhp) and azetidine-2-carboxylic acid (Azc) have been biosynthetically incorporated into CaM and calbindin D,, in order to evaluate these analogs as probes of prolines in proteins. The Dhp- substituted proteins were shown to have interesting NMR properties. Additionally, in NMR studies of tripeptides, Dhp and Azc had opposite effects on the thermodynamics and kinetics of prolyl cis-trans isomerization, which demonstrate the potential for the use of these analogs in work on proteins where prolyl cis-trans isomerization is important. The fluorinated amino acids cis-4-fluoroproline, 5,5,5- trifluoroleucine and S-trifluoromethylhomocysteine (trifluoromethionine) have also been incorporated into CaM and studied by fluorine-19 NMR in an examination of the properties of fluorinated aliphatic, as opposed to aromatic amino acids. The Ca"-dependent conformational change in CaM is seen in 19F-NMR spectra of trifluoroleucine- and trifluoromethionine- substituted CaMs. CaM has also been altered in other ways to study its properties. CaM was cleaved with the protease thrombin to create two fragments, TM1 (1-106) and TM2 (197-1481, which were examined by multinuclear NMR, circular dichroism, and gel bandshift assays. TM1 and TM2 can associate in the presence of metal ions to form a structure with enhanced metal ion affinity and more a-helical structures. In the presence of CaM-target peptides, TM1 and TM2 can form an even tighter complex with properties very similar to CaM-peptide complexes. In another study, CaM was dissolved in a 358 2,2,2-trifluoroethd (TFE) solution. Nitrogen15 NMR relaxation studies of CaM in 35% TFE showed that the central linker portion of the protein was more stable and ordered than in CaM in water, but the presence of alcohols is not sufficient to explain why this region of the molecule is a-helical in the crystal structure. The binding of target peptides to CaM was also studied by isothermal titration calorimetry; the enthalpy of binding and the heat capacity change upon binding varies among the peptides studied, which relate to the type of interactions involved in CaM-peptide binding. Acknowledgements

There are a great many people who have given me support and encouragement during my time at the Vogel lab. There is no doubt that this list is incomplete. First, I would especially like to thank my supervisor, Dr. Hans J. Vogel. He has always been incredibly supportive and understanding during the ups and downs of my time as a graduate student. He always gave me the distance to try my own things, but also had the insight to realize when I was on to something interesting. The help from all the other members of the Vogel lab is also acknowledged, including Dr. Tao Yuan, who first taught me the ropes of the calmodulin system when I was starting out, Dr. Elke Lohmeier-Vogel, for her sense of humor and enthusiasm, Dr. Deane McIntyre for his tireless help in equipment maintenance and NMR experimentation, Kirsten Bagh for keeping the lab in order and well-stocked with reagents, Dr. Hui Ouyang for working side-by-side with me, giving me something to measure myself by, Craig Shepherd for putting up with me as an office mate, Dave Schibli and Teresa Clarke for putting up with me as a roommate, Peter Hwang for his help with NMR data analysis, and all the other members of the Vogel lab, both past and present, including Dr. Jim Aramini, Dr. Rob Penner, Jill Saponja, Alexis David (current Vogel Tankard record holder), Aalirn Weljie, Phoebe Franco, Dr. Vladimir Leontiev (Simpsons Trivia runner-up), Dr. Ning Zhou, Dr. Tung Hoang (trout zen master), and Dr. Ryan McKay. Andriyka Papish is especially acknowledged for her help with the work of Chapter 4, and Ahmad Azarnousch is also acknowledged for his help with the work on the incorporation of fluorotyrosines into schistosomal glutathione-s- transferase, although that work is not presented in great detail in this thesis. People in other groups in the department, including Dr. Gene Huber, Dr. Les Tari, Dr. Ray Turner, and Dr. Barry Phipps are also thanked for sharing ideas and equipment. The guidance of my PhD advisory committee is also acknowledged. Like my supervisor, they too gave me the space to do my own thing but also grabbed on when they found something interesting. Dr. Robert Edwards and Dr. Morley Hollenberg have been with me since the beginning, Dr. Gene Huber and Dr. Raghav Yamdagni are thanked for going easy on me during my PhD candidacy exam. Dr. Brian Keay is acknowledged for being on my PhD oral examination committee, and I would especially like to thank Dr. Cheryl Arrowsmith (University of Toronto) for being my external examiner; perhaps we can work together on a project in the future. For financial support, I would like to thank the Natural Sciences and Engineering Research Council of Canada and the Alberta Heritage Foundation for Medical Research for providing me with studentships. Dr. John Honek (University of Waterloo) is thanked for the gift of trifluorornethionine, and Dr. Ileane McIntyre is thanked for the synthesis of cis-4-fluoroproline used in Chapter 4. Dustin Lippert (University of Victoria) and Dr. Gilles Lajoie (University of Waterloo) are thanked for mass spectrometry analysis of protein samples, and Dr. Don McKay (Department of Medical Biochemistry) is thanked for amino acid analysis. Dr. Ruud Scheek (University of Groningen, the Netherlands) and his group are thanked for the 600 MHz NMR spectrometer time and initial help with the work in Chapter 6. Dr. George Makhatadze (Hershey Medical Center, Penn State University) and his group are thanked for the use of their isothermal titration calorimeter and their collaboration with the work in Chapter 7. There is also space here for people who had nothing directly to do with my project. My soccer team (Crispin Jordan, perennial captain) kept me in shape in the summer, and all present and past members of the University of Calgary Curling Club are thanked for the good times in the winter. My buddies Matt, Chad, Kent, and Gord were always fun to be with on weekends. As far as other distractions go, I guess I should thank all of the people who ever poured a beer for me, and the developers of all of the video games I ever played while I was here, but I'm sure if I tried to comprise a list of either of these it would soon get very long and terribly embarrassing. Lastly, of course, I would like to thank my family. My parents have provided me with all kinds of support from afar, both emotional and financial. It has also made me feel really good to know they are so proud. My brother, Walter, made me realize that life should be enjoyed to the fullest, and it's important to define yourself not only by what you do for a living, but also what you do outside of work. My identical twin brother, Stephen, has always been like a best friend, someone to be with and talk to. Talking to him was helpful because even though we do almost exactly the same thing we still found plenty of time to talk about things other than science. To him and everyone else on this list I wish a very bright future.

vii Table of Contents

.. Approval page 11 Abstract iii Acknowledgements v ... Table of Contents Vlll .*. List of Tables Xlll List of Figures xiv List of Abbreviations xvii

CHAPTER ONE: INTRODUCTION Calcium in Biology Calmodulin The Central Linker of Call The Hydrophobic Patches of CaM Prolines and Protein Folding Non-Natural Amino Acid Analogs Introduction to NMR Scope of this Dissertation

CHAPTER TWO: INCORPORATION OF PROLINE ANALOGS INTO CALMODULIN AND CALBINDIN D,, Abstract Introduction Materials and Methods Materials Construction of Mutant pCaMs

viii Protein Expression Purification of Proteins Calcium-Dependent Bandshifts Calcineurin Assays NMR Spectroscopy Results Discussion

CHAPTER THREE: NMR DETERMINATION OF THE KINETIC AND THERMODYNAMIC PARAMETERS OF CIS-TRANS ISOMERIZATION OF PROLINE ANALOGS Abstract Introduction Inversion Transfer - Theory Kinetics and Thermodynamics - Theory Materials and Methods Materials NMR Spectroscopy T, Measurements Inversion Transfer Measurements Results 1D and 2D 'H NMR Spectroscopy and Thermodynamic Measurements Inversion Transfer Measurements Discussion

CHAPTER FOUR: BIOSYNTEETIC INCORPORATION OF FLUORINATED ALIPHATIC AMINO ACIDS INTO CALMODULIN Abstract Introduction Materials and Methods Materials Expression and Purification of Fluorinated CaMs Native Gel of Fluoroproline-CaMs NMR Spectroscopy Results Expression of Proteins Fluoroproline-CaM Trifluoroleucine-CaM Trifluoromethionine-CaM Discussion

CHAPTER FTVE: PEPTIDE AND METAL ION DEPENDENT ASSOCIATION OF THROMBIC FRAGMENTS OF CALMODULIN Abstract Introduction Materials and Methods Materials Preparation of Thrombic Fragments of Calmodulin UV Absorption Spectroscopy Gel Bandshift Assays Circular Dichroism Spectroscopy Enzyme Assays Proton and Carbon-13 NMR Spectroscopy Cadmium-113 NMR Spectroscopy Results W Absorption Spectroscopy Gel Bandshift Assays Enzyme Activation Assays Circular Dichroisrn Spectroscopy Proton and Carbon-13 NMR Spectroscopy Cadmium- 113 NMR Spectroscopy Discussion

CHAPTER SIX: THE BACKBONE DYNAMIC PROPERTIES OF THE CENTRAL LINKER REGION OF CALMODULIN IN 35% TRIFLUOROETHANOL Abstract Introduction Materials and Methods Materials Nitrogen- 15 Uniform Labeling of CaM NMR Spectroscopy Results Discussion

CHAPTER SEVEN: ENERGETICS OF PEPTIDE BINDING TO CALMODULIN STUDIED BY ISOTHERMAL TITRATION CALORIMETRY Abstract Introduction Materials and Methods Materials Isothermal Titration Calorimetry Results Discussion

CHAPTER EIGHT: CONCLUSIONS

REFERENCES

xii List of Tables

1.1 CaM-binding proteins 8 1.2 CaM-target peptides 19 2.1 Amino acid analysis results for Dhp-CaM 71 3.1 Thermodynamic parameters for prolyl cis-trans isomerization in tripeptides 108 3.2 Kinetic parameters for prolyl cis-trans isomerization in tripeptides 114 6.1 r, values for CaM in water and in 35% TFE 237 7.1 AH and AC, for peptides binding to CaM 265 List of Figures

Structures of CaM 10 Primary sequence of CaM 14 Structure of CaM-dependent protein kinase I 18 CaM-bound structures of target peptides 24 Cis and trans isomers of peptide bonds 45 Structrures of CaM and calbindin D, with proline residues highlighted 52 Proline analogs 56 Schematic of mutagenesis of pCaM 59 Mass spectrometry of proline-substituted CaMs 72 Ca2+-dependentbandshifts of proline-substituted CaMs 75 Calcineurin activation by proline-substituted CaMs 76 ID 'H NMR spectra of Dhp-CaMs 77 TOCSY spectra of Dhp-CaMs 78 NOESY spectrum of Dhp-CaM 79 1D 'H NMR spectra of Dhp-calbindin D,, 80 TOCSY spectra of Dhp-calbindin D,, 81 Schematic of an inversion transfer experiment 102 ID 'H NMR spectra of tripeptides 104 Temperature dependence of cis:trans ratio for tripeptides 106 Inversion transfer raw data 110 Temperature dependence of cis-trans rate for tripeptides 112 1D "F NMR spectrum of glutathione-S-transferase 126 Fluorinated amino acids incorporated into CaM 129 Mass spectrometry of fluorinated proteins 134

xiv 1D 19FNMR spectra of cis-4-fluoroproline 137 'HY19F HETCOSY spectrum of cis-4-fluoroproline 138

'H, 19F' HETCOSY spectrum of fluoroproline-CaM 139 Native PAGE gel of fluoroproline-CaMs 140 ID 19F NMR spectra of apo-trifluoroleucine CaMs at two levels of incorporation 142 1D l9F NMR Ca2+-titrationof trifluoroleucine-CaM 143

'Hy19F HETCOSY spectra of trifluoroleucine-CaM 144 1D 19F NMR spectra of of trifluoroleucine-CaM with CaMKI peptide 147 'H,19F HETCOSY spectrum of trifluoroleucine-TRlC 148 1D "F NMR Ca2+-titraionof trifluoromethionine-CaM 150

ID 19F NMR spectrum of trifluoromethionine-CaM with CaMKI peptide 151 Schematic of CaM with proteolytic cleavage sites 166 W absorption spectra of CaM thrombic fragments 178 Gel bandshifi assays of CaM thrombic fragments 179 Enzyme activation assays for CaM thrombic fragments 182 Far-UV CD spectra of CaM thrombic fragments in the presence of Ca2+ 185 Far-W CD spectra of CaM thrombic fragments in the absence of Ca" 188 Near-W CD spectra of CaM thrombic fragments 192 1D 'H NMR spectra of CaM thrombic fragments 195 NOESY spectra of CaM thrombic fragments 197

'H,13C HMQC spectra of methyl-%Met labeled CaM thrombic fragments 199 'H, 13C HMQC spectra of methyl-13C-Met labele6 CaM thrombic fragments '13Cd-NMR spectra of CaM thrombic fragments l13Cd-NMR titration of CaM thrombic fragments with trifluoperazine Mass spectnun of "N-labeled CaM 'N, 'H HSQC spectrum of of lSN-CaMin H,O 'N, 'H HSQC spectrum of I5N-CaM in 35% TFE NMR relaxation decay curves of 15N nuclei "N-NMR relaxation data for CaM Alignment of several CaM-binding peptides Isothermal calorimetric titration of CaM with CaMKI peptide at 25 OC Isothermal calorimetric titration of CaM with cNOS peptide at 5 OC Bufier dependence of CaMKI peptide binding to CaM Plot of binding enthalpy vs. temperature for CaM- binding peptides Isothermal calorimetric titration of TM2 into TM1 Isothermal calorimetric titration of CaMKI peptide into mixture of TM1 and TM2 Abbreviations

1D one-dimensional 2D two-dimensional Ac acetyl group Azc azetidine-2-carboxylic acid CaM calmodulin CaMKI (CaM-binding peptide from) calmodulin dependent protein base I (CaM-binding peptide from) calmodulin dependent protein kinase kinase circular dichroism (CaM-binding peptide kom) constitutive (rat neuronal) NOS COSY correlation spectroscopy CSA chemical shift anisotropy CT-CaM CaM with methionines 109, 124, 144, and 145 all mutated to leucines

ACP heat capacity change D~P 3,4-dehydroproline DQF double quantum filtered DSS 2,2-dimethyl-2-silapentane-5-sulfonate DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EGTA ethyleneglycol-bis (P-aminoehtyl ether)-N, N, N', N'- tetraacetic acid ESI-MS electrospray ionization-mass spectrometry

xvii FPro cis-4-fluoroproline FTY~ fluorotyrosine GST glutathione-S-transferase HETCOSY heteronuclear COSY HMQC heteronuclear multiple quantum coherence HPLC high performance liquid chromatography HSQC heteronuclear single quantum coherence iNOS (CaM-binding peptide from) inducible (murine macrophage) NOS IPTG isopropylthio-p-D-galactoside ITC isothermal titration calorimetry LB Luria-Bertani (brothlagar) MLCK myosin light chain kinase MOPS 3-(N-rnorpholino)propanesulfonic acid MPD 2-methylpentane-2,4-diol MWCO molecular weight cutoff NMR nuclear magnetic resonance NOESY nuclear Overhauser effect spectroscopy NOS nitric oxide synthase PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PDEa (CaM-binding peptide from) 3':5'-cyclic nucleotide phosphodiesterase 1A2 (bovine brain) Pipes 1,l-piperazinediethanesulfonic acid ROESY rotating frame nuclear Overhauser effect spectroscopy skMLCK (CaM-binding peptide from) skeletal muscle (rabbit) myosin light-chain kinase (CaM-binding peptide from) smooth muscle (chicken gizzard) MLCK spin-lattice relaxation time spin-spin relaxation time motional correlation time 4-hydroxy-2, 2, 6, 6-t etramethylpiperidine-N-oxyl TFE 2,2,2-trifluoroethanol TFLeu 5,5,5-trifluoroleucine TFMet S-(trifluorornethy1)-homocysteine;trifluoromethionine TFP trifluoperazine thrombic fragment 1-106 of CaM thrombic fragment 107-148 of CaM TOCSY total correlation spectrum TPPI time proportional phase incrementation TRlC tryptic fragment 1-75 of CaM tryptic fragment 78-148 of CaM Tris tris(hydroxymethy1)aminomethane Xxx any amino acid; in this thesis Xxx is generally used to designate the amino acid preceding a proline residue any amino acid; in this thesis Yyy is generally used to designate either proline or an analog of proline Introduction 1

CHAPTER ONE: Introduction

Calcium in Biology.

To the uninitiated, it might seem that the most important role of calcium in biology is a structural one, and indeed it may be argued that this is true. Hydroxyapatite, a co-crystal of calcium, phosphate, and hydroxide ions, forms the matrix of tooth enamel, the hardest substance in the human body. Calcium phosphate is also responsible for the rigidity of bone, and the deposition of this matrix in bone is a very tightly controlled biological process. Poor diet or improper regulation of this process can lead to diseases such as childhood rickets or osteoporosis in older adults. Moreover, calcium is also important structurally in other organisms; for example, calcium carbonate is the major component of egg shells and also of the exoskeleton of animals such as mollusks and barnacles. Nutritionally, calcium is found in many foods, but of course the major source in most diets is dairy products. In milk, a predominant class of proteins is the caseins, which function to solubulize calcium phosphate microgranules 5y surrounding them in a micellular structure (Holt, 19921, thereby providing an important mineral nutrient in liquid form. The role of the Ca2' ion in solution, rather than in a solid deposit, is also important. Calcium triggers the binding of many proteins to biological membranes (Nelsestuen and Gaul Ostrowski, 1999), an important part of many physiological process. One such key role is in the Introduction 2 blood clotting process, where many enzymes involved in the blood clotting cascade have post-translationally modified y-carboxyglutamate (Gla) residues, generally 9-13 of them in an N-terminal Gla domain, that specifically bind calcium. It was originally thought that the Ca2+ion, by binding one per Gle residue, forms a bridge that dlows the proteio to bind to phospholipid membranes. It appears now that that is likely not the case; Ca2+-dependentmembrane binding may have something to do with the exposure of hydrophobic residues on the protein (Sunnerhagen et al., 19951, although this too is open to debate (Nelsestuen and Gaul Ostowski, 1999). The absence of Ca" severely limits blood clotting, and in fact, the removal of free Ca" by the addition of chelators is a popular method of preventing blood samples from clotting in clinical laboratories. Many other extracellular proteins also bind Ca" ions, in which the Ca2+ion plays a structural role. These include the C-type lysozymes and the related protein a-lactalbumin (McKenzie and White, 1991; Aramini et al., 1992; Aramini and Vogel, 19981, a protein involved in lactose biosynthesis, deoxyribonuclease I (Pan and Lazarus, 1999) and the bacterial protease subtilisin (Smith et al., 1999) Interestingly, for subtilisin and the related subtilases, the number of Ca2' ions bound and the tightness of binding correlate well with the themostability of the enzyme (Smith et al., 1999, and references therein). Intracellular enzymes bind Ca" as well, but here the role of the Ca" ion is often more complex. Calpains are cytosolic thiol proteases with substrates involved in many different cell signaling processes. Without Ca2+,the active site of these enzymes is not functional (Hosfield et al., 1999, Strobl et al., 2000), illustrating Ca2+as the activator of the protease rather than a more conventional mechanism of zymogen activation, such as limited cleavage. Introduction 3

The relative abundance and physiological function of the Ca2+ion inside the cell is quite different than in extracellular matrices such as blood plasma, and intracellular Ca2+ concentrations are very tightly controlled. This leads to perhaps the most important biological role of CaZ+: that of a secondary messenger ir? eukaryotic cells. Usurrlly, s ouhzryotic cell exists at a so-called "resting state" of Ca" concentration, where the cytoplasmic level of the Ca2+ion is quite low, typically lo6 - 10" M (Vogel, 1994), compared to an extracellular concentration of -10' M. This low cytoplasmic concentration of Ca2+is maintained by the activity of ATP- dependent Ca" pumps (Ca2+-ATPases),which pump Ca2+out of the cell or into specialized organelles such as the sarcoplasmic reticulum in muscle cells. However, external stimuli, such as hormonal signals or neural impulses, cause an increase in cytoplasmic Ca2+to an activated state of -10' M (Means et al., 1991; Vogel, 1994). This activation is short-lived as the cell usually quickly returns to resting Ca" levels through the action of the aforementioned Ca2+-ATPases. This "spike" in cytoplasmic CaY' is sometimes termed a "calcium transient." This calcium transient is a very important cellular signal, and thus Ca" is an essential secondary messenger. It is curious why it is calcium, and not some other ion, which plays this pivotal role in signal transduction (Falke et al., 1994). Many of the reasons have to do with the chemistry of calcium. Ca2+belongs to the "hard" class of metal ions (for literature on the biochemistry of metal ions see Frausto da Silva and Williams, 1991; and Lippard and Berg, 1994). It favors oxygen ligands such as the carbonyl and carboxyl groups of proteins. The most obvious candidate to play the role of Ca2+would be Mgl+, another readily available, soluble, divalent metal cation of the alkaline Introduction 4 earth metal group. Like Ca", it favors oxygen ligands but an important difference is that Mg" has much slower rate for loss of water molecules from its hydration shell (Frausto da Silva and Williams, 19911, and its complexes are generally only 6 co-ordinate, with water molecules occupying at least two sites. M$+, then, is not favorable for the high co- ordinate, irregular binding sites of ca"-binding proteins. It is these types of sites which produce the greatest structural change upon metal binding, which enables Ca" binding to act as a conformational trigger. Other ions may also be considered for this type of signaling role. Monovalent ions such as Na', K, or Cl*,are readily bioavailable, but they generally act only as bulk ions and are not considered to be important protein ligands. Other di- or trivalent metals such as Zn", Mn", Cu", or Fe3+,can certainly bind to proteins, but their off-rate from proteins is usually too slow to be effective as a reversible conformational switch. These ions generally play the role of structural stabilization or act as Lewis acid or redox catalysts in enzymatic reactions. Moreover, the solubility of these larger metal ions is often low, making them difficult to obtain in sufficient amounts to play a signaling role. As an aside, Ca" insolubility may be problematic in the cell as well. Although Ca2+exists at an extracellular concentration of 10" M, inorganic phosphate and phosphate-containing ligands such as phosphoproteins and nucleic acids, as well as other groups in the cell, would form insoluble complexes with Ca2+ at this high a concentration (Vogel, 1994). Consequently, even during a Ca" transient the cytosolic level of Ca2+ generally only raises ten-fold or so to a concentration of -10' M and never approaches the level of that outside the cell. Although this is only a modest increase in intracellular Ca2+levels, Introduction 5 many very important events occur in the cell during a CaL+transient. Many of these changes are mediated through regulatory Ca"-binding proteins, which become saturated with Ca" during the activated state of the cell. Like the vitamin-K-dependent blood clotting factors found in blood plasma, there are Ca2+-dependentmembrane binding proteins in the cytoplasm of cells, including the pentraxins (Nelsestuen and Gaul Ostrowski, 1999), proteins of unknown function, and the annexins (Nelsesteuen and Gaul Ostrowski, 1999; Dubois et al., 1999), which play many roles in cellular signaling. There are often water molecules in the Ca2'-binding sites of these proteins, which are thought to be displaced by the phospholipid head groups as the proteins bind to membranes. The annexins, in turn, are substrates for another class of Ca2+-binding proteins, the protein kinases C (Dubois et al., 1999). Protein kinases C bind Ca2+through their C2 domain calcium binding motifs; it is now known that C2 domains exist in nearly 100 known proteins of several different classes (reviewed by Nalepski and Falke, 1996; Rizo and Siidhof, 1998). The basic C2 domain consists of an eight stranded antiparallel P- sandwich of approximately 130 amino acid residues; up to three CaZ+ions are bound by connecting loops on one side of the sandwich. Like the pentraxins and annexins, C2 domains bind phospholipids in a CaZ+- dependent manner; in the case of protein kinases like the protein kinases C this enables localization to the substrate proteins, which are often membrane-associated. Many lipid modifying enzymes such as the phospholipases C also have C2 domains for the same reason. In the case of other proteins, Ca2+is responsible for localization of the proteins to the cell membrane where signaling events take place. Moreover, many C2 domains are also responsible for binding other substrates, including other Introduction 6 proteins, making these domains quite versatile and complex. Another very important class of Ca2'-binding proteins it the "EF- hand" family (Strynadka and James, 1989; Falke et al., 1994; Ikura, 1996; Kawasaki et al., 19981, a name used to describe the structural characteristics of the calcium binding sites of these proteins. This term was coined by Kretsinger and Nockolds (1973) when they determined the structure of carp panalbumin, and it refers to the conserved heliu-loop- helix Ca2+-bindingmotifs found in these proteins. The EF-hand family includes "structural" Ca2+-bindingproteins (also called Ca" "buffersn) such as parvalbumin, the sarcoplasmic Ca2'-binding proteins (Vijay-Kumar and Cook, 19921, and the vitamin D-dependent intestinal Ca2+-bindingproteins (calbindins). Other EF-hand helix-loop-helix Ca2'-binding proteins include the aforementioned calpains (Hosfield et al., 1999; Strobl et al., 2000). Another interesting EF-hand protein is the retinal protein recoverin (Tanaka et al., 19951, in which Ca" binding causes the exposure of the N- terminal myristoyl group (which is buried in the Ca2+-freestate of the protein), thereby enabling targeting to membranes. There is also an important class of regulatory helix-loop-helix Ca2+-bindingproteins which bind to other target proteins in response to the transient increases in Ca2' concentrations. These include the SlOO proteins, (Smith and Shaw, 1998; Hiezmann and Cox, 1998; Donato, 19991, some of which can bind other metals like Zn2+ and Cu", a host of neuronal Ca2+-bindingproteins (Haeseleer et al., 2000, and references therein), and a newly discovered calmoddin-like protein from human epidermis (MBhul et al., 2000). Two of the more important regulatory Ca2+-bindingproteins of this family include troponin-C, the calcium binding component of the troponin complex in muscle cells (for reviews see Farah and Reinach, 1995; Gagn6 et al., 1998; Introduction 7 and Filatov et al., 19991, and the structurally analogous protein calmodulin.

Calmodulin.

Calmodulin (CaM)is a ubiquitous, acidic protein found in almost all eukaryotic cells, in organisms ranging &om yeast to plants to humans (for recent reviews on calmodulin see Vogel, 1994; Ikura, 1996; Zhang and Yuan, 19981. In response to elevated intracellular Ca" levels, CaM can bind four Ca" ions, one in each of its EF-hand calcium binding motifs. In turn, CaM binds and activates a range of different target proteins (Means et aZ.,1991; Vogel, 1994; Crivici and Ikura, 1995, Table 1.1/, enabling it to affect numerous cellular processes, such as muscle contraction, biosynthesis of other messenger molecules, protein phosphorylation and dephosphorylation, gene expression, and cell-cycle control points. Thus, CaM has a pivotal role in cellular metabolism. The X-ray crystal structure of Ca2+-saturatedCaM (Babu et al., 1988; Chattopadhyaya et al., 1992) reveals a largely a-helical structure with an elongated dumbbell shape with two lobes, each containing a pair of EF-hand motifs, separated by a long, central a-helical linker (see Figure l.lb). Through the results of solution structural studies of CaM, such as small-angle X-ray scattering (Heidorn and Trewhella, 1988) and NMR (Ikura et al., 1991; Barbato et al., 19921, it has been proven that this central linker of CaM is actually quite flexible. CaM belongs to the EF-hand superfamily of calcium binding proteins. There is considerable homology between calcium binding motifs, both internally amongst the motifs in a single protein, and externally Introduction 8

Table 1.1.

Function Protein

Muscle contraction Myosin light-chain kinase (smooth & skeletal) Caldesmon Calponin

Protein phosphorylation/ CaM-dependent protein kinases dephosphorylation Calcineurin (protein phosphataseIIb) Phosphorylase kinase

Cell messengers 3%'-Cyclic nucleotide phosphodiesterase Adenylate cyclase Inositol trisphosphate kinase Constitutive nitric oxide synthase (endothelial and neurond) Inducible (macrophage) nitric oxide synthase Calcium ATPase Calcium and other ion channels

Nuclear proteins Basic helix-loop-helix transcription factors CaM-dependent endonuclease RNA helicase, transcription elongation factor la

Other activities Plant glutamate decarboxylase Dystrophin Multidrug resistance P-glycoprotein HIVISIV glycoprotein Introduction 9

Table 1.1 (continued)

Apo-CaM binding Neuromodulin (GAP43/P-57) proteins: Neurogranin Inducible (macrophage) nitric oxide svnthase 3'5'-Cyclic nucleotide phosphodiesteras Phosphorylase kinase

Table 1.1. List of the major CaM-binding proteins. This list is by no means complete, and many more CaM-binding proteins are surely still to be discovered. Adapted from Vogel (1994) and Rhoads and Friedberg (1997). amongst various proteins in the EF-hand family. For the primary amino acid sequence of bovine CaM, see Figure 1.2. Sequences have been compared and examined extensively in the literature (Strynadka and James, 1989; Marsden et al., 1990; Falke et al., 1994). A typical Ca2*- binding loop from an EF-hand helix-loop-helix motif has 12 residues. Crystallographic evidence now indicates that the last three residues in this 12-residue sequence actually comprise the N-terminal part of the helix following the Ca2+-bindingloop (Strynadka and James, 1989). There are six residues involved in Ca2+liganding, located at positions 1, 3, 5, 7, 9, and 12 of the loop. Originally, it was thought that these ligands formed an octahedral arrangement around the Ca2+ion, but it is now known that there are in fact seven liganding interactions with the Ca2+ion, and that the site resembles a distorted pentagonal bipyramid. All of the ligand atoms are oxygens, contributed either by sidechains of Asp, Asn, Glu, Gln, or Ser residues, main chain carbonyls, or in some cases by water oxygens. In trod uction 10

Figure 1.1. a) Introduction 11

Figure 1.1. b) Introduction 12

Figure 1.1. c) Introduction 13

Figure 1.1. d) Introduction 14

Figure 1.1. (Previous pages) Ribbon representations of representative structures of the four basic states of calmodulin. a) NMR structure of Ca2+-freeCaM (Zhang et al., 1995b, PDB accession code idmo). b) Crystal structure of calcium-saturated calmodulin (Chattopadhyaya et al., 1992, PDB accession code lcll). c) NMR structure of calmodulin with a target peptide bound at the C-terminal lobe (Elshorst et al.. 1999, PDB accession code lcm; the a-helical peptide is seen with its axis perpendicular to the page at the bottom of the structure. d) NMR structure of Ca2+-CaM complexed with a peptide from the CaM-binding domain of skeletal muscle light-chain kinase (Ikura et al., 1992, PDB accession code 2bbm). The peptide is seen as an a-helix with its axis perpendicular to the page in the center of the structure. These figures were generated with the MOLMOL molecular representation program (Koradi et al, 1996).

Figure 1.2. The primary sequence of bovine calmodulin. Amino acids are designated in 1-letter format. The four Ca2+-bindingloops are in bold-face. Bovine CaM is acetylated at its N-terminus and lysine-115 is trimethylated; bacterially expressed CaM is missing both of these post- translational modifications. Introduction 15

Sequence comparisons among calcium-binding loops reveal a high degree of identity at various positions (Marsden et al., 1990). Position 1 is almost invariably an aspartate residue, and position 3 is most often an Asp or an Asn. The residue at position 12 is almost always a glutamate; it binds t-he Ca2+ ion through both of its sidechain carboxyl oxygens ir? s "bidentate" fashion, making it responsible for donating the extra, seventh oxygen atom in the liganding sphere. Moreover, there are also conserved nonliganding residues within the calcium-binding loop. Position 6 is very often occupied by a glycine residue due to the fact that only Gly, with its absence of a sidechain, has the conformational freedom to form the proper EF-hand geometry. Position 8 is also important; it is most often isoleucine, or perhaps some other hydrophobic amino acid (Val or Leu). This residue forms part of the small but important two-stranded P-sheet that exists between opposite Ca2+-bindingloops in a pair of EF-hands. The pairwise existence of these motifs is found across all classes of EF-hand proteins and is very important for their function. Interactions between the individual motifs in the pair allows for tight, co-operative binding of Ca2+ions. If only a single helix-loop-helix domain were found in a protein, it would bind Ca" very poorly. This has been shown through studies of synthetic peptides of helix-loop-helix domains (Reid et al., 1981; Gariepy et al., 19821, and proteolytic fragments of EF-hand proteins (Andersson et al., 1983). In Chapter 5 of this thesis there is a detailed examination of the spectroscopic and biochemical properties of thrombic fragments of calmodulin. With all the homology amongst various EF-hand calcium binding proteins, it is difficult to understand why there is such diversity in their Ca2+-bindingproperties and physiological functions. Some EF-hand Introduction 16 proteins, such as the panralbumins and the calbindins (vitamin D- dependent intestinal calcium-binding proteins), seem to act only as Ca2+ "buffers". All of their EF-hand sites bind Ca" with similar affinity, and little structural change occurs upon Ca" binding by the protein. They egst on!y to aid in absorption of the importmt Ce" ion from the intesthe! lumen, or to prevent potentially harmful free Ca2+ions from forming precipitates with DNA, phosphoproteins, or other compounds. By contrast, the regulatory EF-hand proteins, including CaM, troponin-C and the SlOO proteins, all exhibit important conformational changes upon Ca" binding, that causes the exposure of hydrophobic patches on the surface of these proteins. It is through these patches which these proteins in turn interact with their respective target proteins. This exposure of a hydrophobic surface is the result of the movement of helices within a pair of EF-hand motifs, a conformational change which was revealed by determining the structure of apo- (Ca2+- free) CaM (Figure l.la; Kuboniwa et al., 1995; Zhang et al., 199513). Interhelical angles in the four EF-hand motifs of CaM are altered by Ca2+binding (Zhang et al., 1995b), causing a change from a "closed" to an "open" conformation. This results in the exposure of two hydrophobic clefts on the surface of CaM, one on each of the N- and C-terminal lobes. It is through these hydrophobic patches that CaM binds it target proteins. Although CaM targets are numerous and varied, they share a general feature of a small, contiguous amino acid sequence of 17-25 amino acids in length, usually at or near the C-terminus, which comprises the CaM- binding domain of the protein. These CaM-binding domains share very little sequence homology, although they all do contain a large number of basic residues and have the propensity to form an amphiphilic a-helix, Introduction 17 with the positively charged residues on one face and hydrophobic residues on the other (Rhoads and Friedberg, 1997). Also, there seems to be the important feature of two major hydrophobic "anchor" residues, usually either Trp, Phe, or a bulky aliphatic residue such as Leu, spaced either 9 or 13 residues apart. A structure of an example of a calmodulin-activated protein, calmodulin-dependent protein kinase I (CaMKI), is shown in Figure 1.3. Trp303, the major hydrophobic anchor residue in CaMKI, points away from the rest of the protein into the solvent, ready to be bound by CaM. The binding of this Trp residue by CaM is thought to be the primary event in activation of CaMKI by CaM.. Chemically synthesized peptides comprising the CaM-binding domains of target proteins can also independently bind CaM,and, as well, many naturally occurring CaM-binding peptides (CaM peptide inhibitors) exist. Melittin is a model CaM-binding peptide derived from components of bee venom, and mastoparan is from the venom of wasps. For a list of the synthetic CaM-binding peptides that have been studied in our laboratory, see Table 1.2. The peptides are usually 20-30 amino acids long and bind simultaneously to both domains of the protein. Shorter peptides with partial CaM-binding domains can often form 2:l complexes with CaM, with one peptide bound to each of the two lobes of the protein (Zhang and Vogel, 1997; Yuan et al., 2000b). Another distinct CaM-binding peptide is the CaM binding domain from plant (Petunia) glutamate decarboxylase (PGD). Although PGD is a full-length peptide, it too binds with 2:l stoichiometry, again via both domains of CaM (Yuan and Vogel, 1998). A few structures of complexes of these peptides with CaM have been solved, including a complex of CaM with a synthetic skeletal muscle myosin light- Introduction 18

Figure 1.3. Introduction 19

Figure 1.3. (previous page) The X-ray crystal structure of recombinant truncated rat brain calcium/calmodulin-dependent kinase I (CaMKI; Goldberg et al., 1996, PDB accession code 1A06), in ribbon format. The activation domain of the protein is shown in black, with the CaM-binding domain at the top-right of the figure. Trp303, the important CaM-binding tryptophan residue. is shown in ball-and-stick format. This figure was generated with MOLMOL (Koradi et al., 1996).

Table 1.2.

CaM-target peptides used in this thesis: Peptidea Sequence References

CaMKI AKSKWKQAFNATAWRHMRKLQ Gomes et al., 2000

cNOS KRRAIGFKKLAEAVKFSAKLMGQ Zhang et al., 1995a

Mel GIGAVLKVLTTGLPALISWIKRKRQQ Yuan et al., 2000

PDEa Ac-QTEKMWQRLKGILRCLVKQL-NH, Yuan et al., 1999b

A skMLCK KRRWKKNFIAVSAANRFKKISS Zhang et al., 1993 Introduction 20

Table 1,2. (continued)

Some other CaM-binding peptides used in this lab:

Peptidea Sequence References

CaDl GVRNIKSMVVEKGNVFSS Zhang and Vogel, 1994b

CaD lshort KSMWEKGNVFS-NH, Zhang and Vogel, 1997

CaD2 NKETAGLKVGVSSRINEWLTKT Zhou et al., 1997

CaD2short SMWLTK Zhou et al., 1997

W-2 GFWGTLGQIGRGILAVPRRIRQGAEIAL Ouyang, 2000

NOS RRREIRFRVLVKWFFASMLMRKVMAS Yuan et al., 1998

MelN GIGAVLKVLTTGLP-NH, Yuan et al., 2000E

MeiC Ac-PALISWKEKRQQ Yuan et al., 2000t

PGD HKKTDSEVQLEMITAWKKF'VEEKKKK-NH2 Yuan and Vogel, 1998

STV DLWETLRRGGRWILAIPRRIRQGLELTL-NH2 Yuan et al., 1995

SWN DLWETLRRGGRWI-NH, Yuan et al., 2000a

SIV-C Ac-ILAIPRRIRQGLELTL-NH, Yuan et al., 2000a Introduction 21

Table 1.2. (Previous pages) An overview of the CaM-binding peptides used in our laboratory. 'Abbreviations for proteins from which CaM- binding sequences are taken: Cam: CaM-dependent protein kinase I; cNOS: rat cerebellar (constitutive) nitric oxide synthase; Mel: melittin; PDEa: CaM-binding site A from bovine brain 3':5'-cyclic nucleotide phosphodiesterase 1A2; skMLCK: rabbit skeletal muscle myosin light- chain kinase; CaDl and CaD2: chicken gizzard caldesmon (sites 1 and 2, respectively); HIV-2:human immunodeficiency virus 2 (peptide derived from isolate ST); iNOS: murine macrophage (inducible) nitric oxide synthase; PGD: petunia glutamate decarboxylase; SIV: simian immunodeficiency virus transmembrane glycoprotein 41.

chain kinase (skMLCK) peptide solved by NMR (Ikura et al., 19921, which is shown in Figure l.ld. The structures of CaM complexes with a chicken smooth-muscle MLCK (smMLCK) peptide (Meador et al., 19921, and a brain CaM-dependent kinase IIa (CaMKIIa) peptide (Meador et al., 1993) have been solved by X-ray crystallography. They are both quite similar to the skMLCK complex structure. The CaM-peptide structures reveal that the peptides, normally non structured in solution, adopt an a-helical conformation in the complex. The central helix of CaM unwinds as itengulfs its target molecule, allowing for more intimate contact between CaM and the target peptide. There is a great deal of interaction between the hydrophobic face of the amphiphilic target peptide and the hydrophobic patches on the CaM surface. All contacts are through sidechains, and not the main chain groups, of CaM and the peptides, which is unique for a protein:protein complex. The anchor residues of the Introduction 22 peptide are very important, as they interact extensively with the hydrophobic patches of CaM (in fact, the hydrophobic surfaces on CaM are more similar to a Upocketninto which these anchor residues insert, but the term hydrophobic patch is commonly used to describe these surfaces in CaM). More recently, the structures of two other complexes have been solved by NMR, namely those of CaM with a peptide from the Ca2+pump (Elshorst et al., 1999; Figure 1.1~)and a peptide from CaM-dependent protein kinase kinase (CaMKK; Osawa et al., 1999); the CaM-bound structures of these two peptides, as well as skMLCK and CaMKIIa, are shown in Figure 1.4, with the hydrophobic anchor residues highlighted. The pump peptide is atypical in that it only binds to the C-terminal lobe of Ca2'-CaM; this is not surprising given the fact that the second hydrophobic anchor residue is missing, although it could have been conceivable that the pump peptide bound with a 2:l stoichiometry. Because it only binds to one lobe, the pump peptide is also unable to compact CaM into a globular structure; this structure is also seen as being representative of CaM-binding by more canonical target peptides, where binding to the C-terminal lobe is thought to be the initial step in the interaction. The CaMKK peptide is unique in that the CaM-bound conformation has a hairpin-type structure. What is the same, though, is that the two hydrophobic anchor residues are still in the correct spatial orientation, and the overall shape of the complex is globular, similar to most other CaM-peptide complexes. Additionally, there are many CaM-binding peptides where the structure of them in complex with CaM is unknown. In Chapter 7 of this thesis, the enthalpy of binding for four CaM-binding peptides is determined by isothermal titration calorimetry (ITC). ITC is a very useful Introduction 23 method in that it is the only technique by which the enthalpy of this type of interaction is measured directly. With this information in hand, one can determine the relative contribution of enthalpic factors, such as hydrogen bonds and van der Waals forces, and entropic factors, such as hydrophobic interactions, in the binding equilibrium. Moreover, some information about the degree of compactness of the complex can also be found. The results are discussed primarily in terms of the two most important features of CaM which enable it to bind such a large variety of target molecules. These two features are the methionine-rich hydrophobic patches and the flexible central linker.

The central linker of CUM. The structure of the central linker of CaM has been the subject of much study. Originally, structural determination of Ca2+-CaMthrough X- ray crystallography (Babu et al., 1988; Chattopadhyaya et al., 1992; Figure l.la) led to the assumption that this region was a long, solvent-exposed a- helix. However, even these structures gave some inkling that the central region was not an ideal a-helix. Babu et al. (1988)concluded that CaM was a-helical from residues 65-92, but that residues 79-81 showed "significant deviations from ideal a-helical geometry," as well as high temperature factors for these residues, suggesting strain in this region. They incorrectly theorized, however, that the presence of a target molecule might stabilize an a-helical structure in the central linker. The central helix of the structure of Chattopadhyaya et al. (1992) was significantly straighter around residues 79-81, but, again, temperature factors were high in that region of the protein. Structural studies of CaM in solution revealed still more flexibility Introduction 24

Figure 1.4.

N'

d) CaMKK

Figure 1.4. Ribbon diagrams of the Cf+-CaM-bound structures of CaM- binding target peptides from a) skeletal muscle myosin light-chain kinase (Ikura et al., 19921, b) CaM-dependent protein kinase I1 (Meador et al., 19931, c) CaM-dependent Ca2+-ATPase(Ca2+ pump; Elshorst et al., 1999), d) CaM-dependent protein kinase kinase (Osawa et al., 1999), with the two major hydrophobic "anchor" residues shown in ball-and-stick format. The first two peptides (skMLCK, CaMKII) bind CaM (shown schematically above the structures) in an antiparallel fashion, with the N-terminal Introduction 25 anchor residue burying in the C-terminal hydrophobic patch of CaM. The pump peptide, which only binds to the C-terminal lobe of CaM, only has one hydrophobic anchor residue. The CaMKK peptide binds to CaM in a parallel fashion, with its tryptophan residue buried in the N-terminal lobe of CaM. This figure was generated with the MOLMOL molecular rendering program (Koradi et al.. 1996).

of the central linker in the solution state. Small-angle X-ray scattering (SAXS)studies originally led to some debate about the structure of the central helix. Seaton et al. (1985) found a radius of gyration for Ca2+-CaM of 21.5 A, which is consistent with the dumbbell model. Although the results from the SAXS study of Heidorn and Trewhella (1988) were similar, their more rigorous calculations suggested a bent structure in solution. The important "N-NMR relaxation study of Barbato et al. (1992) showed that residues 77-81 of Ca2+-CaMhad lower "N-nuclear Overhauser enhancements and lower order parameters, showing a high degree of mobility. The secondary structure assignment of CaM by NMR (Ikura et al., 1992) also clearly showed the absence of a-helical structure between residues 78 and 81. In a computer simulation of residues 64-93 of CaM (van der Spoel et al., 19961, unwinding occurred from residues 76-79 after starting from the co-ordinates obtained from the crystal structure (Babu et al., 1988). As more information about how CaM interacts with its target molecules has been uncovered it is now understood that the flexible nature of the central linker of CaM is very important for its activity. An NMR study of amide hydrogen exchange rates (Spera et al., 1991) showed high Introduction 26 exchange rates for Ca"-CaM from residues 76-82. Moreover, the exchange rates of residues 75-79 increased significantly upon complexation with a target peptide derived from the rabbit skMLCK CaM-binding domain (Spera et al., 1991). In the solution-NMR structure of Ca2+-CaMwith the skMLCK peptide (Ikura et al., 1992): CaM unwound into a flexible loop between residues 74 and 82 in order for CaM to "wrap" around the target peptide. In a crystal structure of Ca2'-CaM complexed with a smooth muscle MLCK peptide (Meador et al., 19921, changes occurred between residues 73-77, and in a complex with a peptide from CaM-dependent protein kinase IIa, CaM was found to be quite flexible between residues 73 and 83 (Meador et al., 1993). In light of the varying conformations of the central linker as CaM complexes with different peptides, the central linker has been called an "expansion joint" (Meador et al., 19931, which can unwind to differing degrees to accommodate different target molecules. This varying degree of expansion can be seen in the differing abilities of CaM central helix mutants to activate various target enzymes. Insertions and proline mutations in the central helix did not affect the ability of CaM to activate calcineurin or Ca2+-ATPase(Putkey et al., 1988). Deletions in the central helix only slightly affected the ability of CaM to activate MLCK, significantly affected CaM kinase I1 activation, but substantially reduced the efficiency of CaM in activating phosphodiesterase (VanBerkum et al., 1990). Moreover, a CaM in which the N- and C- terminal lobes were joined by chemical cross-linking activated MLCK with equal ability to native CaM (Persechini and Kretsinger, 1988). These data suggest that the central helix unwinds when CaM binds many targets, but likely remains relatively rigid when binding others, like phosphodiesterase. If the helix were to remain rigid, Introduction 27 then a deletion would change the relative orientation of the two crucial hydrophobic patches of CaM, as shown by the crystal structure of a two- residue deletion mutant (Tabernero et al., 19971, thus severely affecting the activation of some enzymes.

The hydrophobic patches of CaM. Very important are the interactions between the hydrophobic face of the helical target peptide and the exposed hydrophobic patches of Ca2+- CaM. The main-chain confirmation of the two lobes of Ca2+-CaMdoes not change drastically upon binding of a target molecule; rather, it is small changes in the side chain angles of the hydrophobic residues, especially Met, of CaM that are responsible for binding such a diverse range of target molecules (Osawa et al., 1998). Dynamic NMR studies have shown that, although the side chains of the CaM are very mobile in the Ca"-saturated, unbound state, when the protein binds a peptide from smooth muscle myosin light chain kinase the motions of many hydrophobic side chains become highly restricted (Lee et al., 2000). These patches are rich in methionine residues, of which CaM has an anomalously high number; nine of the 148 residues are methionines with eight of them being important components of the hydrophobic patches. The Met sidechains are hydrophobic in nature, yet their polarizeable sulfur atoms lend some polar character as well. Thus, the Met residues are very important to the "promiscuous" nature of CaM, and its ability to bind a wide range of protein targets. Indeed, the CaM-binding domains of these target proteins have very little homology other than the propensity to form a basic, amphiphilic a-helix. The Met residues provide a flexible, malleable surface for these targets to bind. Protein engineering studies Introduction 28 carried out by our lab (Vogel and Zhang, 1995; Yuan et al., 1999a) have demonstrated the importance of the Met residues of CaM. Some experiments have included site-directed mutagenesis (Siivari et al., 1995), and the replacement of Met residues by biosynthetic incorporation of non- natural Met analogs in CaM, including selenomethionine (Zhang and Vogel, 1994a; Yuan et al., 1998a), ethionine and norleucine (Yuan and Vogel, 1999), and fluoro-methionines (Yuan,1998; Chapter 4). This thesis expands the method of non-natural amino acid bioincorporation to proline analogs.

Prolines and Protein Folding.

Proline is a unique amino acid that has an interesting role in protein folding. Among the natural amino acids, it is the only secondary amino (imino) acid, due to the fact that its nitrogen atom is connected to two other carbon atoms as part of the pyrrolidine ring. This distinctive structure leads to several important characteristics. First, the absence of an amide proton in an Xxx-Pro peptide bond (where Xxx could be any other amino acid) means that prolines cannot act as hydrogen bond donors. This makes their occurrence relatively rare in secondary structures such as a- helices; rather, they are often found in N-capping positions (Chakrabartty and Baldwin, 1995). Moreover, when proline residues do occur in a- helices, specific conformational constraints of the peptide backbone occur, as a consequence of the severely limited rotation about the Ca-N bond of Pro due to the ring structure (Barlow and Thornton, 1988). The result is a "kink" in the polypeptide chain; these kinks are highly regular and the prolines within a-helices are highly conserved, suggesting that these Introduction 29 distortions are functionally important (Barlow and Thornton, 1988). Proline plays a much more important role in protein turn structures. Indeed, p-turns are classified according to the position and geometry of the highly conserved prolines within them (Wilmot and Thornton, 1988). Apart From geometric and steric constraints, proline residues also impart a unique conformational freedom to the peptide bond. Normally, peptide bonds are planar. Delocalization of the lone-pair electrons of the amide nitrogen atom into the C-N bond of the peptide group results in a x system with a partial double-bond character of the C-N bond. Consequently, the six atoms of the peptide group (C, 0,N, H, and the two a carbons) all lie in the same plane and the group adopts either a cis (o=OO) or a trans (co=180°) conformation, with a rotational barrier between the two conformations. Almost all peptide bonds in proteins are in the trans conformation due to the fact that, in the cis conformation, unfavorable steric interactions occur between the carbonyl oxygen of an amino acid and the a-carbon of the next amino acid. However, replacement of the amide proton in proline by the bulky 6CH2 group makes the trans conformation of Xxx-Pro peptide bonds less favorable. Consequently, there is a relatively high incidence of the cis conformation of Xm-Pro peptide bonds, as has been shown by statistical analyses of the crystal structure database (Stewart et al., 1990; Wu and Raleigh, 1998). Moreover, in the unfolded state of proteins, or in small peptides without any defhed structure, the cis state of Xxx-Pro peptide bonds exist at a relatively high population relative to other peptide bonds. In these cases, population values for the cis conformation generally range from about 10% to 35% (Brandts et al., 1975, Arnodeo et al., 1994), depending on the protein and the conditions studied. Generally, with the larger Introduction 30 proportion of the Xxx-Pro peptide bonds in the trans state, folding is not a great problem if the state is also trans in the native conformation. However, if a peptide bond is cis in the native structure, attaining the cis conformation from the more favorable trans conformation in the unfolded state represents a considerable energy barrier: due to the restricted rotation about amide bonds. Indeed, early on it was postulated that the slow step in many protein folding reactions was due to prolyl cis-trans isomerization (Brandts et al., 1975; Creighton, 1978). Typically, the energy barrier for rotation about an amide bond is in the order of -20 kcal*mol-' (-85 kJ=m01-~).Work in this area first concentrated on small amide compounds, such as formamides, which were studied both by NMR (Stewart and Siddall, 1970; Drakenberg and Forsen, 1971; Drakenberg et al., 1972) and quantum mechanical calculations (Perricaudet and Pullman, 19731, as well as N-acylprolines (Maia et al., 1971). More recent studies have examined more relevant compounds such as peptides and proteins (Grathwohl and Wiithrich, 1981; Zhou et al., 1991; Shin et al., 1993; Larive and Rabenstein, 1993; McInnes et al., 1994). In viva, prolyl cis-trans isomerization is accelerated by enzymatic catalysis, through the action of peptidyl prolyl cis/trans isomerases (PPIases; for reviews on PPIases see Schmid, 1993 and Fischer, 1994), enzymes which are found in almost every organism. In humans, two major PPIases exist that both bind immunosuppressive drugs. They are cyclophilin, which binds cyclosporin A (CSA), and FKBP, which binds FK506. In fact, cyclophilin was originally discovered only as the cytosolic receptor for CsA (Handschumacher et al., 1984); its action as a PPIase and the inhibition of its activity by CsA was not shown until later (Takahashi et al., 1989; Fisher et al., 1989). The PPIase-inhibitory activity of CsA is Introduction 31 not likely to be the sole reason for its immunosuppressive activity, but rather it is the CsA-cyclophilin complex that has cytotoxic properties (Tropschug et al., 1989). It should also be noted that PPIases, as enzymes, do not alter the cis:trans equilibrium of prolyl peptide bonds in any protein; rather, they allow cisrtrans equilibrium to be reached more quickly and thereby assist proteins in rapidly folding to their native state. Statistical analyses of the protein structure database (Stewart et al., 1990; Reimer ef al., 1998; Pal and Chakrabarti, 1999) show that prolyl peptide bonds have the greatest occurrence of the cis conformation when the residue preceding the proline is aromatic (i.e. Tyr, Phe, Trp, His). Earlier examinations of the protein databank (Stewart et al., 1990) suggested that Tyr-Pro peptide bonds had an anomalously high preponderance of the cis conformation in protein structure. Today it seems that all aromatic residues are effective.

Non-natural amino acid analogs.

As previously mentioned, I chose the use of non-natural amino acids to study the phenomenon of prolyl cis-trans isomerism. As part of my research, proline analogs were incorporated into calmodulin. Non-natural amino acid bioincorporation into the proteins of an organism has been well known for some time, and, in fact, before the technique of site-directed mutagenesis was developed, amino acid analogs were used to study the role of certain residues in proteins. The phenomenon of non-natural amino acid bioincorporation was originally discovered when many amino acid analogs were explored as possible antimetabolites, or inhibitors of protein synthesis, and it was Introduction 32 found that many of them were incorporated into the host cell's proteins in place of their natural counterparts (for reviews see Richmond, 1962 and Meister, 1965). More recently, amino acid analog incorporation has been used in attempts to learn more about the structure and function of proteins. The unnatural proteins, called "alloproteins" (Koide et nl., 2988) enable the researcher to draw conclusions about the cclntribution of one type of amino acid to the properties of the protein and, when used in conjunction with site-directed mutagenesis, can also provide information about specific residues in the protein of interest. These studies exploit bacterial expression systems to biosynthetically incorporate the nonprotein amino acids. In many cases, Escherichia coli strains which are auxotrophic for the amino acid for which the analog is to be incorporated, are used. These provide the lowest background levels of the natural amino acids in the expressed proteins. One amino acid which has garnered a lot of study via analogs is methionine. Apart from the studies by our lab with calmodulin substituted with selenomethionine (Zhang and Vogel, 1994a), ethionine and norleucine (Yuan and Vogel, 19991, and fluoro-methionines (Yuan, 1998; Chapter 41, norleucine has been incorporated into many other proteins, including human epidermal growth factor (Koide et al., 1988). Here, substitution of the S atom of the single Met residue by the methylene group of norleucine had no significant effect on the activity of the protein. Telluromethionine (TeMet), in which the sulfur atom of methionine has been replaced by a tellurium atom, has been incorporated into proteins (Budisa et al., 1997). This is very promising because it, like selenomethionine, presents a specific way to incorporate heavy atoms into proteins for the purpose of structure determination by X-ray Introduction 33 crystallography. Interestingly, TeMet was found to be selectively incorporated into buried locations in dihydrofolate reductase (Boles et al., 1994). The authors theorized that this may have been due to selective incorporation of TeMet during the translation process. However, mass spectrometry results indicate that, in our hands, amino acid analogs are incorporated completely at random. A more plausible explanation may be that the TeMet was incorporated randomly, but proteins with TeMets on the surface were either unstable or not able to be purified. Apart from Met analogs, a whole host of other amino acid analogs have been incorporated into proteins. The acidic 1,2,4-triazole-3-alanine (TAA), a histidine analog with three ring nitrogens, was incorporated in place of histidine into porcine pancreatic phospholipase A? (Beiboer et al., 19961, and the mutant was found to have increased activity at a low pH. Thus, analogs such as TAA may be used to alter enzyme activity for purposes such as industrial applications. Thiaproline (1,3-thiazolidine-4- caboxylic acid) was incorporated in place of proline into annexin V (Budisa et al., 1998). The authors suggested that perhaps proteins such as this could be used as targeted carriers of bioactive amino acids, contained within their sequences, for pharmaceutical purposes. Another advantage of amino acid analog bioincorporation is the potential to incorporate novel NMR-active nuclei, such as the aforementioned selenium, which was incorporated into CaM in the form of selenomethionine (Zhang and Vogel, 1994a). Fluorine is the most useful NMR-active nucleus to be placed into proteins in the form of non-natural amino acids. Fluorine-19 has many distinct advantages which enable it to be easily studied by NMR (Danielson and Falke, 1996). Fluorinated amino acids incorporated into CaM in our lab include fluoro-phenylalanines and Introduction 34 fluoro-tyrosines (David, 19971, fluoro-methionines (Yuan,1998; Chapter 4), trifluoroleucine (David, 1997; Chapter 41, and cis-4-fluoroproline (Chapter 4). Fluorine substitution can also result in drastic changes to the polarity of amino acids due to the high electronegativity of this atom. Apart from the advantages of non-natural amino acids, there is the disadvantage that their bioincorporation into a protein results in the substitution of all amino acids of a given type in a "wholesale" rnutagenesis. For example, this incorporation of, say, selenomethionine into CaM results in the substitution of all nine Met residues, producing nine peaks in a "Se NMR spectrum. This is where site-directed rnutagenesis can work as a perfect foil. Each new signal can be assigned through the use of individual mutant genes in which each residue is mutated separately to another amino acid. Production of non-natural amino acid-containing proteins from these mutants will yield NMR spectra with one peak missing, corresponding to the mutated residue. Apart from combinations of mutagenesis and non-natural amino acid bioincorporation, there are also ways to site-specifically incorporate non-natural amino acids at a given position. This involves creating a nonsense (stop) mutation at the desired site. Then one can "expand the genetic code" (Bain et al., 1992, Mendel et al., 1995) by synthesizing a corresponding suppressor tRNA for the stop codon, to which the non- natural amino acid of choice is ligated. The gene and the suppressor tRNA are then mixed in with a cell-free (in vitro) translation system, such as an E. coli extract, to produce the protein. However, yields are typically very low, making this method prohibitively expensive for producing proteins for general use or NMR study. Moreover, this method is confined only to soluble proteins. There is an example of a site-specific incorporation of Introduction 35 non-natural amino acids into nicotinic acetylcholine receptors in intact Xenopus oocytes through microinjection of the mutant gene and suppressor tRNA into single cells (Novak et al., 1995). However, this obviously only produced minute quantities of the receptor protein. In this dissertation, 1 have used an E. coli expression system for biosynthetic substitution of amino acids into proteins. Although substitutions were not site-specific by this method, it did provide large quantities of protein suitable for NMR experiments.

Introduction to NMR.

Nuclear magnetic resonance (NMR) is an extremely powerful tool to study the structure and dynamics of proteins and other macromolecules. Several recent reviews (Gronenborn and Clore, 1994; Kay, 1997; Wider, 1998) and textbooks (Wiithrich, 1986; Hore, 1995; Cavanagh et al., 2000) exist on the subject. Since the NMR spectrum of ribonuclease by Emst and co-workers (Saunders et al., 1957), NMR of proteins and macromolecules has experienced several quantum leaps in method development. The construction of superconducting electromagnets in the mid-1970s enabled the production of higher-field instruments that made NMR analysis of large macromolecules possible. The application of the Fourier transform to NMR data by Richard Ernst (Ernst, 1992) was also a great advance, and today all protein NMR spectra are acquired in this way. The two-dimensional (2D)NMR experiment (Aue et al., 1976) represented another significant development which took advantage of the high-powered electromagnets, spreading NMR spectra into a two- dimensional plane, facilitating the analysis of complicated NMR spectra of Introduction 36 large molecules such as that of proteins. With the advent of 2D-NMR experiments such as COSY (Aue et al., 1976, Rance et al., 1983) and TOCSY (Bax and Davis, 1985a) to determine through-bond couplings and NOESY (Jeener et al., 1979) to determine through-space dipolar interactions of NMR-active nuclei, complete NMR assignmefit 2nd structural analysis of proteins as large as 10 kDa became possible. Another great quantum leap in NMR methodology has been the development of proton, carbon-13, and nitrogen-15 triple-resonance experiments of isotopically labeled proteins. This has expanded NMR into the third and even fourth dimension (Kay et al., 1990, Clore and Gronenborn, 1991). Through experiments such as the HNCA and the HNCO (Kay et al., 19911, one can sequentially assign all the resonances in proteins without resorting to often-ambiguous sequential NOE data. The spreading of NMR data into further dimensions in 3D and even 4D NMR experiments enables researchers to tackle proteins as large as 25 kDa. Moreover, with the techniques of partial deuteration and perdeuteration of proteins (LeMaster, 1990; Pachter et al., 1992; Venters et al., 1996) proton spin-diffusion problems can be appeased, which pushes the envelope of structural assignment to proteins and complexes 30 kDa and larger. Finally, the use of new gradient probes and pulsed-field gradient techniques (Kay, 1995; Sattler et al., 19991, and NMR cryoprobes (Hajduk et al., 1999; Serber et al., 2000) have led to the improvement of spectra in a variety of NMR experiments. However, "bigger is bettern should not be the only concern when tallring about NMR. Although increases in the size of proteins which can be structurally solved by NMR is an important advance, plenty of other useful biological information can be found by NMR methods. In vivo NMR, Introduction 37 especially of very important nuclei such as phosphorus, like that in phosphate, nucleotides, and high-energy phosphorylated compounds, as well as carbon and nitrogen, is a very useful tool in studying cellular metabolism (Lundberg et al., 1990). As well, other NMR methods can be applied to studying proteins, without necessarily completely solving the structure of the protein. Spin-'/, nuclei like cadmium-113 can provide biologically relevant information about the metal-binding sites of calcium- binding proteins and other metal-binding proteins (Summers, 1988). Moreover, NMR of quadrupolar nuclei (those with a spin greater than 'I2) opens a whole other avenue to study metal-binding sites (Aramini and Vogel, 1998). Important quadrupolar metal ions include A13+,Sc3+, and Ga3+,which can be substituted for iron in the study of iron-binding proteins (Aramini et al., 1993). In many cases, these quadrupolar nuclei can enable the researcher to examine extremely large proteins which cannot be studied by conventional spin-'/, NMR methods. The extremely sensitive spin-'I2 nucleus fluorine-19 can be introduced into proteins through the biosynthetic incorporation of fluorinated amino acids (see Chapter 41, or with the introduction of fluorinated ligands to the protein (Danielson and Falke, 1996). Moreover, NMR is a very important tool in structural biology because it, unlike X-ray crystallography, can reveal very important information about protein motions (Kay, 1997). For example, lSNexperiments such as the 15N heteronuclear NOE can be used to study backbone dynamics in proteins (Kay et al., 19891, and magnetization-transfer NMR experiments can yield kinetic data such as rate constants for a variety of slow-exchange reactions at equilibrium (Alger and Prestegard, 1977; Bain and Cramer, 1996). So, NMR spectroscopy is a powerful and versatile tool for studying Introduction 38 various aspects of structural biology. The work presented in this thesis takes advantage of many NMR methods, as well as other spectroscopic techniques, to learn about protein folding and structure.

Scope of this dissertation.

In working on my thesis, I have become aware that the material presented herein does not exactly follow a linear path. It has been difficult to organize the material of this thesis into a cohesive work, with a beginning, a middle, and an end. Along the way, I have become jealous of some other graduate students whose projects are much more chronological, in that all the research is about one topic, one event follows the other, and each aspect of the project is the product of building on previous results. The chronology of the work in this thesis is that it began with an idea that calmodulin, being a well-understood and well- characterized protein, could be exploited and used as a model to demonstrate the feasibility of biosynthetic incorporation of unnatural amino acids for the purpose of protein engineering and structure-hnction studies. This is how my work began, with the studies of the proline analogs of Chapters 2 and 3. Fluorinated amino acids were also included later, which are the subject of Chapter 4. As my research progressed, however, it became apparent that there is still much to learn about this interesting protein, calmodulin. The questions posed were: "Why do "EF- hand" helix-loop-helix calcium binding domains exist in pairs in proteins such as CaM?" and Thy is the central linker of CaM an a-helix in the crystal structure but flexible in solution?" and, finally, "Are the energetics of binding by CaM the same for all target peptides?" These are all Introduction 39 important questions, and the ideas involved have already been introduced here. Basically, they all deal with the properties of CaM in the presence of various ligands and other molecules. Along the way, my research has developed concepts that could be useful in protein engineering. Thus, my research in this laboratory has ccncerned two rnajor areas. The first is the biosynthetic incorporation of unnatural amino acids into CaM. The major portion of my work concerns proline analogs. As has already been discussed in this chapter, proline has a unique and important role in protein folding and structure. Proline analogs were chosen as a probe to study prolines in proteins because the analogs enable the all-important ring structure of the proline to be maintained. Thus, it was our intent to show them as better tools to perturb and/or evaluate prolyl cis-trans isomerization. Chapter 2 describes the biosynthetic incorporation of the proline analogs 3,4-dehydroproline (Dhp) and azetidine-2-carboxylic acid (Azc) into CaM and bovine calbindin D,,, and the biochemical and spectroscopic properties of these substituted proteins. In Chapter 3, the properties of Dhp and Azc, namely their cis-trans isomerization properties relative to Pro, are determined by NMR methods, and the potential for the incorporation of these analogs into other proteins for the study of prolyl cis-trans isomerization is discussed. Chapter 4 deals with biosynthetic incorporation of non-natural fluorinated amino acids into CaM. The fluorine atom has many properties that make it amenable to NMR study, especially concerning proteins. Most of the fluorine studies of proteins to date have considered fluorinated aromatic amino acids, but in Chapter 4, the properties of fluorinated aliphatic amino acids are examined in order to determine if this class of amino acids could also be generally applicable in fluorine-19 NMR studies Introduction 40 of proteins. The amino acids cis-4-fluoroproline (FPro), 5,5,5- trifluoroleucine (TFLeu) and trifluoromethionine (TFMet) are substituted into CaM for their respective natural counterparts, and the use of these amino acids as probes for the function of CaM are examined. The second ?nsjor srea of my research exarriaes tho eoergetics an3 dynamics of folding and target recognition by CaM. In Chapter 5 I have used the protease thrombin to cleave CaM into two fragments, each of which has an isolated helix-loop-helix Ca2+-bindingdomain. However, the cleavage of CaM by thrombin does not occur exactly between two helix- loop-helix domains, but rather within one of the helices flanking a Ca2+- binding loop. The properties of these fragments, both alone and in association, were studied by a variety of biochemical and spectroscopic methods in order to determine if these fragments can still associate to form a native-like structure even though the site of cleavage is disproportionate. The results demonstrate that the fragments can indeed associate and, moreover, it was also determined that the presence of CaM- binding peptides can also strengthen the association, forming a tight complex with the fragments. In Chapter 6, I have used nitrogen-15 labeled CaM to examine the backbone dynamics of CaM in a solution of 2,2,2- trifluoroethanol (TFE)that mimics the crystallization conditions. It is known that cosolvents such as TFE can stabilize protein a-helices, and this effect has been postulated to be the reason for the stability of the a- helical structure in the central linker portion of CaM in the crystal structure of the molecule. Through nitrogen-15 NMR studies the effect of TFE on the central linker was evaluated. My results indicate that TFE does decrease the flexibility of the central linker of CaM,but the presence of organic solvents is not the sole reason for the stability of this region in Introduction 41 the crystal structure of CaM. In Chapter 7, the thermodynamics of binding to CaM for four different target peptides are investigated by isothermal titration calorimetry (ITC) in order to determine the contributions of entropy and ethalpy to the binding interactions. It was found that binding of peptides to CaM can occur under different regimes, with different relative contributions of entropic and enthalpic factors. Finally, in Chapter 8 I conclude this dissertation and propose some future work concerning non-natural amino acids, and the structure and dynamics of CaM. Proline analogs in calmodulin and calbindin D.. 42

CaAPTER TWO: Biosynthetic incorporation of proline analogs into calmodulin and calbindin D.,

ABSTRACT.

Proline variants (alloproteins) of the eukaryotic Ca"-binding proteins calmodulin (CaM),and calbindin D,, have been produced through biosynthetic incorporation of the proline analogs azetidine-2-carboxylic acid (Azc) and 3,4-dehydroproline (Dhp) by bacterial overexpression of the proteins in chemically defined media. Levels of substitution of Azc or Dhp for Pro were found to be approximately 75%. Calcium-dependent SDS- PAGE bandshift assays, enzyme activation assays, and NMR spectra all demonstrated that Azc or Dhp incorporation did not greatly affect the overall structure or biochemical properties of CaM. However, the NMR properties of Dhp-substituted CaM were unique in that H3 and H4, the olefinic protons of the Dhp residues, were found in a very clear region of the proton-NMR spectrum of CaM between 5.7 ppm and 6.2 ppm. Incorporation of Dhp into calbindin D, again resulted in a new set of NMR peaks from the Dhp residues. Wild-type calbindin D,, demonstrates cis- trans isomerization of Pro43 in the native state, but spectral overlap of the peaks from the four Dhp residues made this difficult to observe in Dhp- substituted calbindin D,,. Nevertheless, Dhp is a potentially very useful probe to study the structural and functional properties of proline residues in proteins. Moreover, the ease of incorporation of Aze and Dhp into CaM Proline analo~sin calmodulin and calbindin D., 43 demonstrate potential of the use of these proline analogs in studies of prolyl cis-trans isomerization in other proteins.

INTRODUCTION.

Of the twenty standard amino acids, groline has a unique structure and role. It is the only amino acid to have its side chain linked to the amide nitrogen atom, resulting in its characteristic five-membered pyrrolidine ring structure. This strictly hinders rotation about the C,-N ($1 bond, lending a conformational constraint to the polypeptide backbone at positions occupied by prolyl residues. Consequently, prolines play a very important role in p-turns, where they induce a well-defined kink in the polypeptide backbone. In fact, p-turns can be classified according to the location of the prolines within them (Wilmot and Thornton, 1988). The Linkage of the nitrogen atom of proline to two carbon atoms in a secondary amino (imino) group also means that there is no amide hydrogen atom in the prolyl (Xux-Pro) peptide bond. Therefore, prolyl residues cannot donate an amide proton in a hydrogen bond. For this reason prolines are infrequently found in secondary structures such as a-helices or P-sheets. However, there are some instances where proline residues are highly conserved in certain a-helices (Barlow and Thornton, 1988). It may be that these prolines participate via donating a C-H proton in a unique hydrogen bond (Chakrabarti and Chakrabarti, 1998). Prolines are especially consewed in the membrane-spanning regions of membrane transport proteins and ion channels (Woolfson et ul., 1991). It is thought that the basic character of the carbonyl group in the Xxx-Pro peptide bond contributes in creating an electron-rich "pocket" which aids in the Proline analogs in calrnodulin and calbindin D, 44 translocation of positive ions across the membrane, or perhaps also that cis-trans isomerization of these prolines is part of the "switch" that opens and closes the channel in response to external stimuli (Brandl and Deber, 1986; Deber et al., 1990). Importantly, a-helices with prolines are always kinked, or bent, due to the conformationd constraints of the proline ring. Moreover, prolyl residues often exist in a-helices at N-capping positions, where their secondary aide groups reduce the unfavorable effects of unpaired hydrogen bond donors (Chakrabartty and Baldwin, 1995). As well as restricting the conformational complexity of the polypeptide backbone about the $ bond and reducing the capacity for hydrogen bonding, proline also imparts a unique rotational freedom to polypeptides. The C-N bond of a peptide group, though formally singular, does have partial double-bond character due to delocalization of the lone- pair electrons of the nitrogen atom, and thus all six atoms in a peptide group (C,0, N, H, and the two a carbons) are planar (see Figure 2.11, with hindered rotation about the peptide bond. Normally, the trans configuration of the peptide bond is highly favored, due to unfavorable steric interactions between the two side chain groups and the extending polypeptide chains when in the cis configuration. However, when the amide nitrogen is itself linked to the side chain, as is the case for proline, new steric hindrances exist in the trans conformation, and the energy difference between the cis and trans conformers is relatively low. This is manifested as a higher incidence of cis prolyl peptide bonds compared to those with other naturally occurring amino acids in protein structures and a high population of cis Xxx-Pro conformers in unst~cturedpeptides in solution. When a prolyl peptide bond in the native structure of a protein is cis, Proline analogs in calmodulin and calbindin D, 45

Figure 2.1.

trans cis

Figure 2.1. Schematic of the trans and cis isomers of a) a non-prolyl peptide bond and b) a prolyl peptide bond. The extending polypeptide chains are depicted as open circles. Proline analogs in calmodulin and calbindin D, 46 a different folding problem is presented. Even though the energy difference between a cis and a trans peptide bond is low, there is a considerable energy barrier of rotation about the peptide bond from the trans conformation, which is the default in ribosomal synthesis of nascent polypeptide chains, to the cis state. The commonly accepted value for the energy barrier of prolyl cis-trans isomerization, and rotation about any amide bond for that matter, is about 20 kcal*mol", or 80 kJ*rnol-' (Harrison and Stein, 1992). This works out to a lifetime of approximately 1-10 s, a considerable length of time when considering the folding of a protein. Therefore, if a protein has a cis peptide bond in it, the overall rate of folding would be extremely slow due to the amount of time involved in this step. The hindrance of prolyl cis-trans isomerization to protein folding has been examined since the 1970s, (Brandts et al., 1975; Creighton et aL, 1978) and today, it is accepted that prolyl cis-trans isomerization is the rate-Limiting step in the folding of many proteins. Moreover, prolyl cis-trans isomerization may also have important functional roles in many proteins, such as, for example, membrane transport proteins (Brandl and Deber, 1986; Deber et al., 1990), C-type lectins (Ng and Weis, 19981, and lysis protein E of bacteriophage 6x174 (Witte et al., 1997). In viuo, there is a class of enzymes that is capable of increasing the rate of cis-trans isomerization during folding of nascent proteins. Peptidyl prolyl isomerases (PPIases; for review see Schmid, 1993; Fischer, 1994; and Fischer et al., 1998) occur in practically every living organism. In 1989, it was discovered that cyclophilin, the cytosolic receptor for the immunosuppressive drug cyclosporin A, is in fact also a PPIase (Takahashi et al., 1989; Fischer et al., 1989). Since then, the group has expanded to include three major classes of PPIases: the cyclophilins, the Proline analoes in calrnodulin and calbindin D., 47

FK506 binding proteins (Kay, 19961, which bind a similar immunosuppressive drug called FK506, and the parvulins (Rahfeld et al., 1994). PPIases all serve to accelerate the rate of prolyl cis-trans isomerization in protein and peptide substrates, thus accelerating the rate of folding for many proteins. Evidence suggests that catalysis is simply through binding the substrate such that the prolyl peptide bond is twisted out of planarity (Harrison and Stein, 1990, 1992); when the substrate is released, it is free to revert to either the cis or the trans ground state. Additional evidence suggests that desolvation is important for catalysis: although the free energies of the cis and the trans state are similar in most cases (Radzicka et al., 1988) the transition state is thought to be considerably less polar (Eberhardt et al., 19921, and so the hydrophobic environment in the active site of PPIases can aid cis-trans isomerization. PPIases, being enzymes, do not alter the cis-trans equilibrium of a prolyl peptide bond. Instead, that is governed by other amino acids in the vicinity of the proline residue. Of great importance is the residue preceding the proline - that is, residue Xw in the Xux-Pro peptide bond. This was originally examined by Stewart et al. (1990) in a statistical analysis of the occurrence of cis peptide bonds in crystal structures of proteins in the Brookhaven Protein Data Bank. The authors noted that a great deal of these cis peptide bonds were found in bends and turns, and suggested that there may be an important role for cis Xxx-Pro peptide bonds in these structures. Interestingly, a high proportion of Tyr-Pro peptide bonds are cis (25% in the study of Stewart et al., 1990), the highest for any amino acid combination. This appeared to be not simply due to the bulk or aromaticity of the Tyr side chain; Phe-Pro was cis in only 10% of the cases. However, in a more recent look at the protein structure Proline analoes in calrnodulin and calbindin D, 48 database that examined more structures (Reimer et al., 1998), tyrosine was less conspicuous in its apparent ability to stabilize cis prolyl peptide bonds. Tyr-Pro peptide bonds were cis in 9.7% of the cases, compared to 7.2% for Phe-Pro, and Trp-Pro bonds had a higher incidence of the cis conformatior? (12.4%). Ir? light of these more recer?t observations, it seems likely that the aromaticity of the side chain of the amino acid Xxx, rather than the specific properties of tyrosine, is important in determining the ability of an Xux-Pro peptide bond to form the cis conformation. The preponderance of aromatic amino acids in Xux-Pro peptide bonds may have something to do with high occurrence of cis prolyl peptide bonds in bends and turns. Solution NMR studies of sequence variants of immunogenic peptides derived from influenza virus hernagglutinin, carried out by Wright's group (Dyson et al., 1988), showed that small peptides, such as Tyr-Pro-Tyr-Asp-Val, can form a relatively stable reverse turn structure in solution with the Tyr-Pro peptide bond in the cis conformation. Interestingly, the third amino acid in this sequence also had a significant effect on the cis:trans ratio of the prolyl peptide bond, with aromatic amino acids at this position also highly favoring the cis conformation. Structural determination of the hexapeptide Ser-Tyr-Pro- Phe-Asp-Val (Yao et al., 1994) revealed a folded form in high amounts (about 70% of the population) with the Tyr-Pro peptide bond in the cis state, stabilized by stacking of the three rings of the Tyr, Pro, and Phe residues. The authors suggested that sequences such as these represent possible nucleation sites in protein folding, stressing the importance of further study in this area. The importance of an aromatic amino acid not only preceding but also following Pro in these nucleation sequences suggests other amino Proline analoas in calmodulin and calbindin D, 49 acids in the vicinity of the proline may also have something to do with dictating a cis peptide bond. Frommel and Pliessner (1990) examined the six residues on either side of cis prolines in the protein structure database. They found several groups of related sequences, including one where the residue preceding the praline was a hydrophobic aromatic residue. They concluded that the only requirement for the residue following the proline was that it was not itself a prolyl residue. They also noted "polarity changing" around the cis prolines in this family, perhaps suggesting the involvement of salt bridges or hydrogen bonds in stabilizing turns in these sequences. In light of more recent investigations of the protein structure database, it would be interesting to revisit this study and examine the existence of aromatic residues surrounding cis prolines, and the occurrence of cis prolines in tun structures. In addition to statistical studies of protein databases, site-directed mutagenesis has been used in a lot of other cases to examine the contribution of cis prolines, and prolines in general, to the stability of proteins. Mutation of a cis proline to another residue often has drastic consequences due to the change from a secondary amide of the prolyl peptide bond to a primary peptide group. For example, the slow kinetic phase in the refolding of thioredoxin, presumably a cis-trans isomerization event, is eliminated by the mutation of a proline to an alanine (Kelley and Richards, 1987). Mutations of the cis prolines in ribonuclease A are strongly destabilizing (Schultz and Baldwin, 19921, and, again, mutations of the cis prolines in this protein also affect the folding kinetics (Schultz et al., 1992a, b; Dodge and Scheraga, 1996). Sometimes, mutation of a cis proline generates a non-prolyl cis peptide bond of the amino acid in its place, as was found for ribonuclease A (Dodge and Scheraga, 1996), Proline analogs in calmodulin and calbindin D., 50 ribonuclease T, (Mayr et al., 1994; Odefey et al., 1995), and TEM-1 P- lactamase (Vanhove et al., 1996). These cis proline mutant proteins are generally unstable, stressing the importance of a cis proline at these positions. Site-directed mutageneeis of cis grolines produces nnstabln proteins because, by default, the mutation removes the all-important ring structure of the proline residue. An alternative means for probing the role of proline residues in proteins is obviously highly desirable, and such a method is the biosynthetic replacement of structurally related non- natural or nonstandard amino acids for proline. Originally, amino acid analogs were examined as possible antimetabolites. For example, it was found that some plants accumulated high amounts of azetidine-2- carboxylic acid (Azc), the lower homolog of proline, in their tissues, and that this compound was toxic to many other species (Peterson and Fowden, 1963). It was found that this toxicity was due to incorporation of Azc into proteins, which impaired their function. Many other nonstandard amino acids, some natural and some synthetic, were tested as antimetabolites and were also found to be incorporated into the subject's cellular proteins (for reviews see Richmond, 1962; and Meister, 1965). In addition to Azc, the proline analogs 3,4-dehydroproline (Smith et al., 1962; Fowden et al., 1963), 4-fluoroproline (Gottlieb et al., 1965; Chapter 4), and 1,3- thiazolidine-4-carboxylic acid (thiaproline; Budisa et al., 1998) can also be incorporated into proteins. Today, bioincorporation of nonstandard amino acids, both natural and synthetic, is similar to the technique of residue-specific isotope labeling of proteins and involves high-output bacterial expression systems. Often, bacterial strains which are auxotrophic for the amino Proline analo~sin calmodulin and calbindin D, 51 acid of interest are used (Waugh, 19961, because they provide the lowest background levels of the natural amino acid in the expressed protein. The technique has been used successfully in our lab for calmodulin (CaM), a ubiquitous calcium-binding messenger protein found in almost all eukaryotic cells. halogs of amino scids such ss methionine (Zhmg Vogel 1994a; Yuan and Vogel, 1999; Weljie and Vogel, 2000; Chapter 41, leucine (David, 1997; Chapter 41, phenylalanine, and tyrosine (David, 1997) have been successfully incorporated into CaM in our lab. This chapter extends the research on non-natural amino acids to proline analogs. Calmodulin has two proline residues in its primary sequence (Figure 1.2). The position of the two prolines in the structure of CaM is shown in Figure 2.2a; Pro43 forms part of a turn that links EF-hand domains I and I1 in CaM, whereas Pro66 is found in the second Ca2+- binding site, perhaps playing an N-capping role for helix 4 in the protein (for the location of the proline residues in the primary sequence of CaM, see Figure 1.2). These two Pro residues probably do not play that important of a role in dictating the structural stability or function of the protein; in fact, they are not highly evolutionarily consenred amongst CaMs and CaM-like proteins in different species (Strynadka and James, 1989; Marsden et al., 19901, and mutation of the two Pro's in vertebrate CaM to the corresponding residue in the internally homologous C- terminal lobe did not significantly affect the activity of the protein (Persechini et al., 1996). In addition to incorporating proline analogs into CaM, it would be very interesting to incorporate Dhp into a protein that has a cis Pro in its native state, or in which prolyl cis-trans isomerization plays an important role in its function. A suitable choice for this thesis is bovine calbindin D,,, Proline analogs in calrnodulin and calbindin D, 52

Figure 2.2. a) Proline analogs in calrnodulin and calbindin D, 53

Figure 2.2. b)

Figure 2.2. a) Crystal structure of the Ca2+-saturatedcalmodulin (Chattopadhyaya et al., 1992; PDB co-ordinates lcll), with the proline residues highlighted. The side chains of the two proline residues, are shown in space-filling format. b) The X-ray crystal structure of Ca2+- saturated bovine calbindin D,, (Svensson et al., 1992; PDB co-ordinates 4icb), with the proline residues highlighted. The cis and the trans isomers of Pro43, both of which occurred in the crystal structure, are superimposed (lower right). Figure 2.2a was generated with MOLMOL (Koradi et al., 19961, and Figure 2.2b was generated with SETOR (Evans, 1993). Proline analogs in calmodulin and calbindin D, 54 because it is a related protein to calmodulin. Calbindin Dgk,also previously called intestinal calcium binding protein (ICaBP), is a vitamin-D inducible protein which plays a role in absorption of calcium from the digestive tract. Calbindins from mammals have two "EF-hand" helix-loop-helix Ca2+-bindingdomains with a molecular weight of around 9 000 (calbindins from birds are approximately 28 kDa with six Ca2+-bindingsites), making bovine calbindin D,, an ideal subject for NMR studies. Although the X-ray crystal structure of calbindin D,, was successfully obtained in 1986 (Szbenyi and Moffat, 1986), it became apparent later on, demonstrated by NMR spectroscopy, that calbindin D,, actually had two native conformations in solution, as a result of isomerization of the Gly42-Pro43 peptide bond (Chazin et al., 1989a; Kordel et al., 1990). More recently, both the cis and trans isomers of calbindin D,, have also been observed concurrently by crystallography (Svensson et al., 1992). The existence of both cis and trans conformers under a given set of solution conditions means two separate sets of NMR peaks that interconvert on the slow- exchange time scale, making NMR spectra quite complicated, such that now NMR studies most often use the Pro43Gly mutant (see, for example, Akke et al., 1991, 1995), which has previously been demonstrated to be very similar to the wild-type protein (Kordel et al., 1990). Since this chapter concerns proline cis-trans isomerization, it uses the wild-type calbindin gene. Bovine calbindin D,, has four proline residues, one of which, Pro43 displays cis-trans heterogeneity in the native state of the protein (residue 42 of calbindin is a glycine). As shown in Figure 2.2b, Pro43 is in the linker region between the two helix-loop-helix Ca2+-bindingdomains of the protein, in an analogous position to proline-43 of vertebrate calmodulin. There are three other prolines in bovine Proline analogs in calmodulin and calbindin D, 55 calbindin D,,; Pro3 N-caps the fist a-helix, Pro20 is found in the first Ca2+- binding loop, and Pro37 is found in a kink in the second a-helix of the protein. Figure 2.2b depicts the crystal structure of Ca2+-saturated calbindin D,,, in which both the cis and the trans isomers of Pro43 were superimposed (Srcnsson et al., 1992). In this structure, the workers observed that the cis and the trans states were approximately equally populated. The perturbations due to the isomerization were highly localized in that structural differences of the two states were found only between Lys41 and Pro43, with the biggest differences being around Gly42. By contrast, in the NMR observation of simultaneous isomers (Chazin et al., 1989a), the major isomer (the trans) was found in a 3:l ratio over the minor isomer. Effects of the two different states were observed on chemical shifts of about one third of the protein, mostly in the region between Ser38 and Asp54. Once again, the largest effects on the backbone chemical shifts were around Pro43 (between Gly42 and Thr45), demonstrating that it is the Gly42-Pro43 peptide bond that is undergoing conformational change. This chapter describes the bioincorporation of 3,4-dehydroproline (Dhp) and azetidine-2-carboxylic acid (Azc; see Figure 2.3) into CaM and some properties of these mutant proteins. Also, 3,4-dehydroproline is incorporated into bovine calbindin D,, and the Pro43Gly mutant of bovine calbindin D,,, and these proteins have also been characterized. The rationale behind the choice of these two analogs is that, as already mentioned, they have been known to be biosynthetically incorporated into E. coli proteins. In addition, Azc has one less carbon than Pro, which might affect the backbone geometry and the cis-trans isomerization properties of the protein. Dhp, although having a structure very similar Proline analogs in calmodulin and calbindin D, 56 Figure 2.3.

IH

coo

3,4-dehydro- L-proline (Dhp)

L-azetidine-2- L-pipecolic carboxylic acid (Azc) acid (Pip)

Figure 2.3. Proline analogs used in this chapter. The numbering of the ring carbons in Dhp is indicated. Dhp and Azc were both able to be incorporated into CaM in place of Pro, whereas Pip was not.

to Pro, does have an olefinic carbon-carbon double bond, and the protons attached to these carbons have quite unique spectroscopic properties. Proline analogs in calmodulin and calbindin D, 57

MATERIIALS AND METHODS.

Materials. The Escherichia coli strains Mb1294 (wild-type) W2961 (proline auxotrophic) were obtained from the E. coli genetic stock center at Yale University (http://cgsc.biology.yale.edu). The plasmid pCaM was a gift from Dr. T. Grundstrom (University of Umel, Sweden), and has been described elsewhere (Waltersson et al., 1993; Zhang and Vogel, 1993a); pBM+ is a plasmid modified from Stratagene's pBS+ plasmid (see below). The plasmid pRCBl (Brodin et al., 19891, which contains a synthetic wild- type bovine calbindin D,, gene, and the plasmid encoding the Pro43Gly mutant (Kordel et al., 1990), were both generous gifts from the laboratory of Prof. Sture Forsen (University of Lund, Sweden), and have been described elsewhere. They were supplied within the wild-type E. coli strain MM294. Deuterium oxide (D,O;D, 99.9%) was obtained from Cambridge Isotope Laboratories, Cambridge, Mass. 3,Cdehydro-(DL)- proline and (L)-azetidine-2-carboxyBcacid were obtained from Sigma. Isopropylthio-P-D-galactoside(IPTG) was obtained from Gibco. All other chemicals were obtained from reputable sources.

Construction of mutant pCaMs. The mutagenic primers Pro43Gly (5'-CTGCTTCTGTACCGTTCT- GACCAAGAG-3') and Pro66Glu (5'-TTTTGCGCGCCATCATTGTCAGAG- ACTCTTCGAAGTC-3') were constructed in the reverse orientation and were synthesized by the laboratory of Dr. M. M. Moloney, University of Calgary. A three-primer PCR-based approach (Landt et al., 1990) was used for mutagenesis, with the plasmid pBM+ as the cloning vector. pBM+ is Proline analogs in calmodulin and calbindin Do, 58 modified from Stratagene's pBS+ plasmid and contains the KpnI-Sac1 fragment of the CaM gene (from Gly26 to the end of the gene); for details on its construction see Zhang, 1994, pp. 35-39. Along with the mutagenic primer, two universal primers, one, CaM-I?, a forward primer before the beginning of the gene, and the other, CaM-R, a reverse primer after the end of the gene, were used to clone the entire portion of the CaM gene in the pBM+ vector (Figure 2.4). Briefly, the first round of PCR created a fragment that extended from the mutagenic primer to one end of the gene. This fragment was then used as a primer for the second round, which extended the product to the other end of the gene such that a fragment covering the entire gene, with the mutation, was synthesized. The PCR reactions used Vent DNA polymerase (New England Biolabs) with its accompanying ThermoPol buffer. The first round of PCR contained 1X ThermoPol buffer, 0.2 mM of each of dATP, dTTP, dGTP, and dCTP, 1 pM of each of the CaM-F and mutagenic primers, 1 nM of the pBM+ template, and 1 unit Vent DNA polymerase in a total volume of 50 pL. Prior to the addition of Vent polymerase, the samples were incubated in a preheated Techne Progene thermocycler (0.5 mL tube, 16 tube capacity) at 94 OC for 5 min. Polymerase was added, and then the samples were subjected to 30 cycles of 94 "C for 1 min, 50 OC for 1 min, and 72 OC for 11/,min. After 30 cycles, samples went through a final extension step of 72 OC for 5 min, and then they were kept at 4 "C until recovery. The resulting product was separated on an agarose gel (Sigma low gelling temperature agarose, type XI) in 0.5 X TBE buffer with 1 pg/mL ethidium bromide. An agarose piece containing the product band was visualized with an ultraviolet lamp, then excised with a razor. After weighing the agarose piece in a microcentrifuge tube, 3 volumes of TE buffer (Sambrook et al., 1989) was Proline analogs in calmodulin and calbindin D, 59

Figure 2.4.

Mutagenic 7-

is Round 1 product Mutant PCR product to be 'mgaprimer" nady to be cloned. used for mund 2.

Figure 2.4. Schematic of mutagenesis of the CaM gene. The protocol uses three primers: two universal primers and one mutagenic primer (Landt et al., 1990), and takes place in two separate PCR reactions (rounds 1 and 2). Proline analogs in calmodulin and calbindin D,, 60 added. This was heated to 65-70 "C in a water bath for 5 min to melt the agarose. The solution was mixed vigorously and then rapidly frozen in liquid nitrogen, then hand-thawed quickly. The tube was mixed vigorously again to resuspend the agarose, and then was spun down in a microcentrifuge at 1.7 000 rpm for 2 min to pellet the agarose. The supernatant, containing the PCR product, was carefully drawn off. To precipitate the DNA, two volumes of 95 % ethanol were added and the tube was kept at -20 "C for at least 1 h. The tube was spun down in a microcentrifuge at 4 "C for 10 min to pellet the DNA. The pellet was washed twice with cold (-20 "C)70 % ethanol to remove salt, then air-dried. A small amount of this pellet (-15 %) was saved in order to compare to the round 2 product; the rest was used as a primer for round 2. In addition to the round 1 product, the round 2 PCR reaction also contained 1 pM of the CaM-R primer. The concentrations of the template and all other reagents in the tube, as well as the reaction conditions, were the same as for round 1. The round 2 product was purified from low gelling temperature agarose in the same fashion as the round 1 product. For restriction digestion, this round 2 product was dissolved in 30 pL 1X One-Phor-All buffer PLUS (Pharmacia) with 10 units each of KpnI and SacI. Some pCaM vector was also digested. The digested PCR product and vector were purified from low gelling temperature agarose. For ligation, the mixture contained a roughly 10:l ratio of insert to vector in 30 pL of 1X T4 DNA ligase buffer (Pharmacia), 0.5 mM ATP, 5 mM dithiothreitol, and 1 unit T4 DNA ligase. Samples were incubated at a constant temperature of 15 "C in a Techne Progene Thermocycler. Competent MM294 cells for transformation were prepared by a procedure modified from the standard calcium chloride method (Sambrook Proline analogs in calmodulin and calbindin D,, 61 et al., 1989). All solutions and centrifuge tubes used were sterile. Briefly, cells were grown in a 40 mL LB culture until mid-log phase (OD,,, - 0.4), and then harvested in a 50 mL centrifuge tube at 5000 rpm. They were resuspended in -25 mL 10 mM MgSO, and incubated on ice for at least 30 min. Cells were then collected again bv centrifugation and resuspended in -25 mL 50 mM CaCl,, and incubated on ice for at least 30 min. Cells were spun down once more and carefully resuspended in 5 mL ice-cold 50 mM CaCl,. To freeze the cells, 400 pL of the cell suspension and 100 pL of 80 % glycerol were gently mixed in a microcentrihge tube, and stored at -80 OC. For transformation, 1 pL of a wild-type pCaM preparation, or 30 pL of a mutant pCaM ligation mix, was incubated on ice with 200 pL of competent cells for at least 30 min. A ligation mix of cut vector only, with no insert, was used as a negative control. After incubation on ice, cells were heat shocked at 42 "C for 90 sec in a PCR thermocycler. The cells were then transferred to a sterile culture tube with 2 mL LB medium and grown for

1-2 h, with shaking, at 30 OC. After collecting the cells in a microcentrifuge tube, they were spread out on LB plates with ampicillin (100 mg/L) and colonies were counted after incubation of the plates overnight at 30 OC. The efficiency of the ligation was estimated by comparing the plates from the ligation mixes with the control ligation mix of cut vector only. If the ligation was deemed to be successful, colonies were saved by subculturing on fresh LB-ampicillin plates. Cultures for plasmid purification were grown by inoculating a 2 mL culture in LB-ampicillin media and growing at 30 OC until mid-log phase (OD,,, = 0.4-0.6) was reached. Since pCaM is a temperature sensitive plasmid, derived from pBEU50 (Uhlin et al., 19831, the temperature was raised to 37 "C for -1 h to cause "run-away" production of the plasmid. Proline analogs in calmodulin and calbindin D, 62

The plasmid DNA was then purified by using Pharmacia's "Easy Prepn unit with the DNA plasmid purification kit, following the accompanying instructions. The unit facilitates rapid purification of plasmid DNA by forcing solutions through small columns at high pressure. Plasmids were checked for the correct size and presence of the insert by performing a KpnUSacI restriction digestion followed by agarose gel electrophoresis. For the larger amounts of DNA necessary for sequencing, a 40 mL LB- ampicillin culture was grown, using the same temperature-induction method. Plasmid DNA was purified from the 40 mL culture by using Qiagen's Plasmid Midi-Prep kit, following the accompanying instructions, and sent for sequencing at the University of Calgary's automated sequencing laboratory. Plasmids were sequenced to confirm the mutation, and to assure that the PCR process did not introduce any other mutations to the CaM gene. Once it was certain that the proper mutant pCaM plasmid had been constructed, it was used to transform competent W2961 (mo)cells by the procedure described above. In addition, pRCBl and Pro43Gly-pRCB1 were also used to transform W2961.

Protein expression. Wild-type CaM and calbindin D,, were prepared by inoculating a 2.8 L Fernbach culture flask containing 800 mL of LB broth with ampicillin (50 mgL) with a colony of MM294 cells with the appropriate plasmid from a fresh LB-ampicillin plate. The culture was grown overnight at 30 OC with shaking (until O.D,,,.. = 0.9 for Ca.; 0.7 for calbindin), at which

point an additional 200 mL of hot (50-60 OC) LB medium and 10 mg Proline analogs in calmodulin and calbindin D, 63 ampicillin was added and the temperature was raised to 37 "C to allow "runaway" production of the plasmid, independent of protein production. ARer 0.5-1 h (O.D,,,,, = 1.1 for CaM; 0.9 for calbindin), IPTG (100 mg/L) was added to induce protein production. After an induction of 4 h for CaM, or 3 h for calbindin, cells were harvested by centrifugation at 7 000 g for 5 min. The proline analogs 3,4-dehydroproline (Dhp) and azetidine-2- carboxylic acid (Azc) were biosynthetically incorporated into CaM. Dhp was also incorporated into Pro43Gly-CaM, Pro66Glu-CaM, calbindin D,,, and Pro43Gly-calbindin D,,. The method of bioincorporation has been used in our laboratory for other amino acid analogs, including selenomethionine, ethionine, and norleucine (Zhang and Vogel, 1994a; Yuan and Vogel, 1999; Weljie and Vogel, 2000). The first few steps are the same as for the wild-type proteins, until the point after the initial 37 OC incubation in LB media without IPTG. After this time, the cells were harvested by centrifugation at 7 000 g for 5 min; the cell pellets were resuspended in 1 L of a prewarmed (37 "C) chemically defined MOPS buffer-based medium (Niedhardt et al., 1974) supplemented with 1.32 mM KHPO,, 22 mg uracil, 22 mg cytosine, 5 mg d-biotin, 10 mg pantothenate, 3 mg p-hydroxybenzoic acid, 3 mg p-aminobenzoic acid, 3 mg 2,3- dihydroxybenzoic acid, 7 mg thiamine-HC1, 20 mg adenine, 10 mg guanosine, 100 mg of each amino acid except proline, and 50 mg ampicillin. After the cells were resuspended, Dhp or Azc (to 25 rngL concentration of the L-enantiomer) and 160 mg IPTG were added and cells were shaken at 37 "C for 4 h (for CaM) or 3 h (for calbindin D,,) to express protein. Cells were harvested by centrifugation at 7 000 g for 5 min. To guard against the loss of the three C-terminal residues of CaM, cells containing CaM Proline analogs in calmodulin and calbindin D, 64 proteins were washed with 100 mL 50 mM Tris-HC1, pH 7.5, 150 mM NaC1, 2 mM EDTA, 17.5 pM DTT and recentrifuged prior to freezing. Cells containing calbindin were washed similarly with 20 mM imidazole, 150 mM NaCl, 1 mM EDTA, pH 7.0 before being frozen.

Purification of proteins. Wid-type CaM, Azc-CaM, Dhp-CaM and the mutant Dhp-CaMs were purified according to the same methods used for wild-type CaM (Gopalakrishna & Anderson, 1982; Vogel et al., 1983; Putkey et al., 1985). They take advantage of the Ca2+-dependentexposure of hydrophobic patches in CaM and purify the protein by a hydrophobic affinity chromatography procedure. Briefly, cells were resuspended in a lysate buffer (50 mM Tris-HC1, pH 7.5, 2 mM EDTA, 17.5 pM DTT) and passed twice through a French press at 1000 psi. The lysate was spun down at 15 000 g for 25 min to pellet cell debris. The supernatant was loaded onto a 50 mL phenyl8epharose CL-4B (Pharmacia) column equilibrated with buffer E (50 mM Tris-HC1, pH 7.5 , 100 mM KCl, 300 pM DTT) and the eluate, containing apo-CaM, was collected. A further wash of 50 mL buffer E was collected as well. CaCl, (to 10 mM) was added to this pooled eluate in order to expose the hydrophobic patches in CaM. This was then loaded onto another 50 mL phenyl-Sepharose CL-4B column equilibrated with buffer A (50 mM Tris-HC1, pH 7.5, 1 mM CaCl,, 1 mM MgC1,); under these conditions CaM bound to the column. The column was washed with -1 L buffer A (until A,,,, < 0.05). Next, the column was washed with -500 mL buffer B (buffer A with 0.5 M NaCl) until A.,,,, < 0.01, and finally with -200 mL buffer C (50 mM Tris-HC1, pH 7.5, 100 mM NaC1) until A, c 0.01. Pure CaM was eluted with buffer D (50 mM Tris-HC1, pH 7.5, 100 mM Proline analops in calmodulin and calbindin D, 65

NaC1, 1 mM EDTA). Column pre-equilibration, loading, washing, and eluting steps were all generally done at -1.5 mumin. Fractions containing CaM (A, > 0.1) were pooled, dialyzed extensively against 7.5 mM WHCO, at 4 "C,and subsequently lyophilized. If necessary, metal ions were removed from protein samples by passing a solution of the protein through a 5 mL Chelex-100 (BioRad) column equilibrated with 50 mM m4HC03. The eluate containing the metal-free protein was lyophilized and stored at -20 "C. Wild-type calbindin D,,, Dhp-substituted calbindin D,,, and Dhp- substituted Pro43Gly-calbindin D,, were purified from E. coli cells by published methods (Brodin et al., 1986; Chazin et al., 1989b). Frozen cell pellets were resuspended in -80 mL calbindin buffer A (20 mM imidazole, 20 mM NaC1, I mM EDTA, pH 7.0) and passed twice through a French press at -1000 psi. The cell lysate was centrifuged to remove cell debris, and the supernatant was loaded onto a DEAE Sephadex A-25 (Pharmacia) column (2.5 cm x 40 cm) equilibrated with buffer A. The column was washed with buffer A until at -2 mumin A,,,<0.05. Then a 500 mL gradient of buffer A to buffer A with 0.5 M NaCl was applied to elute the proteins. Fractions were checked for calbindin by running on a 20% SDS- PAGE gel according to standard methods (Sambrook et al., 1989); fractions containing calbindin were pooled, dialyzed at 4 OC against 5 mM NH,HCO, with a 3500 molecular weight cutoff (MWCO) membrane, and lyophilized. The lyophilized sample was redissolved in a small volume (a few mL) of 50 mM ammonium acetate and loaded onto a Sephadex G-50 (Pharmacia) column (1.5 cm x 100 cm) equilibrated with 50 mM ammonium acetate. Proteins were eluted with 50 mM ammonium acetate at -0.35 dmin. Fractions were checked for calbindin D, by UV-absorbance spectroscopy Proline analogs in calmodulin and calbindin D, 66 and SDS PAGE (20% gel), and fractions containing calbindin D,, were pooled and lyophilized. Over the entire purification, prolonged exposure to room temperature was avoided in order to prevent nonenzymatic deamidation of the proteins at Am56 (Chazin et al., 198913). Determination of the percent incorporation of the proline analog into the protein was done by amino acid analysis, performed by Dr. Don McKay, Peptide Sequencing Facility, University of Cdgary. Both Azc and Dhp are unstable under acid hydrolysis conditions; the efficiency of incorporation was determined by the amount of residual Pro left in the samples. Alternatively, incorporation was determined by electrospray ionization-mass spectrometry, performed by Dustin Lippert (University of Victoria) or Dr. Jim Chen (University of Waterloo).

Calcium-dependent bandshifts. Ca2+-dependentSDS-PAGE bandshift assays for CaM and CaMs with proline analogs were performed similarly to Klee et al. (1979). A 158 SDS-PAGEgel was prepared according to standard methods (Sambrook et al., 1989). A 1 mghL solution of each of WT-CaM, Azc-CaM, and Dhp-CaM were prepared in H,O, with either 1 mM CaC1, or 1 mM EDTA. Small amounts of dilute NaOH were added to aid in dissolving the protein. These solutions were diluted 1:10 in SDS sample buffer, and 5 pL of each sample (0.5 ng of protein) were loaded per lane.

Calcineurin assays. Assays for activation of calcineurin were performed similarly to Newton et al. (1984). Assay mixtures contained 20 mM Tris-HCl (pH 8.O), 0.1 M NaC1, 6 mM MgCl,, 18 pM MnCl,, 1.5 mM CaCl,, 0.1 mg/mL bovine Proline analogs in calmodulin and calbindin D.. 67 serum albumin, 0.45 mM ditihiothreitol, 34 nM calcineurin, and various concentrations (0.6-18 nM) of CaM, Azc-CaM, or Dhp-CaM. Reactions were started by the addition of p-nitrophenylphosphate (pNPP) to 2.5 mM. Hydrolysis of pNPP to p-nitrophenol was followed by monitoring A,,,., for 15 min. Activities were calculated by subtracting s baseline slope (calcineurin with no CaMs added) from the actual slope. Percent activations were estimated by comparing the activity relative to that of calcineurin in the presence of 18 nM CaM, which was set at 100%.

NMR spectroscopy. Samples for NMR spectra were prepared by dissolving the proteins in 99.9% D,O with 100 mM KC1, 0.01% NaN,, and 1 mM 2,2-dimethyl-2- silapentane-5-sulfonate (DSS). Sample pH was checked with a thin-stem pH electrode (Aldrich), adjusted to 7.4 with dilute solutions of deuterated HC1 or KOH, and were not adjusted for the isotope effect. Sample concentrations were typically 1.5 mM (12.5 mg of CaM or 7 mg of calbindin) in a 500 pL sample volume. All NMR experiments were carried out on a Bruker AMX5OO NMR spectrometer, running at a lH frequency of 500.139 MHz, equipped with either a 5 rnm broadband probe or a 5 mm triple-resonance z-axis gradient probe. One-dimensional proton-NMR spectra with water presaturation were acquired according to standard methods. Typically, 256 scans were acquired per spectrum, with a relaxation delay of 1.5 s. COSY (Rance et al., 1983), TOCSY (Bax and Davis, 1985a), and NOESY spectra (Jeener et al., 1979) were acquired according to standard methods. Mixing times were 40 ms for TOCSY spectra and 100-200 ms for NOESY spectra. Spectra were acquired in the pure phase absorption mode by using the Proline analogs in calmodulin and calbindin D, 68 time-proportional phase incrementation (TPPI) method (Marion and Wuthrich, 1983); they generally consisted of 512 (Fl) by 2048 (F2) data points. All proton NMR spectra were referenced to internal 2,2-dimethyl- 2-silapentane-5-sulfonate (DSS) at 0 ppm. One-dimensional spectra were processed with the SwaN-MR processing softu.ere !Balacco, 1994) m_n_aing on a Power Macintosh 6100/60 personal computer. Two-dimensional spectra were processed using the NMRPipe software (Delaglio et al., 1995) running on a Silicon Graphics Indy R5000 workstation, and were viewed with the accompanying NMRDraw graphics package.

RESULTS.

A typical purification of wild-type CaM from MM294-pCaM cells grown in 2 L LB medium yields -80 mg of protein; purification of CaM containing proline analogs from W2961 (APro) cells using 2 L of the MOPS- based expression medium yielded 25 mg of protein on average. Typical yields from 2 L for calbindin D,, were -10 mg (wild-type) or - 5 mg (Dhp- substituted). The Pro43Gly mutant of pRCBl generally yielded more protein, most likely because the plasmid was present in higher copy number. Amino acid analysis results (Table 2.1) showed -75% replacement of the two prolines in CaM by Dhp or Azc, and a similar amount of incorporation by Dhp in calbindin (data not shown). Pipecolic acid, the higher homolog of proline (Figure 2.3) was not able to be substituted for Pro into CaM with our expression system (results not shown). Mass spectrometry results (Figure 2.5) showed that the incorporation of Dhp or Azc was at random; instead of there being a substantial peak for the unsubstitituted protein and a peak for the fully Proline analogs in calrnodulin and calbindin D, 69 substituted protein, there was a peak for the fully substituted protein and a smaller peak arising from proline being substituted at either one of the two sites. Random substitution has been obsewed in all other cases of biosynthetic incorporation in our laboratory, including multiple substitutions such as the nine Met residues in CaM. The effects of the incorporation of Dhp and Azc on the overall structure and activity of CaM were examined by a Ca2+-dependentSDS- PAGE bandshift assay (Figure 2.6) and a calcineurin activation assay (Figure 2.7). The SDS-PAGE gel shows that the mobilities of Dhp-CaM and Azc-CaM are retarded in the absence of Ca2+ions in a similar fashion to WT-CaM. The calcineurin activation assay results show that neither Azc-CaM nor Dhp-CaM are significantly different from WT-CaM in their ability to activate calcineurin, a Ca2+-CaM-dependent tyrosine phosphatase. One-dimensional 'H NMR spectra of CaM, Dhp-CaM, and the two mutant Dhp-CaMs are shown in Figure 2.8, and the 1D 'H NMR spectra of calbindin, Dhp-calbindin, and Pro43Gly-Dhp calbindin are shown in Figure 2.11. 'H NMR spectra of Azc-CaM (not shown) are qualitatively similar to those of WT-CaM. The incorporation of Dhp results in the appearance of new peaks in the region of 5.5-6.5 ppm, which arise from H3 and H4 of Dhp. As, well, the a-proton (H2) of Dhp43 is apparent at 5.52 ppm. Assignment of the signals from each Dhp residue was done by examining the spectra of the mutant Dhp-CaMs and concluding which peaks were absent as a result of the mutation. Assignment of the other protons in the Dhp ring was done by COSY (not shown) and TOCSY spectra; see Figure 2.9 for TOCSY spectra of Dhp-CaM and Pro66Glu-Dhp- CaM, and Figure 2.12 for TOCSY spectra of Dhp-calbindin D,, and Proline analogs in calmodulin and calbindin D, 70

Pro43Gly-Dhp-calbindin D,,. This was facilitated by the clear window in which to visualize the peaks in the 2D spectra. The NOESY spectrum of Dhp-CaM is shown in Figure 2.10; it is apparent that the clear window also facilitates the assignment of the Dhp residues in Dhp-CaM through sequential NOEs, and furthermore, through longer range NOEs the environment around these Dhp residues can be determined. There are several strong NOE crosspeaks to H3 and H4 of Dhp43, including a series of NOEs to what appears to be a leucine residue; this has been labeled Leu48 (see Discussion). Also visible in the region of the spectrum that is plotted are NOES between the 6,6' protons of

Dhp43 and the o! proton of Asn42. This a-6 interaction indicates a trans peptide bond (Wiithrich, 1986; Figure 2.1). There are, however, few NOEs to Dhp66; this may be because the side chain of Dhp66 is directed to the solvent in Ca2+-saturatedCaM (Babu et a1 ., 1988; Figure 2.2a). There is an NOE between H4 and what appears to be one (or both) of the 6 protons (H5) of Dhp66 at 4.50 ppm, a connectivity which was not detected in the TOCSY spectrum (Figure 2.9). The NOESY spectrum of Dhp-calbindin D,, is not shown because it actually showed fewer crosspeaks than the TOCSY spectrum run at the same conditions. This may be because, as with Dhp66 of Dhp-CaM, the side chains of the Dhp residues extend out into solvent (Svensson et al., 1992; Figure 2.2b) and thus are not close enough to other amino acid side chains to display any NOEs. Proline analogs in calmodulin and calbindin D, 71 Table 2.1.

Amino acid pmolesb Mole % Expected I I AA comn.

Thr Ser Glxa Pro G~Y Ala Val Met Ile Leu TY~ Phe His LYS I

I Total AA = 147.604 I 148

Table 2,l. Typical amino acid analysis results for a preparation of Dhp- substituted CaM, as performed by Dr. Don McKay, peptide sequencing facility, University of Calgary. Notes: 'Asx = Asp and Asn, there are 17 Asp and 6 Asn in CaM; Glx = Glu + Gln, there are 21 Glu and 6 Gln in CaM; bcalculated after a standard addition of 5 nmol norleucine; 'calculated relative to kg,which was normalized to 6 residues. The result obtained was approximately 77% (*I%)substitution of Dhp for Pro. Proline analogs in calmodulin and calbindin D, 72

Figure 2.5. a) Proline analogs in calmodulin and calbindin D., 73

Figure 2.5. b) Proline analogs in calmodulin and calbindin D, 74

Figure 2.5.

o+, . . , I, a I , ,. , . , , , .. , , , . . , , ., ., , , , . I , , , . , , , , . , , , . . . 4 Mass 1EiaIl 15!ml 16OClll 'll%UI 17mll l7$;d ' ' ladm

Figure 2.6. Electrospray ionization-mass spectrometry of a) wild-type CaM, b) Azc-CaM, and c) Dhp-CaM. The theoretical mass for wild-type CaM is 16 702, the expected mass for CaM with two prolines substituted by Azc is 16 674, and the expected mass for CaM with two Pro's substituted by Dhp is 16 698. The peak at 16 398 amu in c) is probably from Dhp-CaM with the final three amino acids missing. Removal of these amino acids has been observed in other CaM preparations and was confirmed by C-terminal amino acid sequencing (data not shown). Modification of the purification procedure in future preparations has eliminated this unwanted reaction (see Methods section). Proline analogs in calmodulin and calbindin D,, 75

Figure 2.6.

Figure 2.6. Calcium dependent bandshifts of WT-CaM,Dhp-CaM, and Azc-CaM. Lanes: 1: CaM + EDTA, 2: CaM + CaCl,, 3: Dhp-CaM + EDTA,4: Dhp-CaM + CaCl,, 5: Azc-CaM + EDTA,6: Azc-CaM + CaCl,. Proline analogs in calnodulin and calbindin D,, 76

Figure 2.7.

Figure 2.7. Calcineurin activation by wild-type CaM, Azc-CaM, and Dhp- CaM. Percent activation is relative to that by 18 nM WT-CaM, which is set at 100%. Where two points for WT-CaM exist at a single concentration, this is the result of duplicate trials. Error bars are 95% confidence levels which were determined from the calculation of the slope of &,,., vs. time (not shown). Proline analogs in calmodulin and calbindin D, 77

Figure 2.8.

Figure 2.8. One-D 'H NMR spectra of a) WT-CaM, b) Dhp-CaM, c) Pro43Gly-Dhp-CaM, and d) Pro66Glu-Dhp-CaM, in 99.9% D,O, 100 mM KCI, pH 7.4. All samples contained 1.5 mM protein and 10 mM Ca2+,and spectra consisted of 256 scans, except for a), which was 64 scails. Proline analogs in calmodulin and calbindin D,, 78

Figure 2.9.

Figure 2.9. TOCSY spectra of a) Dhp-CaM and b) Pro66Glu-Dhp-CaM. Sample conditions were as in Figure 2.8. Spectra consisted of 80 scans and had a 40 ms mixing time. Peaks arising from various protons on the Dhp residues are labeled. The upfield resonances out of the chosen chemical shift window are the S,6' (H5, H5') of the Dhp residues. This region of the spectrum of WT-CaM is devoid of any signals (see Figure 2.8). Proline analogs in calmodulin and calbindin D, 79

Figure 2.10. Proline analogs in calmodulin and calbindin D, 80

Figure 2.10. (previous page) NOESY spectrum (96 scans; 100 ms mixing time) of Dhp-CaM. The H3 and H4 protons of Dhp43 show NOE's to what appears to be two different amino acid residues; these have been labeled as Asn42 and Leu48 (see discussion).

Figure 2.11.

Figure 2.11. One-dimensional 'H NMR spectra of a) WT-cdbindin Dgk,b) Dhp-calbindin D,,, and c) Pro43Gly-Dhp-calbindin D,,, in 99.9% D,O, 100 mM KC1, pH 7.4. The peaks labeled with arrows in b), which are not present in c), arise from Dhp43 of calbindin D,,. The other three Dhp residues are tentatively assigned as 1, 2, and 3 (the peaks for residues 2 and 3 overlap). Protein samples were -1.5 mM and contained 5 mM Ca2+. 256 scans were acquired for each spectnun. Proline analogs in calmodulin and calbindin D, 81 Figure 2.12.

a)

Figure 2.12. TOCSY spectra (80 scans, 40 ms mixing time) of a) Dhp- calbindin D,, and b) Pro43Gly-Dhp-calbindin D,,. Sample conditions were the same as in Figure 2.11. The peaks connected with lines in spectrum a) are from Dhp43. Also in a), the peak labeled with an arrow is a Dhp a proton (it is not present in the spectrum of WT-calbindin D,,; see Figure 2.11), although no crosspeaks with any other Dhp protons are seen. There are, however, distinct crosspeaks between this proton and the rest of the side chain of its Dhp residue in Pro43Gly-Dhp-calbindin D,, (spectrum b). Pe82

DISCUSSION.

Calmodulins with its two proline residues substituted by azetidine- 2-carboxylic acid (Azc) or 3,4-dehydroproline (Dhp) have been successfully produced in high amounts using a proline auxotrophic E. coli strain and a high-yield expression system. As well, Dhp has been substituted into the related Ca2+-bindingprotein calbindin D,,, which has four proline residues, one of which, proline-43, co-exists as the cis and trans isomers in the native state (Chazin et al., 1989a; Svensson et al., 1992). Interestingly, pipecolic acid, the higher homolog of Pro (Figure 2.3), could not be incorporated into CaM in place of Pro using this expression system. This is likely because pipecolic acid is too large to fit into the active site of the prolyl-tRNAPrOsynthetase, or perhaps because there is no efficient method for uptake of pipecolic acid by E. coli cells. The extent of incorporation of Dhp and Azc was approximately 75%, comparable to the 85-90% levels of substitution which are possible with substitution of the methionines in CaM by the Met analogs selenomethionine (Zhang and Vogel, 1994a), ethionine and norleucine (Yuan and Vogel, 1999), and difluoromethionine (Yuan, 1998) and the substitution of Phe and Tyr by their fluorinated analogs (David, 1997). Ideally, for the purposes of functional studies, such as enzymatic assays, and structural studies, such as NMR and X-ray crystallography, the level of incorporation of the analog should be as high as possible in order to avoid heterogeneity problems in the sample. It may be possible to raise the level of incorporation by including more of the nomaturd amino acid in the expression media, but many amino acid analogs are toxic at higher concentrations (Liu and Schultz, 19961, so there becomes a trade off between expression levels and cell viability. The Proline analogs in calmodulin and calbindin D2 83 beauty of an inducible expression system, however, is that one can control cell growth and protein expression independently, by choosing when to transfer cells from the rich growth media to the defined expression media, and by choosing when to add the inducer. Another practical problem with adding more of the fiormatural amino scid is the cost fsctor; many of these compounds are quite expensive so they are used sparingly (e.g. 25 mg/L of L-Dhp in expression media vs. 100 mg/L of the "natural" amino acids). Even under these conditions, though, the level of incorporation of Dhp was sufficient to provide excellent NMR spectra. The effects of the proline substitutions to the overall structure of CaM and calbindin are likely to be quite minimal; Dhp and Azc substitutions did not significantly affect the activity of CaM in activation assays for the tyrosine phosphatase calcineurin (Figure 2.7), which is a canonical CaM-activated enzyme. As well, the 'H NMR spectra of Azc- CaM (not shown), Dhp-CaM (Figures 2.8-2.10), and Dhp-calbindin (Figures 2.11, 2.12) are all qualitatively similar to those of their corresponding wild-type proteins. An exception is the properties of the Dhp-substituted proteins, which have one unique aspect: in the 1D 'H NMR spectra of WT-CaM (Figure 2.81, there is an area between the a- proton peaks and the aromatic proton peaks, at around 5.5-6.5 ppm, that does not have any signals. This is typical for most proteins, especially ones that do not have a great deal of P-sheet structure. Alpha-protons from P- sheet residues tend to be shifted downfield relative to other a-protons such that they can infringe upon this area (Cavanagh et al., 2000). The relative abundance of a-helical structures and scarcity of P-sheets in CaM (there are only two small two-stranded P-sheets in the protein) means that this clear area in the spectrum is quite substantial. Incorporation of Dhp into Proline analms in calmodulin and calbindin D, 84 the protein results in new NMR signals that arise in this area, which are from protons H3 and H4 of the Dhp residue. These are "olefinic" protons, attached to carbon atoms which are involved in a C=C double bond. There are no naturally occurring olefinic protons in proteins, so the incorporation of Dhp int~proteins results in the introduction of a unique chemical group. The appearance of the signals from these protons in a clear window in 'H NMR spectra gives the researcher a unique probe to specifically study the proline residues in proteins. It is almost like labeling Pro residues with a completely novel NMR-active nucleus. However, since this nucleus is simply a chemically unique proton, NMR experimentation remains quite simple with only routine homonuclear spectra being necessary, and no need for more complicated heteronuclear experiments. The ease of finding the olefinic protons of Dhp is evident when examining 2D NMR spectra as well. TOCSY spectra (Figure 2.9) show clear connectivities between the protons in the Dhp ring. Normally, TOCSY spectra are used in conjunction with COSY spectra to assign the protons within a particular spin system (i.e. an amino acid side chain). They are complementary because COSY spectra only detect, at most, three-bond couplings and thus only show crosspeaks between protons on neighboring carbon atoms. TOCSY spectra, on the other hand, generally show crosspeaks between all the protons in a spin system. However, olefinic protons, such as those of Dhp, are a special case because they are held in a rigid conformation as a result of the C=C double bond. Moreover, the double bond is somewhat shorter than a single bond. As a result of these factors, it is possible to see couplings of greater than three bonds with olefinic protons in COSY spectra. This made assignment of H3 and Proline analoas in calmodulin and calbindin D,, 85

H4 of the Dhp residues in Dhp-CaM and Dhp-calbindin D,, difficult, because both H3 and H4 generally showed the same pattern of COSY crosspeaks. However, through careful inspection of the relative intensities of the crosspeaks in COSY (not shown) and TOCSY spectra (Figures 2.9 and 2.111. is was possible to assign H3 and H4 of the Dhp residues in these proteins. Through NOESY spectra (Figure 2.10) it is very simple to see other protons which are close to H3 and H4 of the Dhp residues in the three- dimensional structure of the molecule. This enables assignment of the Dhp residues, as well as some conclusions about the tertiary structure around the Dhp residues. Interestingly, Dhp43, the proline residue found in a flexible connecting loop in CaM, actually has more NOES to it than Dhp66, which is involved in a tightly defined structure in a Ca"-binding loop. Dhp43 shows a fair number of contacts with what is likely a leucine residue. Distance picking (not shown) of the Ca"-CaM crystal stmcture (Chattopadhyaya et al., 1992) with the RasMol v. 2.6 molecular renderer program (ORoger Sayle 1992-95)showed that Pro43 is in the vicinity of Leu48, which may be the leucine in question, and is thus labeled as such in Figure 2.10. There is also an NOE crosspeak between the S,6' protons of Dhp43 and the a proton of Asn42. This a-6 (i, i+l) through-space connectivity involving a Pro residue indicates that the peptide bond is trans (Wiithrich, 1986), which is confirmed by the X-ray crystal structure of the protein (Babu et al., 1988; Chattopadhyaya et al., 1992). If there were a cis peptide bond, then there would be an a-a(i, i+l)connectivity instead (Wiithrich, 1986; see Figure 2.1). Distance picking also showed that the side chain of Pro66 does not have a great deal of contacts with other residues in Ca2+-CaM. Instead, as is demonstrated in the X-ray Proline analogs in calrnodulin and calbindin D, 86 crystal structure (Babu et al., 1988; Chattopadhyaya et al., 1992; Figure 2.2a) is pointed out towards the solvent where it contributes to the hydrophobic patch of the N-terminal lobe. One NOE with H4 of Dhp66 that is labeled in Figure 2.10 is most likely an intraresidue NOE to the 6 and/or 6' protons at 4.50 ppm, and is labeled tentatively as such. This connectivity could not be seen in the TOCSY spectrum of the same sample, however, and there does exist the possibility that this could be an NOE to another residue. Although no other protons from other residues in the vicinity of Dhp66 resonate at 4.50 ppm, there is a certain caveat when assigning protons neighboring to Dhp residues via NOESY spectra. The n- electrons of the double bond in Dhp have similar effects to the side chains of aromatic amino acids in that they can induce ring-current shifts. The result is that chemical shifts of nuclei around Dhp residues may be different than the published values for wild-type proteins. However, since the Dhp ring is not aromatic, the shifts induced are small compared to those generated by phenyl rings in proteins. Thus Dhp is an excellent probe for the structure around proline residues in proteins. The NOESY spectra of Dhp-calbindin and Pro43Gly-Dhp-calbindin are not shown here, because they actually showed fewer crosspeaks than the corresponding TOCSY spectra. This could be because the four Dhp residues of Dhp- calbindin are in flexible regions and are thus subject to conformational averaging, or possibly simply that the side chains of these Dhp residues do not make any close contacts with any other amino acid side chains. In Figure 2.2b it does seem that the side chains of the prolines in wild-type calbindin D,, extend towards the exterior of the protein, so perhaps it is not surprising that the Dhp residues of Dhp-calbindin do not display any NOES. Proline analogs in calmodulin and calbindin D2 87

Dhp is also potentially useful for examining prolyl cis-trans isomerism in proteins. The appearance of the peaks for H3 and H4 of Dhp in a clear window in protein NMR spectra makes them easily discernible, with a relatively low chance of problems associated with overlapping peaks in the region. Thus,these peaks are ideal candidates not orrly for the determination of cis:trans isomer ratios by peak integration, but also determination of the kinetics of prolyl cis-trans isomerization through experiments such as NMR lineshape analyses (Hiibner et al., 1991, Kern et al., 1997) or magnetization transfer experiments (Alger and Prestegard, 1977, Bain and Cramer, 1993). In our case, Figure 2.8 shows minor peaks in the vicinity of H3 and H4 of the Dhp residues in Dhp-CaM, especially in the Pro43Gly mutant (Figure 2.8~). These may be due to a minor conformation, such as a cis isomer. However, magnetization transfer experiments with inversion of these minor peaks at elevated temperatures (data not shown) did not demonstrate any conformational exchange between these isomers. It may be that these minor peaks are not exchange-related, or that exchange is too slow to be detected. In calbindin D,,, there is a prolyl cis-trans isomerization event, involving the Gly42-Pro43 peptide bond, in the native state of the protein that is well-characterized both by NMR (Chazin et al., 1989a) and X-ray crystallography (Svennson et al., 1992). Incorporation of Dhp into calbindin D,, results in the appearance of four new sets of peaks from the four Dhp residues (see Figures 2.11 and 2.12). Through comparison with spectra of Pro43Gly-Dhp-calbindin, it was possible to assign the peaks of Dhp43 in Dhp-calbindin (indicated with arrows in Figure 2.11 and with lines in Figure 2.12); the other three Dhp residues are labeled as 1, 2, and 3. However, there is only one set of peaks visible for Dhp43. This is in Proline analogs in calmodulin and calbindin Do, 88 contrast to WT-calbindin, where the Gly42-Pro43 peptide bond can exist as either the cis or the trans isomer in the native state, with the levels of the cis isomer being between 25% (Chazin et al., 1989a) and 50% (Svensson et al., 19921, depending on the conditions. It could be possible that the set of peak for the minor !i.e. cis! conformstion of Dhp43 is buried under the peaks for the other Dhp residues; the area of the peak for Dhp43 at 5.93 ppm in spectrum 2.11b is 0.80 while the area for the peak for Dhp residue 1 is 1.20 (relative to the peak at 5.86 ppm which is set at 2.00); this could be because the peak for the cis isomer of Dhp43 is buried under the peak for residue 1, or perhaps this is due to some other exchange event or incomplete relaxation. Another possibility is that the cis and the trans H3 and H4 peaks of Dhp43 are degenerate. However, 1D 'H NMR spectra acquired under various conditions, including different temperatures, pHs, and ionic strengths (data not shown) did not show minor peaks for any of the Dhp residues. It could also be that the substitution of Pro by Dhp stabilizes the trans conformation of the Gly42-Pro43 peptide bond of calbindin. In the tripeptide studies of the next chapter, Dhp does favor the trans conformation more than Pro in the sequence Acetyl-Tyr-Pro-Ser. The same might also be happening in calbindin. In addition to Dhp, the substitution of Azc for Pro has significant effects on the rate and equilibrium of prolyl cis-trans isomerization, as has been shown by a detailed study of the tripeptides presented in the next chapter. These properties of Dhp and Azc, as well as the ease of their biosynthetic incorporation into proteins in place of Pro as has been shown in these studies of calmodulin and calbindin D,,, make these analogs ideal substitutes for the study of the role of prolines in the folding and function of proteins. NMR studies of nroline analom in tri~e~tides 89

CaAPTERTHRBE: NMR determination of the kinetic and thermodynamic parameters of cis-trans isomerization of proline analogs

ABSTRACT.

The cis-trans isornerization kinetics and thermodynamics have been studied for the two proline analogs L-azetidine-2-carboxylicacid (Azc) and L-3,4-dehydroproline (Dhp) have been studied by 1D 'H NMR and inversion transfer-NMR methods. The system chosen was the tripeptide Acetyl-Tyr-Yyy-Ser, where Yyy is either Pro, Azc, or Dhp; Tyr was chosen as the amino acid to precede the Yyy residue because it gives a high percentage of the cis conformation. The cis-trans equilibria of the three tripeptides were studied by a Van't Hoff analysis, which revealed that Azc had a higher cis:trans ratio, while Dhp had a lower &:trans ratio, than Pro in this system. The differences between the three tripeptides were greatest at lower temperatures, due to differing contributions by enthalpic and entropic factors. The kinetics of the cis to trans isomerization were examined by inversion transfer-NMR at elevated temperatures and analyzed with the Eyring relationship. While the results for the Dhp- and Pro-containing peptides were similar, the Azc-containing peptide showed a faster rate of cis to trans isomerization, owing to a significantly different (positive) entropy of activation for the process. The results are discussed in terms of the potential for incorporation of these analogs into proteins in which prolyl cis-trans isomerization is important for the folding or the NMR studies of ~rolineanalogs in triwwtides 90 function of the protein.

INTRODUCTION.

Proline has unique roles and characteristics when it comes to the folding and structure of proteins, as was discussed in the last chapter. The ring structure of the prolyl residue not only introduces unique constraints to the backbone, but also provides relative freedom for the peptide bond to adopt the cis conformation. However, as is the characteristic for rotation about all amide bonds, isomerization between the cis and trans isomers of Xm-Pro peptide bonds is very slow relative to the majority of other protein folding events. For some time, experimentalists and theoreticians alike have been trying to determine the kinetic parameters of prolyl cis-trans isomerization. The accepted value for the energy barrier restricting rotation about the C-N bond of amides, which is the result of data from many groups, is in the range of 20 kcal*rnol*l,or 80 kJmol*'. This has been determined predominantly by NMR experimentation for a variety of small compounds, such as N- methylformamides and N, ET-dimethylformamides (Stewart and Siddall, 1970; Drakenberg and Forsen, 1971; Drakenberg et al., 19721, and N- acylprolines (Maia et al., 1971). Molecular dynamics simulations have also been applied in this area (Perricaudet and Pullman, 1973). Many of the experimental methods for measuring isomerization rates have involved perturbing the cis-trans equilibrium by temperature- or pH-jumps (Brandts et al., 1975; Grathwohl and Wuthrich, 1981) or by a solvent change (Kofkon et al., 1991; Reimer et al., 1998) and then measuring the rate of isomerization by NMR or other means as equilibrium is NMR studies of ~rolineanalogs in tri~evtides 91 reestablished. Spectrophotometric assays in which the cis-trans isomerization is monitored by a chymotryptic digestion specific for the trans isomer (Kofron et al., 1991; Keller et al., 1998) have also been used. By contrast, NMR methods can be extremely useful for measuring these types of kinetic processes because they cm do so without disturbi~gthe chemical equilibrium. In particular, magnetization transfer experiments can be used for reactions, such as peptidyl cis-trans isomerization, that are relatively slow on an NMR time scale (Alger and Prestegard, 1977; Bain and Cramer, 1993). In these experiments, a specific radio frequency pulse is applied to one of the exchange-related signals and the cis-trans isomerization is observed as magnetic equilibrium is reestablished. Alternatively, rate constants can be determined by NMR measurements at magnetic equilibrium, such as 1D lineshape analyses (Hiibner et al., 1991; Kern et al., 1997; Brown et al., 1998) and 2D exchange-NOESY spectra (Jeener et al., 1979; Justice et al., 1990). These methods generally work better for faster processes such as prolyl cis-trans isomerization that takes place at higher temperatures or is catalyzed by prolyl cis-trans isomerases. Determination of the thermodynamic and kinetic parameters of prolyl cis-trans isomerization, or any slow exchange process for that matter, ideally requires the presence of two NMR peaks, one for the cis isomer and one for the trans isomer, that are readily discernible from each other as well as from all other peaks in the spectrum. Because of this, most NMR experiments concerning prolyl cis-trans isomerization have used peptides as models, rather than whole proteins, due to the peak overlap problems and the complexity of spectra associated with polypeptides of any appreciable size. One exception is the characterization of a proline NMR studies of ~rolineanalogs in tripeotides 92 isomerization in staphylococcal nuclease by saturation transfer and inversion transfer methods (Loh et al., 1991), which took advantage of the NMR peaks from well-resolved histidine ring protons, which are shifted downfield from all other proton signals in proteins.

It 7vor?ld be very usefu! to have EUI NMR technique which can be used to probe prolyl cis-trans isomerization in a wide variety of proteins. One potentially promising idea would be to incorporate 3,4-dehydroproline (Dhp) in place of proline into proteins, which, as was demonstrated in the previous chapter, introduces new, chemically unique protons into the protein which could fill the criterion of possessing NMR peaks for which the cis and trans isomers are readily discernible from each other as well as from the rest of the peaks in the spectrum. However, it is important to know if Dhp has effects relative to Pro on the relative stability of the cis and the trans isomers, as well as the kinetics of cis-trans isomerization. There may be instances where influences on the rate and equilibrium of prolyl cis-trans isomerization could benefit the researcher. For example if the presence of a small amount of a minor isomer in solution complicates NMR spectra of a particular protein, incorporation of a Pro analog which is highly favors one isomer may eliminate the undesirable minor component. Dhp could potentially benefit the researcher in this respect as well, and, moreover, the biosynthetic substitution of azetidine-2- carboxylic acid (Azc) for proline, which was demonstrated in the previous chapter, could also beneficially affect cis-trans kinetics. To determine the result of substitution of Azc and Dhp for Pro on the rate and equilibrium of prolyl cis-trans isomerization, the NMR experiments described above should provide useful information. An NMR lineshape analysis study of cis-trans isomerization in a set of tripeptides Ala-Yaa-(p-nitroanilide), NMR studies of ~rolineanalogs in tri~e~tides 93 where Yaa is thiazolidine-4-carboxylic acid, oxazolidine-4-carboxylic acid, pipecolic acid, or Azc, and their catalysis by the peptidyl prolyl isomerase cyclophilin, has already been undertaken (Kern et al., 1997). This chapter describes NMR inversion transfer studies of the tripeptides Acetyl-Tyr- Y-y-Ser: where Yw is Pro: Dhp, or Aac. The reason why tyrosine was chosen as the amino acid preceding proline for this study was because statistical studies of protein structure databases show that Tyr-Pro peptides have a very high incidence of the cis conformation (Stewart et al., 1990; Reimer et al., 1998). It now seems that this may be in the context of a larger sequence where cis Tyr-Pro peptide bonds are involved in a reverse turn, with the Tyr aromatic ring interacting with the ring methylene groups of the proline (Yao et al., 1994). This type of interaction has also been noted in non-prolyl cis peptide bonds (Jabs et al., 19991, which also have a high incidence of aromatic amino acids, where there are interactions between the aromatic ring and the p methylene group of the adjacent amino acid sidechain. Whatever the case, we chose Tyr-Pro for this study in order to "stack the deck" and increase the odds in favor of the cis conformation, thus making integration of the minor cis peaks in NMR peaks more reliable. The Ser residue was added to increase solubility of the peptide, and the peptide was N-acetylated in order to reduce charge interactions between the N- and C- termini. In order to determine thermodynamic parameters of the cis-trans equilibrium for these three tripeptides, peaks were integrated at various temperatures similarly to Wu and Raleigh (1998). For determination of kinetic parameters, we chose the inversion transfer technique (Alger and Prestegard, 1977), a technique which has also worked well in many other areas, such as determination of kinetic constants in enzymatic reactions NMR studies of oroline analoes in tripe~tides 94

(Robinson et al., 1984). With the results in this chapter, and data fitting using the proper program (Bain and Cramer, 1996), this method works at least as well as the lineshape analysis methods preferred by others (Hiibner et al., 1991; Kern et al., 1997; Bain, 1998). Quality data was obtained for all three peptidest and the three proline analogs, Azc, Dhp, and Pro, were compared.

Inuersion transfer - theory. Although inversion transfer NMR experiments can determine the kinetic parameters for interconversion between several exchangeable sites (Muhandirarn and McClung, 19871, the theory behind interconversion between only two forms, such as the case for peptidyl cis-trans isomerization, is simpler to understand and will be the focus here. In the inversion transfer experiment, a 180' inversion pulse is selectively applied to one of the exchangeable peaks in an NMR spectrum. After a suitable delay time the usual 90" acquisition pulse is applied to all the protons and the spectrum is acquired in the traditional manner for 1D spectra. This experiment is then repeated over several scans. The peak which has had the inversion pulse applied would be upside-down relative to all other peaks in the spectrum. However, during the variable delay, inversion of magnetization can be relieved from the selected peak in one of two ways. In the absence of exchange, only the first is accessible: this is recovery of inversion through the normal T, relaxation process. However, if the inverted peak is in chemical equilibrium with another peak in the spectrum, such as through peptidyl cis-trans isomerization, inversion of magnetization can be "transferred" to its partner through the exchange process. This would be shown as a faster rate of recovery for the inverted NMR studies of proline analogs in tripeptides 95 peak, and a transient decrease in the area of the exchange-related peak (Figure 3.lb). Eventually, both peaks would recover to their full heights through T, relaxation. The overall process can be depicted as follows (Dahlquist et al., 1975):

Relaxation

If site C (the cis peak) is inverted, then the equations for inversion transfer would be as follows (Mariappan and Rabenstein, 1992):

Mc(t)= c,expOc,t) + c,exp(h,t) + Mce M'(t) = c,~Jk,+ l/r,,)expOc,t) + c2r2(;(-' + llr,,)exp(h?t)+ M', where t is the delay time, Mc(t) and Mt(t) are the magnetizations (peak areas) of the cis peak and the trans peak, respectively, at time t; Mceand Mee are their magnetizations at equilibrium (i.e. at t = -1; r,, and r,, are the effective relaxation times:

lhl,= m*,+ kc

1/71, = VTI, + k (note that k and l/~are interchangeable for a given rate constant k and lifetime r) and A,, &, c,, and c2 are defined by Alger and Prestegard (1977): NMR studies of proline analoes in tri~e~tides 96

where Mc, and Meoare estimates of the magnetizations at t = 0. There are eight variables in these equations: MC0,Mt,, Mee, Mte,T,,, TI,12, and kt. In principle, it is possible to find all eight variables simply from fitting the equations to experimental data. However, it is generally more appropriate for the researcher to supply data values, or at least estimates thereof, for at least some of the parameters Meo,ML,, Mce, Mee, T,,, and T,,,and then fit the data to determine the rate constants kc and kt. The values of magnetizations at equilibrium (Mceand Mte)are straightforward to find; usually this is done by analyzing a spectrum without inversion. The values of the magnetizations at zero time can only be estimated due to the fact that there is a minimum possible delay between pulses, and thus the magnetizations immediately after inversion can never be accurately known. The T, values can be determined from a separate inversion recovery experiment, in which the entire spectrum is inverted. The T,s are then just the time constants for the exponential recovery of the magnetizations to equilibrium values. Only estimates of the T,s can be determined, due to the fact that exchange (i.e. cis-trans isomerization) also NMR studies ofproline analoes in tri~e~tides 97 contributes to recovery of magnetization. However, this is only a problem if the T,s for the cis and trans peaks are very different, or very slow relative to the exchange process. Here this is not the case, so exchange is neglected. Once the data values are supplied. there are a few approaches to determining the rate constants. One way is to supply guesses of the exchange rates along with the other parameters and then use these values to calculate a model set of data. The model set of data is compared to the actual data, and then changes to the parameters are made according to the discrepancies. The new parameters are then inputted to create a new set of model data, and so on. This process is iterated until it converges. The CIFIT data fitting program (Bain and Cramer, 19961, which employs this approach, is used in this study. In practice, generally all the parameters were fixed and only the rate constants were floated.

Kinetics and thermodynamics - theory. Once the equilibrium constants (from peak areas of 1D NMR spectra) and rate constants (from inversion transfer spectra) have been determined, it is simple to determine thermodynamic and kinetic parameters such as energy and entropy differences, and energy and entropy of activation, for the process. What follows are some basic concepts and equations that enable us to determine these parameters. It is possible to calculate the Gibbs' free energy difference between the cis and trans forms from an equilibrium constant at a given temperature by using Gibbs' free energy relationship: NMR studies of ~rolineanalogs in tri~eptides 98

To determine the enthalpy and entropy differences, one creates a plot of lnKp versus Utemperature (in Kelvins). The enthalpy is determined from the slope of the plot, and the entropy from the y-intercept, by the Van't Hoff relationship:

Note that AGO is dependent on temperature and is reported at some standard temperature value, such as 298 K. In this study, we chose to report AGO values at 323 K because at this temperature reliable data could be collected for all three peptides. Determination of kinetic parameters follows similar steps. From a rate constant, it is possible to determine the energy of activation by a plot of ink versus l/T and the Arrhenius relationship:

k = A*exp(-Al3'/RT),or: Ink = -hE'IRT + lnA.

This equation also contains A, the pre-exponential factor. It contains information about the entropy of activation, although generally the Arrhenius relationship is only used to determine activation energies, and the A factor is simply ignored. One can find both the enthalpy and entropy of activation by a similar plot (In k/T vs. UT) and the Eyring equation: NMR studies of proline analoes in tri~ewtides 99 where k, is the Boltzmann constant and h is Planck's constant. AH: is determined from the slope of the plot and AS' is found &om the y-intercept. Alternatively, one may determine the enthalpy and entropy of activation from the following (Brown et al., 1998):

LW = flf- RT;and AS*= R(lnA + ln[hNA1/eRTI), where NA is Avogadro's number, and e is the base of the natural logarithm. In practice AH* and AS: were determined from the Eyring relationship, although following the other route obtained similar results. Finally, it is once again possible to determine Gibbs' free energy of activation from Gibbs' equation:

MATERIALS AND METHODS.

Materials. (L)-Azetidine-2-carboxylicacid (Azc) and (L)-3,4-dehydroproline (Dhp) were obtained from Sigma. Deuterium oxide (99.9% D) was obtained from Cambridge Isotope Laboratories, Cambridge, Mass. The tripeptides acetyl-Tyr-Pro-Ser, acetyl-Tyr-Azc-Ser, and acetyl-Tyr-Dhp-Ser were synthesized at the Queen's University Peptide Synthesis Facility (Kingston, Ont.) according to standard methods, and were purified to >95% by HPLC. Correct mass of the tripeptides was confirmed by ESI-MS. NMR studies of ~rolineanalogs in trine~tides 100

All other chemicals were obtained from reputable chemical sources.

NMR Spectroscopy. For NMR, 0.5 mL samples of the three tripeptides were prepared in 99.9% D,O, 100 mM KC!, 40 mM KDiPO,.The peptide concentration was approximately 12.5 mM. Sample pH was adjusted with appropriate amounts of dilute KOD or DCl to 7.4; pH was checked with a thin-stem pH electrode and was not adjusted for the isotope effect. In order to remove oxygen from the samples, which can act as a relaxation agent, samples were purged with N2gas for -10 min. All NMR spectra were acquired on a Bruker AMX500 NMR spectrometer operating at a proton frequency of 500.13 MHz, equipped with a triple-resonance z-axis gradient-shielded probe. Proton NMR spectra were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) at 0 ppm. 1D 'H NRIR spectra were acquired according to standard methods, and were processed by the SwaN-MR processing program (Balacco, 1994) operating on a Power Macintosh 6100160 personal computer. 2D TOCSY spectra with 40 ms mixing times were acquired according to the methods of Bax and Davis (1985a), and 2D rotating-frame nuclear Overhauser effect (ROESY) spectra (250 ms mixing times) were acquired according to the methods of Bax and Davis (1985b). All spectra were acquired in the TPPI mode (Marion and Wiithrich, 1983) to obtain quadrature detection in the F1 dimension. They were processed with the NMRPipe processing package (Delaglio et al., 1995) running on a Silicon Graphics Indy workstation and were viewed with the accompanying NMRDraw graphics interface. NMR studies of proline analogs in tripe~tides 101

TImeasurements. T, inversion recovery experiments consisted of a 180" hard pulse to the entire spectrum, followed by a variable delay, then the 90" acquisition pulse. Generally 18 delay times were used, ranging from 0.01 s to 40 s. Spectra of 40 scans were acquired per delay time. with delays of 25 s between scans to ensure complete relaxation. Water presaturation was applied during the relaxation delay. 16k data points were acquired per scan. The 18 spectra were collected as a 2D data set, with the delay time being the F1 dimension. The total acquisition time for each experiment was 7.5 h. T, values were determined by analyzing the data with SwaN- MR's (Balacco, 1994) T, data fitting routine. T, data were collected at five different temperature values for each peptide, ranging from 318-338 K (Ac- Tyr-Pro-Ser), 303-323 K (Ac-Tyr-Azc-Ser),or 323-343 K (Ac-Tyr-Dhp-Ser).

Inversion transfer measurements. The inversion transfer experiment is a variation of a simple 1D pulse sequence, with an additional selective inversion pulse in the middle (Figure 3.la). Selected peaks were inverted with a 50-60 ms Gaussian- shaped soft pulse. The inversion pulse is followed by a delay, which is varied, and then the conventional 90" hard pulse for acquisition. Inversion transfer data were collected as a series of ID 'H NMR spectra, each one with a different inversion transfer delay. A total of 16 spectra were generally acquired for each experiment, with delay times ranging from 0.05 s to 12 s. Generally 40 scans were acquired per spectrum, with a 25 s delay between each scan to ensure complete relaxation. Two series of inversion transfer spectra were collected for each peptide, one with inversion of the cis peak and one with inversion of the trans peak. The NMR studies of ~rolineanalogs in trine~tides 102

Figure 3.1.

ACQUIRE

Figure 3.1. a) Pulse sequence for the inversion transfer experiment. The relaxation delay T, should be at least 5x the TI relaxation time for the inverted peak. The 180" soft pulse is selective for the inverted peak, and 7, is the variable delay. b) A typical inversion transfer experiment, as a result of inversion of the cis Tyr3,5 protons of Ac-Tyr-Azc-Ser at 323 K. Delay times are indicated to the right of each spectrum. NMR studies ofproline analoes in trioeptides 103 total time for both series of inversion transfer spectra was -12 h. Data were collected at five different temperatures for each peptide, at the same temperature values as the T, data. Wherever possible, the T, inversion recovery experiments and the inversion transfer experiments were run consecutively to ensure const-ant sample conditions. The inversion transfer spectra were processed with the SwaN-MR processing program (Balacco, 1994) operating on a Power Macintosh 6100/60 personal computer. Integrated peak areas were placed in a text file and imported to a Silicon Graphics Indy workstation, where they were fitted using the CIFIT exchange data fitting program (Bain and Cramer, 1996). The program was supplied with the raw data of peak areas, as well as estimates of the peak areas at time = 0, peak areas at equilibrium (determined by a 1D experiment with no inversion transfer), and the T, values supplied by the separate inversion recovery experiment. Only the values for the rate constants were floated.

ID and 20 'H NMR spectroscopy and thermodynamic measurements. The 1D 'H NMR spectra of Ac-Tyr-Pro-Ser, Ac-Tyr-Azc-Ser, and Ac- Tyr-Dhp-Ser at 298 K are shown in Figure 3.2. There are two sets of peaks in each spectrum, correlating to a major and a minor isomer. In each case the major set of peaks were determined to be from the trans isomer and the minor peaks form the cis isomer as deduced from NOESY and ROESY spectra (not shown). The arrows in the 1D 'H NMR spectra indicate the peaks that were integrated in order to determine the cis:truns ratios, and NMR studies of oroline analogs in trine~tides 104

Figure 3.2. NMR studies of ~rolineanaloes in tri~e~tides 105

Figure 3.2. (previous page) 1D 'H NMR spectra of a) Ac-Tyr-Pro-Ser, b) Ac-Tyr-Dhp-Ser, and c) Ac-Tyr-Azc-Ser. Peptides were dissolved at -12.5 rnM in D20,100 mM KC1, 40 mM KD,PO,, pH 7.4, at 323 K. 256 scans were taken for each spectrum. Peaks labeled with arrows correspond to cis-trans related pairs that were chosen for inversion and integration. For Ac-Tyr-Pro-Ser, one set of peaks was chosen, depending on the position of the residual water peak, which shifts with temperature.

were inverted in the inversion transfer experiments. In the case of Ac- Tyr-Azc-Ser, the degenerate Tyr ring protons H3 and H5 were inverted. For Ac-Tyr-Pro-Ser, either the a-proton of the Tyr residue or the a-proton of the Pro residue was chosen. The residual water peak shifts with temperature in proton-NMR spectra, such that it overlapped with either one of these two peaks at some temperatures. The peak with the least overlap was chosen at each temperature. For Ac-Tyr-Dhp-Ser, the H3 and H4 protons of the Dhp ring provided peaks that were very convenient for integration and inversion. The temperature dependence of the cis:trans ratio for the three tripeptides is shown in Figure 3.3a. Best-fit lines are drawn for each plot. The percentage of the cis isomer for Ac-Tyr-Pro-Ser remains fairly constant, at about 28% over the temperature range studied. By contrast, the Azc-containing peptide has a substantially higher percentage of the cis isomer, nearly 40% at 25 "C,but this ratio decreases with increasing temperature. The Dhp-containing peptide, conversely, has a lower percentage of the cis isomer, only about 19% at 25 OC but that increases with temperature, such that the percentage of cis is similar for all three peptides at around 70 OC. NMR studies of ~rolineanalogs in tri~e~tides 106

Figure 3.3. a)

Temp. (K)

Ac-Tyr-Pro-Ser A Ac-Tyr-Azc-Ser 0 Ac-Tyr-D hp-Ser NMR studies ofproline analogs in tri~e~tides 107

Figure 3.3.

[7 Ac-Tyr-Pro-Ser A Ac-Tyr-Azc-Ser 0 Ac-Tyr-Dhp-Ser

Figure 3.3. a) Temperature dependence of the cis:trans ratio, with the cis plotted as a percentage of the overall population, for the three tripeptides. The data originate from integrated peak areas of ID 'H NMR spectra; each data point is from a single spectrum at the given temperature. Best-fit lines are drawn. b) Van't Hoff plots for the three tripeptides. The equilibrium constant was found by dividing the cis area by the trans area (i.e. the cis isomer was treated as the product). Best-fit lines are drawn. NMR studies of nroline analogs in tri~e~tides 108

Table 3.1.

Peptide AH0 (kJ-mot'1' AS0 (Jemol-'*K1 )' AGO (kJ-mot')d

Ac-Tyr-Pro-Sera 0.1k0.6 -7.7k0.4 2.5k0.7

Ac-Tyr-Azc-Sera -6.7k0.5 -26.6k0.2 1.9k0.5

Ac-Tyr-Dhp-Sera 3.8k0.6 0.9k0.4 3.5k0.8

Gly-Tyr-Pro-Glyb 2.72 -2.25 3.44

Table 3.1. Thermodynamic parameters for the trans <-> cis equilibrium for the three tripeptides in this study, as well as Gly-Tyr-Pro-Gly (Wuand Raleigh, 1996). The uncertainty values reported are 95% confidence levels (calculated from twice the standard error taken from the regression analysis of the Van't Hoff plots). NMR conditions are outlined in the Methods section. a) This study; b) data taken from Wu and Raleigh, 1996; C) From Van't Hoff relationship (lnK,, = -AHO/RT+ ASO/R);d) at 323 K; from Gibbs's equation (AGO = AH0 + TAS*).

The Van't Hoff plot for the three tripeptides is shown in Figure 3.3b. As explained in the Introduction, this enables determination of the three thermodynamic parameters for the trans c-> cis equilibrium. They are listed for all three peptides in Table 3.1. The enthalpy differences between the cis and the trans states are very different for the three peptides. For Ac-Tyr-Pro-Ser, the enthalpy difference between the cis and the trans state is negligible. The trans state, however, is entropically favored. This is similar to Gly-Tyr-Pro-Gly (Wu and Raleigh, 19961, although here the trans state is enthalpically as well as entropically favored. For Azc, a NMR studies of nroline analo~sin tripeatides 109 negative enthalpy difference between the cis and the trans states, at -6.7 kJ*mol-', means that enthalpy actually favors the cis conformation, and it is the large negative entropy difference that is responsible for the trans state being more populated. Lowering the temperature results in an increase in the percentage of the cis conformation. With Dhp, enthalpy favors the trans state such that it is more populated than in the case with Pro, and it is a small favorable entropy change that is responsible for the increase in the percentage of cis with increasing temperature.

Inversion transfer measurements. The kinetic data presented in this chapter are from fitting of data from inversion of the cis peak, which gave more reliable results. This is probably because the rate constant for the cis to trans rotation, and thus the rate of inversion transfer from cis to trans, is larger. The results for the trans to cis conversion would then be expected to have less uncertainty. Figure 3.4 shows the peak areas involved in the inversion transfer as a result of inversion of the cis peak for the three tripeptides at 323 K The pair of data points for each delay time is the result of one 1D spectrum. The experiments were also done with inversion of the trans peak and at several different temperatures, as outlined in the Methods section. The lines are drawn through theoretical data points calculated in the fitting routine of the CIFIT program (Bain and Cramer, 1996). It is evident that there is good agreement between the actual and the calculated values. It is also evident, by visual inspection, that the rate of transfer from the cis to the trans peak is fastest for Ac-Tyr-Azc-Ser and slowest for Ac-Tyr-Dhp-Ser, with Ac-Tyr-Pro-Ser somewhere in the middle, but similar to Ac-Tyr-DhpSer. This is also depicted in the Arrhenius NMR studies of ~rolineanalogs in triveptides 110

Figure 3.4.

0 0 2 4 6 8 10 12 Delay (s) NMR studies of wroline analogs in trioe~tides 111

Figure 3.4. (previous page) Inversion transfer data as a result of inversion of the cis peak at 323 K for a) Ac-Tyr-Pro-Ser, b) Ac-Tyr-Azc- Ser, and c) Ac-Tyr-Dhp-Ser. The open squares are the areas of the trans peak, and the open circles are the areas of the cis peak. As explained in the Methods section, experiments were carried out at a variety of other temperatures and also with inversion of the trans peak.

(Figure 3.5a) and Eyring (Figure 3.5b) plots, where the Pro- and the Dhp- containing peptides are fairly similar, but the plot for the -4zc- peptide is shifted upwards quite significantly, owing to the faster rate. It is notable that this shift upwards is not attributed to a smaller activation energy (Table 3.2). Rather, the entropy of activation is of opposite sign for Ac-Tyr- Azc-Ser than for Ac-Tyr-Pro-Ser, such that entropy favors the transition state for the Azc- peptide but disfavors it for the Pro- containing peptide. This is quite different than the case of Ala-Pro-p-nitroanalide vs. Ala-Azc- p-nitroanalide (Kern et al., 19971, where it was enthalpy, and not entropy, differences that caused the cis to trans isomerization to be faster for the Azc-containing peptide than for the Pro-containing peptide. The transition state is also entropically favorable for Ac-Tyr-Dhp-Ser, but the large enthalpy of activation for the cis to trans isomerization of the peptide means that overall, the isomerization rate for the Dhp- peptide is similar to that for the peptide containing proline. The kinetic plots for the trans to cis isomerization are not shown. However, owing to the differences in the equilibrium constants for the three tripeptides, it can be concluded that the trans to cis isomerization is faster for Ac-Tyr-Azc-Ser than for Ac-Tyr-Pro-Ser, whereas the rate for Ac- Tyr-Dhp-Ser is considerably slower. NMR studies of proline analogs in tri~eptides 112

Figure 3.5. a)

Ac-Tyr-Pro-Ser A Ac-Tyr-Arc-Ser 0 Ac-Tyr-Dhp-Ser NMR studies of vroline analogs in tri~eotides 113

Figure 3.5. b)

Ac-Tyr-Pro-Ser

A Ac-Tyr-Azc-Ser

0 Ac-Tyr-Dhp-Ser

Figure 6. a) Arrhenius plots, and b) Eyring plots for the three tripeptides. Each data point is the result of fitting of a series of inversion transfer spectra, as shown in Figure 3.4. Error bars are from error values supplied by the CIFIT program (Bain and Cramer, 1996). Best-fit lines are drawn. NMR studies of oroline analo~sin tripe~tides 114 Table 3.2.

Peptide hE'(kJ-rn~l-')~&P(kJ-mol-' )" ~S*(J-mol-'-K1 )' AG'(~J-mol")'

Ac-Tyr-Pro-Sera 76*17 73*17 -23*2 80k18

Ac-Tyr-Azc-Sera 8 7t2 84+2 25.3k0.4 76*2 Ac-Tyr-Dhp-Sera 9 126 88*6 23.4~0.9 8126

Ala-Pro-pNAb 78.93t0.2 -6.Ok2.0 80.8k0.8 Ala-Azc-pNA' 67.0*3 -23.3k1.7 74.6*4

Table 3.2. Kinetic data for the cis to trans conversion. NMR experimentation and data analysis was outlined in the Methods section. a) this study; b) data taken from Schutkowski et al. (1994); c) data taken from Kern et al. (1997); d) from Arrhenius relationship (Ink = -AE'/RT + 1nA); e) from Eyring equation (ln(k/T) = -(AH:/R)(YT) + ASVR + ln(k,h)?;f) at 323K, from Gibbs's equation (AG: = &EP - TAW. Uncertainty values for this study are 95% confidence levels (calculated from twice the standard error resulting from regression analyses).

DISCUSSION.

The unnatural amino acids 3'4-dehydro-L-proline and L-azetidine-2- carboxylic acid have been compared to the natural amino acid L-proline in terms of their NMR properties and the kinetics and thermodynamics of cis-trans isornerization. The peptides Acetyl-Tyr-Yyy-Ser, where Yyy is Pro, Azc, or Dhp, were chosen for comparison because tyrosine can increase the ratio of the cis isomer of prolyl peptide bonds. The goal of this NMR studies of oroline analoes in trioeotides 115 study was to determine what effects these analogs have on cis-trans kinetics and thermodynamics when they are biosynthetically incorporated into proteins. As was the case for Dhp-CaM (Chapter 21, the peaks for H3 and H4 of Dhp in the 1D 'H NMR spectra of Ac-Tyr-Dhp-Ser were in a ucique region, removed from any of the other peaks. Moreover, the cis and the trans peaks for these protons were also quite resolved from each other, making peak integration and inversion transfer studies easy. This raises the possibility of biosynthetic incorporation of Dhp into proteins for the purpose of examining cis-trans isomerization, eliminating the problems of peak overlap and crowded spectra. Although the spectra of Ac-Tyr-Dhp-Ser show promise for the use of Dhp as a probe for prolyl cis-trans isomerization in proteins, the real goal of this chapter was to determine the influences of substitution of Dhp, as well as Azc, for Pro on the kinetics and thermodynamics of prolyl cis-trans isomerization. It is obvious from the 1D 'H NMR spectra that neither Azc nor Dhp favors one conformation exclusively over the other. This is somewhat unfortunate for the NMR spectroscopist, because it would be extremely desirable to have a proline analog that exclusively favors one conformation in order to eliminate confbsing minor components in NMR spectra. However, Dhp does favor the trans conformation more than Pro (Figure 3.31, such that incorporation of Dhp into proteins does show some promise in terms of Yocking" prolyl peptide bonds in a protein in the trans conformation. With Azc, the opposite was observed, such that the cis conformation was favored more than Pro. The opposite effects of Dhp and Azc were both exaggerated at lower temperatures. This is due to different enthalpic and entropic contributions, as is depicted in Table 3.1. The NMR studies ofproline analogs in triaeptides 116 enthalpy difference between the cis and trans states is negligible for Ac- Tyr-Pro-Ser, and so the cis:trans ratio of this peptide displays little dependence to temperature. By contrast, enthalpic factors actually favor the cis state for Ac-Tyr-Azc-Ser, and entropic factors are responsible for the trans stste being more favored st higher temperatures. For Ac-Tyr- Dhp-Ser, enthalpic factors favor the trans state, but are compensated somewhat by entropic factors at higher temperatures. Thus, it is possible to alter the cis-trans ratio of a prolyl peptide not only by changing Pro for one of these analogs, but also by altering the temperature as well. It is unclear whether or not these same effects could be seen for these analogs in a protein, but it is conceivable that, with the right choice of Pro analog and manipulation of temperature, biosynthetic incorporation of Dhp or Azc into a protein may eliminate or at least alleviate cisltrans heterogeneity problems for structural studies. Apart from the cis:trans ratios, the analogs Azc and Dhp also have different rates of cis-trans isomerization than Pro, as determined by inversion transfer NMR. The results obtained by this method, shown in Figure 3.5 and Table 3.2, are quite reliable and, moreover, inversion transfer is quite convenient compared to other methods of measuring prolyl cis-trans isomerization. The traditionally used chymotrypsin- coupled assay (Fischer et al., 19841, or more newly developed fluorometric uncoupled assays (Garcia-Echevenia et al., 1992) have disadvantages in that they require chromogenic peptide substrates which can be poor mimics of a native protein (Scholz et al., 1997). In some cases the cis and trans isomers of peptides can be separated by HPLC methods, facilitating kinetic studies (Jacobson et al., 19841, but this probably only has limited applications. Other NMR methods of measurement of rate constants NMR studies ofproline analogs in trioe~tides 117 besides inversion transfer exist (Baine, 1986), such as NMR lineshape analyses and two-dimensional methods. These methods are also very useful, although the results obtained by inversion transfer NMR in this chapter have been quite reliable. The results of the inversion transfer studies indicated an energy of activation for the cis to trans isomerization of about 80 kJmol" (about 20 kcalmol-') for the tripeptide Acetyl-Tyr-Pro-Ser, which is in agreement with the commonly accepted values for prolyl cis-trans isomerization in peptides (Harrison and Stein, 1992). The activation parameters we determined (Table 3.2) correspond to a rate constant of about 0.05 s" at room temperature (25 OC), which is quite slow on an NMR time scale. This explains why we performed our inversion transfer experiments at elevated temperatures (between 30 and 70 "C). The Azc-containing peptide had a faster rate of isomerization than that with Ro, even though the activation energy was higher. This was owing to a significantly different entropy of activation (25 J*mol*l*K1for Azc compared to -23 Jemol-I-K1for Pro), which favored formation of the transition state. This is easily seen on the Arrhenius and Eyring plots for the three tripeptides, where the lines are more-or-less parallel, put the Ac-Tyr-Azc-Ser line is offset significantly higher than the other two lines. Ac-Tyr-Dhp-Ser has a higher enthalpy of activation than Ac-Tyr-Pro-Ser, but again there is a significantly different entropy of activation than Pro, similar to Azc. The end result is a free energy of activation for Ac-Tyr-Dhp-Ser that is similar to Ac-Tyr-Pro-Ser at 323 K (50 OC). The lower proportion of the cis state for Ac-Tyr-Dhp-Ser compared to Ac-Tyr-Pro-Ser likely results from a slower rate of trans to cis isomerization. Ac-Tyr-Azc-Ser has a higher proportion of the cis state than Ac-Tyr-Pro-Ser, which means that the difference in rates of trans to NMR studies of proline analogs in trine~tides 118 cis isomerization between the two peptides must be even larger than for cis to trans isomerization, with the Azc-containing peptide being even faster. This raises the possibility of biosynthetic incorporation of Azc into proteins in which conversion from the trans to the cis state is important for the folding of the protein (i.e. in proteins which have a cis prolyl peptide bond in the native structure). There are also proteins where prolyl cis- trans isomerization is important for the function of the protein, such as, for example, the coupling of prolyl isomerization to Ca" binding in C-type lectins (Ng and Weis, 19981, or the coupling of prolyl isomerization to induction of the lysis process in lysis protein E (Witte et al., 1997). With Azc in place of Pro, the rate of cis-trans isomerization, and thus the rate of the coupled process, would be increased. With Dhp in place of Pro, the researcher would be provided with a convenient handle, to be monitored by conventional NMR methods, for the isomerization process in proteins where it is important for folding or function. One could envision future studies in this area which exploit the properties of Azc or Dhp in a variety of protein systems. There are a few caveats for the researcher when evaluating Azc or Dhp as potential Pro substitutes, however. The influences of Azc or Dhp on the kinetics and thermodynamics of prolyl cis-trans isomerization seem to depend highly on the system in which they are incorporated. One need only to look at Table 3.2 to see this. This chapter determined that the substitution of Azc for Pro, in the peptide Ac-Tyr-Pro-Ser, had the effect of lowering the Gibbs' free energy of activation. The study of Kern et al. (1997) compared Ala-Azc-(p-nitroanalidelto Ala-Pro-(p-nitroanalidel and found similar effects on the free energy of activation. However, their NMR studies of ~rolineanalogs in trioeotides 119 effects were largely the result of enthalpy differences (bH' was 67 kJ-mol-I for Azc vs. 79 kJ*mol" for Pro), and the entropy differences worked in the opposite direction. In this chapter, the opposite was true, where it was very significant differences in the entropy of activation that were responsible for the increased rate of cis-tran. isornerizetion of Ac-Tyr-hc- Ser vs. Ac-Tyr-Pro-Ser. One might envision cases where Azc is incorporated into proteins and the effects could be largely different than that observed for the peptides. A similar argument could be made for Dhp. However, with all this considered, it is very important to note that the effects of Azc on AG', the free energy of activation, were very similar in our study and in the study of Kern et al. (1997). This is very encouraging because it is this value which has the most reliability (Brown et a1 ., 1998), since it is determined most directly from experimental data. It is this value which should be most important because it is the one which directly reports the differences in isomerization rates. Therefore, even though there are significant discrepancies between the results of this study and those of Kern et al. (19971, the potential for use of Azc and Dhp in influencing prolyl cis-trans isomerization thermodynamics and kinetics is very promising. The hture goal of this line of research is to combine the techniques of non-natural proline analog bioincorporation, outlined in the previous chapter, and the inversion transfer techniques demonstrated in this chapter, and apply them in a system where prolyl cis-trans isomerization is an important aspect of the folding or function of the protein. Fluorinated ali~haticamino acids in calrnodulin 120

CHAPTER FOUR: Biosynthetic incorporation of fluorinated aliphatic amino acids into calmodulin

ABSTRACT.

Fluorinated amino that are incorporated into proteins are generally aromatic because of their ease of incorporation and the disperse fluorine- 19 NMR spectra of the substituted proteins. This chapter describes an examination of the properties of fluorinated aliphatic amino acids into proteins, and evaluates the potential for their use in biological applications. The fluorinated amino acids cis-4-fluoroproline (FPro), 5,5,5- trifluoroleucine (TFLeu), and S-trifluoromethylhomocysteine (trifluoro- methionine; TFMet) have been biosynthetically incorporated into the ubiquitous and important Ca2+-bindingprotein calmodulin (CaM) through a bacterial expression system and methods described in Chapter 2. The levels of incorporation were approximately 60% for FPro and 20% for TFLeu and TFMet, as was determined by mass spectrometry and amino acid analysis. It was found that these amino acids were not as useful as the fluorinated aromatic amino acids because of their low incorporation, and the instability of the substituted proteins; the dispersity of their 19F NMR spectra was also found to be poor, but through the two-dimensional NMR experiment the 19F, 'H heteronuclear COSY (HETCOSY) it was possible to obtain good spectra in some cases. Moreover, TFLeu-CaM and TFMet both reported changes in the hydrophobic patches of CaM as a Fluorinated aliphatic amino acids in calmodulin 121 result of Ca2+-bindingand the binding of target peptides.

INTRODUCTION.

The discussions on NMR in this thesis thus far have focused solely on NMR of more conventional nuclei, namely protons, carbon-13, and nitrogen-15. However, there are many other NMR-active nuclei that are quite useful in biological NMR, including cadmium-113, an NMR-active calcium substitute used in the studies of thrombic fragments of calmodulin presented in the next chapter, phosphorous-31, which is widely used in NMR of nucleic acids and phosphorylated metabolites, and a range of quadrupolar metal ions which are useful in NMR studies of metalloproteins. Fluorine-19 is the NMR-active nucleus which is the subject of this chapter, and it has many applications in NMR. Fluorine-19 has many advantages which make it very suitable for NMR in biological systems (reviewed by Gerig, 1989; and Danielson and Falke, 1996):

1. First, lgF is a highly sensitive, receptive NMR-active nucleus. In fact, its sensitivity rivals that of protons. It is also spin-U2, like 'H, 13C, and 15N, which makes it possible to study by conventional NMR techniques. 2. It's natural abundance is 100%, so expensive isotopic enrichment, like that needed for 13C and 15N (uniform I3C-labeling of proteins for NMR can often run into the hundreds or even thousands of dollars; specifically labeled amino acids are also quite expensive), is not necessary. 3. The van der Waals radii of fluorines and hydrogens are quite similar Fluorinated aliphatic amino acids in calrnodulin 122

(1.35 A for F vs. 1.20 A for H; Pauling, 1960) so substitution of a proton for a fluorine atom is isosteric and there are no major structural perturbations. 4. There are almost no fluorine atoms naturally occurring in biological systems. so the background signal in 19F NMR spectra is essentially zero. This means that spectra of proteins are much simpler, which opens the possibility for NMR analysis of larger proteins (Gettins, 1994). 5. Fluorine-19 also has a wide chemical shift range, about 100 times that of 'H, as a result of paramagnetic shielding generated by the fluorine lone-pair electrons (Danielson and Falke, 19961, which means that 19F signals in proteins are most often well resolved. Additionally, this large range means that the chemical shifts of lgF nuclei are extremely sensitive to their environment. Thus, they can report changes to their local environment, such as those in tertiary packing, ligand binding, electrostatic effects, pH changes, and changes to solvent exposure. 6. Fluorine is much more electronegative than hydrogen (in fact, it is the most electronegative atom in the periodic table), which can be advantageous in some instances. Fluorines can be used to alter the polarity of various amino acids, examining the role of hydrophobic effects in proteins, or alter the pK, of amino acid sidechains such as tyrosine (Sykes et al., 1974; Ring et al., 1985; Parsons and Armstrong, 1996) and histidine (Jackson et al., 19941, which can change the pH profile of some enzymes.

Apart from the advantages of fluorine NMR, there are also some Fluorinated ali~haticamino acids in calmodulin 123 disadvantages. First, the electronegativity of fluorine already mentioned can be a problem if it affects the folding or stability of a protein. Ideally, there should be no structural changes as the result of the introduction of fluorine atoms to a protein in order for the results of 19F NMR spectra to be interpretable, but sometimes this is not the case. However, surprisingly, fluorine substitution in proteins, especially in aromatic amino acids, is quite non-perturbing (reviewed by Danielson and Falke, 1996). Another disadvantage of ''F NMR is the large anisotropy of its chemical shift tensor. Consequently, contributions of the chemical shift anisotropy (CSA)relaxation, especially to the transverse (T,)relaxation mechanism, result in increasing line widths as the magnetic field is increased (Smith et al., 1989; Gerig, 1989). Thus, there is often no advantage to obtaining "F NMR spectra at higher fields. This is why many of the 19F NMR spectra in this thesis have been obtained on a 300 MHz spectrometer (I9Fv, = 282 MHz) and none of them have been collected on a 500 MHz spectrometer. However, due to the very large receptivity of lgF,very good spectra can be obtained even on a 300 MHz spectrometer. As already mentioned in this introduction, the background 19F signal in most biological samples is essentially zero, and indeed there are no naturally occurring fluorine atoms in proteins. This, then, leaves the introduction of 19F into a sample entirely to the discretion of the user.

There are as many ways of introducing 19Finto a biological sample as there are biological applications of lgF NMR. Many drugs, including anticancer drugs, contain fluorine, and 19F NMR spectroscopy has been successfully applied to studies of the metabolism of these agents (Robinson et al., 1997; Hanusovska et al., 1998). As an aside, another very important medical application of fluorine is in positron emission tomography (PET) of Fluorinated aliphatic amino acids in calmodulin 124 fluorine-18-labeled compounds, such as 2-fluoro-2-deoxyglucose, where it is the radioactive fluorine-18 that emits the positrons. This has been applied in imaging of many disorders, including heart disease, brain disorders, and various cancers (see, for example Magistretti and Pellerin, 1999). There are also other in vivo applications of IgF NMR, such as the measurement of transmembrane pH gradients with fluorinated vitamin B, derivatives (Mason, 1999), the use of fluorinated proteins to measure intracellular concentrations of metabolites (Williams et al., 19931, and and the measurement of the rotational mobility of fluorinated proteins in vivo (Williams et al., 1997). There are also many other fluorinated ligands and protein modification reagents that can be used for 19FNMR (Gerig, 1989). For our purposes, the most interesting method of incorporation of fluorine atoms into a biological sample is through biosynthetic incorporation of fluorinated, non-standard amino acids. The technique of non-natural amino acid bioincorporation has already been discussed in Chapter 2, which dealt with proline analogs. Its application in fluorine- NMR was pioneered largely by Brian Sykes while at Harvard University and is the subject of numerous reviews (Sykes and Hull, 1978; Sykes and Weiner, 1980; Gerig, 1994; Danielson and Falke, 1996). Much of the work has concentrated on fluorinated aromatic amino acids, because these have produced the most success in bioincorporation into E. coli proteins. Fluorinated tyrosines and phenylalanines have been used in our lab to probe the role of these residues in the hnction of calmodulin (David, 1997). Fluorotyrosines are particularly interesting because fluorine substitution lowers the pK, of the hydroxyl function; 2- and 3-fluorosyrosine (FTyr) incorporation into gluathione-S-transferase (GST),an enzyme with a Fluorinated aliphatic amino acids in caZrnoduZin 125 tyrosine in its active site, affects the pH profile of the enzyme (Parsons and Armstrong, 1996; Brokx, Azarnousch, and Vogel, unpublished results). For the 19F NMR spectrum of 2-FTyr-GST, see Figure 4.1. 3-F"ryr,having the fluorine atom nearest to the hydroxyl group, gave the largest effect. Fluorotyrosine substitution did cause the GST to be less active overall: however; presumably this is due to conformational changes around the many other tyrosines in the enzyme, as was observed in the crystal structure of a rat GST substituted with 3-fluorotyrosine (Xiao et al., 1996). Fluorinated tryptophans were applied to the study of the effects of ligand binding in proteins such as histidine-binding protein (Post et al., 19841, and retinol binding protein (Li et al., 1990). Fluorotryptophans were also used in the characterization of membrane-bound proteins such as bacteriophage M13 coat protein (Wilson and Dahlquist, 1985) and lactate dehydrogenase (Rule et al., 1987; Peersen et al., 19901, demonstrating the applicability of 19F NMR to study large molecular-weight proteins and complexes. In addition, the high sensitivity of 19F also allows for the collection of real-time dynamic information about protein folding through the application of techniques such as stopped-flow NMR (Frieden et al., 1993). Although there is a great deal of literature on 19F NMR of proteins with fluorinated aromatic amino acids, fluorinated aliphatic amino acids have thus far been largely neglected. There may be many reasons for this: one is that fluorinated aromatic amino acids are readily commercially available, while fluorinated aliphatic amino acids are not. Another is that researchers have perhaps been wary of the effects of incorporating fluorinated aliphatic amino acids on the folding and stability of the target protein. Another potential problem is that conformational heterogeneity is Fluorinated ali~haticamino acids in calmodulin 126

Figure 4.1.

Figure 4.1. A representative 1D 19F NMR spectrum of a protein with fluorinated aromatic amino acids. This is the spectrum (v, = 376 MHz) of 2-FTyr-substituted schistosomal glutathione-S-transferase (GST), a 27 kDa protein with 14 tyrosine residues. a) with no glutathione added, b) with 1 equivalent glutathione added. The peak for Tyr7 (labeled), the active-site tyrosine of the protein, shifts upon the addition of glutathione. The other peaks in the spectra are for the other 13 tyrosine residues in the protein. The sharp peak at -44.8 ppm is from a processing artifact. Fluorinated aliphatic amino acids in calmodulin 127 more prevalent in aliphatic side chains; aromatic groups are sterically rigid, so the only potential for conformational heterogeneity in by rotation of bonds about the CP atom. By contrast, aliphatic side chains, such as those of Leu and Met, sample a much wider conformational space. 19F, as already discussed, is highly sensitive to the local environment, so therefore some types of conformational heterogeneity could cause problems in interpreting NMR spectra. Conformational averaging as the fluorinated aliphatic side chains rotate freely may result in more degeneracy in spectra of fluorinated aliphatic amino acids. However, there have been a few examples of fluorinated aliphatic amino acids successfully being incorporated into proteins. One example is that of leucine analogs; 5-fluoroleucine has been used in NMR of dihydrofolate reductase (Feeney et al., 1996) and there, indeed, side chain conformational differences were responsible for there being a large chemical shift range of the incorporated "F atoms. It has long been known that 5,5,5- trifluoroleucine can be incorporated into E. coli proteins (Rennert and Anker, 1963; Fenster and Anker, 19691, although, to my knowledge, a detailed NMR investigation of a 5,5,5-trifluoroleucine-labeled protein has not yet been attempted. In addition, S-(trifluoromethyl)homocysteine,i.e. trifluoromethionine, has been incorporated into bacteriophage h lysozyme (Duewel et al., 19971, and difluorornethionine has been incorporated into CaM in our lab (Yuan, 1998). Chapter 2 of this thesis described studies of proline analogs in calmodulin, and indeed there are also some examples of fluorinated prolines being incorporated into proteins in the literature. Cis and trans 4-fluoroprolines' can both be incorporated into collagen in

' It should be noted here that the terms cis and trans for 4-fluoroproline do not indicate the rotameric state of a proIyl peptide bond, but rather indicate the position of the fluorine on the proline ring relative to the carboxyl group; see Figure 4.2. Fluorinated aiivhatic amino acids in calmodulin 128 guinea pig granuloma minces (Gottlieb et al., 1965). It was also found that the electron withdrawing effects of trans-Cfluorination can increase the rate of prolyl cis-trans isomerization (Eberhardt et al., 1996), in the same way that trans-4-hydroxylation can in collagen. As already mentioned in Chapter 2, CaM has twc? proline residues, proline-43 and proline-66. In this chapter, cis-4-fluoroproline has been incorporated into CaM and its properties have been examined by fluorine- 19 NMR. Unfortunately, the expense of the starting materials, along with the complexity of the synthetic protocol for the amino acid, precluded any studies of trans-4-fluoroproline substituted CaM. The methionine residues in CaM have been discussed in Chapter 1. There are nine methionine residues in CaM (Figure 1.2); Met36, Met51, Met71, and Met72 are in the N-terminal hydrophobic patch of CaM, MetlO9, Met124, Met144, and Met145 are in the C-terminal hydrophobic patch, and Met76 is in the central linker of the protein (Babu et al., 1988; Yuan et al., 1999a). CaM also has nine leucine residues (Figure 1.2); Leu32, Leu48, Leul05, and Leu116 are involved in the hydrophobic patches while Leus4, 18, 39, 69, and 112 pack in the hydrophobic cores of the protein and contribute to the hydrophobic character of the four Ca2+-bindingsites. In this chapter, TFMet and TFLeu are incorporated into CaM and the Caz+-bindingand peptide binding properties of these substituted proteins are examined by I9F NMR. The three fluorinated amino acids in this chapter are shown in Figure 4.2. The results will show that, as could be expected, the "F NMR spectra of these proteins are not as disperse as the spectra of proteins with fluorinated aromatic amino acids. Changes in the hydrophobic patches of CaM were, however, able to be reported by TFLeu and TFMet. Moreover, the 19F NMR spectra of FPro-CaM and TFLeu-CaM were expanded into two Fluorinated ali~haticamino acids in calnodulin 129

Figure 4.2.

H coo-

cisll-f luoroproline 5,5,5-trif I uoroleucine trifluoromethionine (FPro) (TFLeu) (TFMet)

Figure 4.2. Fluorinated amino acids used in this chapter. The fluorine atoms are bolded. As well, the carbon atoms of FPro and TFLeu are numbered. The preparation of TFLeu used in this study is racemic with respect to carbon 4. Fluorinated ahhatic amino acids in caLmoduLin 130 dimensions by the 19F, 'H HETCOSY experiment.

MATERIALS AND METHODS.

Materials. Trans-N-carbobenzyloxy-4-hydroxymethylproline and p-toluene- sulfonyl fluoride were purchased from Sigma. Cis-4-fluoro-L-proline (FPro) was synthesized by Dr. Deane D. McIntyre according to the methods of Patchett and Witkop (1957) and Gottlieb et al. (1965). S- trifluoromethyl-L-homocysteine (trifluoromethionine, TFMet) was a gift from the laboratory of Dr. John Honek (University of Waterloo, Ont.). (4RS)-5,5,5-trifluoro-DL-leucine(TFLeu) was purchased from Lancaster (Pelham, NH, USA). Deuterium oxide (D,99.9%) was purchased from Cambridge Isotope Laboratories. Cambridge, MA, USA. Calcium chloride (gold label) was purchased from Aldrich. 4-hydroxy-2,2,6,6- tetramethylpipendine-N-oxyl(TEMPOL) was purchased from Sigma. The E. coli awotrophic strains W2961 (APro), DL41 (AMet), and NK6732 (ALeu) were obtained from the E. coli genetic stock center at Yale University (http://cgsc.biology.yale.edu). The plasmid pCaM was a gift from Dr. T. Grundstrom (University of Umeb, Sweden), and has been described elsewhere (Chapter 2; Waltersson et al., 1993; Zhang and Vogel, 1993a). The plasmid encoding TRlC, the N-terminal half molecule of CaM (Fabian et al., 1996; Yuan et al., 1999a; Yuan et al., 2000), has been derived from pCaM and has been described previously. Isopropylthio-P-D-galactoside (IPTG)was obtained from Gibco. The CaM-kinase I peptide (CaMKI; sequence AKSKWKQAF'NATAWRHMRKLQ) was synthesized by the Peptide Synthesis Facility, Queens University, Kingston, Ont., Canada, Fluorinated ali~haticamino acids in calmodulin 131 and was judged to be >95% pure by mass spectrometry and HPLC. All other reagents were acquired from reputable chemical sources.

Expression and purification of fluorinated CaMs. The plasmid pCaM was used to transform the E. coli strains W2961, DL41, and NK6732 and NK6732 was transformed with the TRlC plasmid according to the methods of Chapter 2. FPro-CaM, TFMet-CaM, TFLeu- CaM, TFLeu-TRlC and TFLeu-CT-CaM were produced according to the methods of Chapter 2, with substitution of the appropriate amino acid with its fluorinated analog. In addition, a sample of TFLeu-CaM with low incorporation of TFLeu was prepared by growing NK6732-pCaM cells in 1 L LB-ampicillin medium as before. The cells were then peletted and transferred to 1 L MOPS-ampicillin medium without leucine, with 60 mg TFLeu, 25 mg L-leucine, and 100 mg IPTG. Cells were grown for 4 h with shaking at 37 "C to induce protein. The fluorinated CaMs, as well as TFLeu-TR1C and TFLeu-CT-CaM, were purified according to the methods of Chapter 2. The extent of incorporation of the fluorinated amino acids into the proteins were checked by amino acid analysis (performed by Dr. Don McKay, Peptide Sequencing Facility) or by ESI-MS (performed by the laboratory of Dr. Gilles Lajoie, Mass Spectrometry Facility, University of Waterloo).

Native gel of FPro-CaMs. To test for degradation of the FPro-CaM samples, a native PAGE gel was m. Samples from NMR tubes, as well as a sample of WT-CaM, were dissolved to 1mgmL in 50 mM WHCO,. The preparations were split in to two equal portions, one which was boiled for 5 min and one which was Fluorinated ali~haticamino acids in calrnodulin 132 not boiled. One vol of 50% glycerol was then added and 20 p.L (10 pg or 0.6 nmol) of each sample was loaded per lane of a 15% native-PAGE gel with 0.375 M Tris-HC1, pH 8.8 as the buffer. The gel was run at a constant voltage of 100 V for 2 h with a running buffer of 25 mM Tris, 0.192 M glycine, md stsined ~.+.rithCcomassie Blue zccording to standard ~rotocols (Sambrook et al., 1989).

NMR spectroscopy. Samples for 19F NMR spectra were prepared by dissolving the lyophilized protein to a concentration of -1.5 mM in D,O, 100 mM KCl, pH 7.4, according to the methods of Chapter 2. A sample of FPro for NMR was prepared by dissolving FPro (-2.5 mg; -40 mM) in 500 yL D20 and adjusting the pH to 7.4. Sample pHs were not corrected for the isotope effect. For calcium titrations, 1 M and 0.25 M stocks of CaCI, (Gold Label) were prepared in D,O, and appropriate amounts were added to the samples. TEMPOL was added from a 0.25 M stock in D,O. Fluorine-19 NMR spectra were acquired at 37 "C either on a Bruker AM400 wide-bore spectrometer with a fluorine-19 probe operating at a frequency "F v0 = 376 MHz, or on a Bruker AMX2300 spectrometer (Department of Chemistry) with a fluorine-19 probe operating at a frequency 19F v0 = 282 MHz. 1D 19F NMR spectra were acquired with proton decoupling. Typically between 1000 and 2000 scans were collected for each spectrum, with a recycle delay of 1 s. 1D lgF NMR spectra were processed with SwaN-MR (Balacco, 1994) or Bruker's XWINNMR processing software with the application of a line-broadening of 5 Hz. 2D 19F,'H HETCOSY spectra (fluorine-detected) were acquired according to the methods of Collier et al. (1996) and consisted of 512 (lgF,F2) X 128 ('H, Fluorinated ali~haticamino acids in calmodulin 133

F1) data points and 300-1200 scans. HETCOSY spectra were processed either with NMRPipe (Delaglio et al., 1995) running on a Silicon Czaphics Indy R5000 workstation and viewed with the accompanying NMRDraw graphics package, or processed with Bruker's XWINNMR software on the Silicon Graphics Tndy2 console. Tn the 'H dimension, spectra were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate(DSS) at 0 ppm; in the 19F dimension, spectra were referenced indirectly to DSS by applying a conversion factor of 0.940867196 (Maurer and Kalbitzer, 1996).

RESULTS.

Expression of proteins. CaM has been substituted with FPro, TFLeu, and TFMet through the use of a bacterial expression system. Normally 2 L of cell culture yields -80 mg of pure WT-CaM; typical preparations of FPro-CaM were -20-25 mg, while -10-15 mg of TFLeu-CaM and TFMet-CaM could be purified from 2 L. The single preparation of TFLeu-CaM at low incorporation yielded 94 mg from 2 L cell culture. FPro-CaM, TFLeu-CaM, and TFMet-CaM were all checked to determine the extent of incorporation of the fluorinated amino acid into the protein. FPro-CsM had an incorporation level of -60%, as determined by mass spectrometry (not shown). The mass-spectrum of TFLeu-CaM in shown in Figure 4.3a, and from this spectrum the extent of incorporation was estimated to be -25%. From the mass spectrum of TFLeu-CaM at low incorporation (not shown) it was estimated that the incorporation of TFLeu was -10%; most of the population had either 0 or 1 Leu substituted with TFLeu. The mass spectrum of TFMet-CaM is shown in Figure 4.3b and from this spectrum Fluorinated aliwhatic amino acids in calmodulin 134

Figure 4.3. a)

2

16000 16500 17000 - mass (Da) Fluorinated ali~haticamino acids in calmodulin 135

Figure 4.3.

FM8t CaM

.-

mass (Da) u ! --., ....,-*..; ,r,..,, ,.,, .;. ,,..h... ,&., . , ;. ; . , . . , .' 14000 16000 18000

Figure 4.3. a) Mass spectrum of TFLeu-CaM. The theoretical mass of CaM is 16 702 Da, and the increase in mass as the result of one TFLeu substitution is 54 Da. The number of TFLeus substituted for each species is indicated. The family of peaks at a lower molecular mass is due to the loss of the three C-terminal amino acids (see Chapter 2); this has been avoided in more recent preparations. b) Mass spectrum of TFMet-CaM. The increase in mass as the result of one TFMet substitution is 54 Da. The number of TFMet residues substituted is indicated. Fluorinated ali~haticamino acids in calmodulin 136 the extent of incorporation of TFMet was estimated to be -20-25%.

FPro-CUM. The 1D 19F NMR spectra of cis-4-fluoroproline, both with and without proton decoupling, are shown in Figure 4.4. There is a single fluorine peak from the one fluorine atom; it is split into 64 components due to coupling with the six other non-exchangeable protons on the ring, as is demonstrated by the spectrum without proton decoupling (Figure 4.4b). The coupling to the protons in the proline ring is also demonstrated by the 2D 19F,'H KETCOSY spectrum (Figure 4.5), where correlations to all the other protons in the ring are seen but the crosspeaks are very complex due to the high degree of splitting involved. The HETCOSY experiment could also be applied to FPro-CaM, as is depicted in Figure 4.6. Here there are two sets of crosspeaks owing to the two proline residues in CaM, and couplings to the protons in the ring can still be detected in the protein. The presence of only two sets of crosspeaks greatly simplifies the spectrum in the proton dimension (see the ID 'H NMR spectrum at left). However, due to the inherent instability of the protein, a more complete characterization of FPro-CaM, by experiments such as a calcium titration, a temperature titration, and peptide-binding experiments, could not be performed. The native PAGE gel of FPro-CaM, both with and without Ca2+,is shown in comparison to WT-CaM in Figure 4.7. It indicates how prolonged periods of storage can lead to breakdown of the protein, a process which can be accelerated by boiling of the samples. In addition, a sample of Pro43Gly-FPro-CaM is also shown in Figure 4.7; due to the instability of Pro43Gly-FPro-CaM no NMR spectra of this protein are shown in this chapter. After a prolonged period of boiling, all Fluorinated ali~haticamino acids in calmodulin 137

Figure 4.4.

Figure 4.4. 1D 19F NMR spectra of cis-4-fluoroproline (FPro) a) with and b) without proton decoupling. The structure of is also shown for illustration; there are six protons to which the fluorine atom can couple. Fluorinated aliphatic amino acids in calmodnlin 138

Figure 4.5.

19F ppm

Figure 4.5. 2D 19F,'H HETCOSY of FPro (I9Fv0 = 282 MHz),. The vertical line through the center of the spectnun is a processing artifact. Fluorinated ali~haticamino acids in calmodulin 139

Figure 4.6.

19F ppm

Figure 4.6. 2D 19F, 'H-HETCOSY (IgF V, = 282 MHz) of Ca2+-saturated FPro-CaM, with the 1D I9F NMR spectrum with proton decoupling above, and the 1D 'H NMR spectrum at left. Fluorinated ali~haticamino acids in calmodulin 140

Figure 4.7.

Figure 4.7. 15% Native-PAGE gel of CaM and FPro-CaMs. Lane designations are as follows: lanes 1 and 2: Ca2+-WT-CaM,3 and 4: CaZ+- FPro-CaM, 5 and 6: Chelex treated (apo-) FPro-CaM, 7 and 8: Ca2+- Pro43Gly-FPro-CaM. For each pair of lanes the first lane is sample loaded without boiling, and the second lane is sample loaded after a 5 minute boiling treatment. Fluorinated ali~haticamino acids in calmodulin 141 the samples showed the same cleavage pattern as P43G-FPro-CaM (not shown).

TFLeu-CUM. The results of TFLeu-CaM this chapter deal with TFLeu-CaMs at two different levels of incorporation. Figure 4.8 shows the 1D 19F NMR spectra of apo-TFLeu CaM at the two levels of incorporation of TFLeu,which are approximately 10% and 30%. The peaks in the spectrum of TFLeu-CaM at 10% incorporation are more distinct than that of TFLeu- CaM at 30% incorporation; presumably this is because the sample at lower incorporation is less heterogeneous and the chance of a TFLeu residue being in the vicinity of another TFLeu residue is much lower. There also seems to be more than nine peaks in each spectrum (there are nine Leu residues in CaM). This could be due to heterogeneity in the proteins, but this is not likely at the lower level of incorporation. Also, the TFLeu used in these experiments was purchased as a mixture of stereoisomers, and it could be that both (4R)-L-TFleu and (4s)-L-TFLeu were able to be incorporated into proteins. A calcium titration of TFLeu-CaM (10% incorporation) is shown in Figure 4.9. As more Ca" is added, peaks collapse and become less distinct. This is likely because the environments of the TFLeu residues change as a result of the Ca2'-dependent conformational change. This is expected especially for TFLeus 32, 48, 105, and 116, which become solvent-exposed in the hydrophobic patches in the Ca2+-saturatedstate of the protein (David, 1997).

The 19F spectra of TFLeu-CaM can also be spread into two dimensions by heteronuclear COSY experiments. Figure 4.10a is the HETCOSY of apo-TFLeu-CaM (30% incorporation). The crosspeaks are Fluorinated ali~haticamino acids in calmodulin 142

Figure 4.8.

Figure 4.8. 1D 19F NMR (v, = 376 MHz) of apo-TFLeu-CaM at a) -10% incorporation of TFLeu and b) -25% incorporation of TFLeu. Fluorinated aliohatic amino acids in calmodulin 143

Figure 4.9.

Eq. ca2+

Figure 4.9. 1D lgF NMR (vo = 282 MHz) Cab-titration of TFLeu-CaM (-10% incorporation). The number of equivalents of Ca2+ (ratio of Ca2':CaM) is indicated over each spectrum. 1400 scans were acquired per spectrum, with proton decoupling. The spectrum of TFLeu-CaM with 5 equivalents Ca2+(not shown) is not significantly different than that with 4 equivalents Ca2+. Fluorinated ali~haticamino acids in calrnodulin 144

Figure 4.10. a)

6 5 4 19~ppm Fluorinated aliphatic amino acids in calmodulin 145

Figure 4.10.

llllll,llll~.~ll)llll~l...1..1 6 5 4 19~ppm

Figure 4.10. 2D "F, 'H IIETCOSY spectra ("I? v,= 282 MHz) of a) apo- TFLeu-CaM (300 scans) and b) apo-TFLeu-CaM with 4 equivalents TEMPOL (1200 scans). The peaks in a) are assigned with the numbers 1- 9. Fluorinated ali~haticamino acids in calrnodulin 146 between the three equivalent fluorines of the trifluoromethyl group and the proton attached to carbon 4 of TFLeu (see Figure 4.1). Again, es with the 1D spectrum, it shows more than nine sets of crosspeaks, although there are nine major sets of crosspeaks. They are designated with the numbers 1-9. Figure 4.10b shows the HETCOSY of the same apo-TFLeo- CaM sample with 4 equivalents of TEMPOL added. TEMPOL is a soluble nitroxide spin label which has an unpaired electron. It can effectively broaden peaks from solvent-exposed nuclei in NMR spectra (Yuan et al., 1999a). By comparison of Figure 4.10a and Figure 4.10b it is apparent that the addition of TEMPOL can selectively broaden some of the TFLeu peaks, while others remain relatively sharp and intense. The TFLeu residues that are not broadened are likely to be buried in the hydrophobic core of one of the lobes of the protein. In the Ca2+-saturatedform of TFLeu- CaM, it is difficult to obtain good HETCOSY spectra, even without TEMPOL (data not shown). This could be because of spectral overlap, or conformational heterogeneity in the solvent-exposed TFLeu side chains. Protection of the TFLeu residues from exposure to solvent is also seen after the addition of a CaM-binding peptide to Ca2'-saturated TFLeu-CaM. Figure 4.11 is the 1D 19F spectrum of Ca2+-TFLeu-CaMin the presence of 1 equivalent of the CaM-binding peptide from CaMKI, both in the absence and in the presence of 4 equivalents of TEMPOL. There is no significant broadening of the 19F signals upon the addition of TEMPOL, indicating that the TFLeu residues are buried in the TFLeu-CaM:CaMKI complex, presumably because of contacts that are made with the peptide. A way to make 19F NMR spectra of TFLeu-CaM easier to interpret is to use fragments of TFLeu-CaM. Figure 4.12 is the HETCOSY of recombinantly expressed apo-TFLeu-TRlC, fragment 1-75 of CaM, which has six leucines. Fluorinated ali~haticamino acids in calmodulin 147

Figure 4.11.

f ! ; 1 j! I I ,I I I r! ; I1 ,I,+i \, A ;I' 4 i *rr*--*%;--J I 1, :/I : I I I , ij , : I I i : ' '141 ': , .' :;; .: I _ 3 B /+ '; / i2-h !\; * 'cs +.& -.-,4 ----.-d--A-*a---,---- -. -r--+ ',4LA-,,.&*4p*-hr4r r~~.."'1'"""1 ""'"1'"'~"1'-'."'l'~r-7~q1 19~ppm 8 6 4 2 0

Figure 4.11. ID 19F NMR spectra (v, = 282 MHz), with proton decoupling, of a) Ca"-TFLeu-CaM with 1 equivalent of the CaMKI peptide (525 scans), and b) Ca2+-TFLeu-CaMwith 1 equivalent of the CaMKI peptide and 4 equivalents of TEMPOL (1000 scans). The peak marked with an X' at 2.2 ppm is from trifluoroacetic acid, which was present in the CaMKI preparation. Fluorinated ali~haticamino acids in calmodulin 148

Figure 4.12.

19~ppm 6 5 4 Fluorinated ali~haticamino acids in calrnodulin 149

Figure 4.12. (previous page) 2D "F, 'H HETCOSY spectrum ("F v, = 282 MHz) of apo-TFLeu-TRlC (600 scans) with the ID 19FNMR spectrum (1170 scans) above. They are numbered using the same numbering system as for intact TFLeu-CaM (Figure 4.10a).

TFMet-CaM. Figure 4.13 shows a ID 19F NMR Ca2+-titrationof TFMet-CaM, with a TFMet incorporation level of -20%. There are nine Met residues in CaM, and there are nine major peaks in the spectrum of apo-TFMet-CaM (0 eq. Ca2+). There are also some minor peaks, which may be because of heterogeneity of the sample due to the intermediate level of incorporation. After successive additions of Ca" to the sample, the 19F peaks of TFMet CaM collapse into one peak. This reflects the solvent exposure of these Met side chains in the hydrophobic patches of Ca2+-CaM.As with the Leu residues, the TFMet residues in Ca2+-CaMare likely conformationally flexible, and in similar chemical environments. The effect is greater than with TFLeu-CaM because eight of the nine Met residues in CaM are involved in the hydrophobic patches (the exception is Met76). The 1D I9F NMR spectrum of Caz+-saturatedTFMet-CaM with 1 equivalent of the CaMKI peptide is shown in Figure 4.14. There is a better spread of signals than in the spectrum of Ca2+-saturatedTFMet-CaM without any peptide, as the trifluoromethyl groups are shifted as they make contacts with the peptide CaMKI, which is a possible explanation for there being more than 9 peaks in this spectrum. It is not possible to acquire HETCOSY spectra of TFMet-CaM because the fluorine atoms are at least 4 bonds away from any other protons, and are separated from the rest of the side chain from Fluorinated aliphatic amino acids in calmodulin 150

Figure 4.13.

Eq. ca2+ - 0 -

Figure 4.13. 1D 19F NMR (v, = 376 MHz) Ca"-titration of TFMet-CaM with the number of equivalents of Ca" indicated above each spectrum. The number of scans per spectrum is also indicated. The spectrum of TFMet-CaM with 5 equivalents Ca2+ (not shown) is not significantly different than that with 4 equivalents Ca2+. Fluorinated ali~haticamino acids in calrnodulin 151

Figure 4.14.

Figure 4.14. 1D ''F NMR spectrum (v. = 376 MHz) of Ca2+-TFMet-CaM with 1 equivalent of the CaMKI peptide added (20 000 scans). Fluorinated ali~haticamino acids in calnodulin 152

the sulfur atom. Therefore, coupling of the trifluoromethyl group to protons in the side chain does not occur.

DISCUSSION.

In an assessment of the usefulness of the biosynthetic incorporation

of fluorinated aliphatic amino acids into proteins for 19F NMR spectroscopy, the fluorinated aliphatic amino acids FPro, TFLeu, and TFMet have been biosynthetically incorporated into CaM through the use of a bacterial expression system, and the properties of these substituted proteins were examined by NMR. One-dimensional 'H NMR spectra (not shown) demonstrate that these proteins are similar to WT-CaM in their overall three-dimensional structure. This should not be too surprising since, with the exception of FPro, the level of incorporation of these amino acids is fairly low (-20%; Figure 4.3). This low level of incorporation is also the reason why the ability of TFMet-CaM and TFLeu-CaM to activate CaM targets, such as calcineurin (Chapter 2) were not determined. Incorporation of fluorinated amino acids at such low levels would be expected to have a minimal functional impact. In order for one to have confidence in interpreting functional assays, the incorporation level should be about 50% at least. The incorporation of FPro into CaM results in the introduction of only two fluorine atoms to the protein, so again it would not be expected to have a great functional effect. Indeed, FPro-CaM, with a 60% level of incorporation, was not significantly different than WT- CaM in its ability to activate calcineurin (data not shown). The incorporation level of 60% for FRO,which has only one fluorine atom, is similar to the levels seen for Azc and Dhp in Chapter 2. The -20% Fluorinated ali~haticamino acids in calnodulin 153 incorporation rates for TFLeu and TFMet are lower than for any other amino acid analogs which have been incorporated thus far. Fluorine atoms are highly electronegative and have large electron-withdrawing effects; the result is a markedly larger dipole moment for TFLeu than Leu (David, 1997): and similar effects would also be expected to occur for TFMet. The low incorporation of TFLeu and TFMet is probably due to either poor transport of these amino acids into the E. coli cells and/or poor recognition of the compounds as substrates for their respective aminoacyl- tRNA synthetases. Recognition of aliphatic amino acids like Leu or Met by either their transporter or their aminoacyl-tRNA synthetases is likely to be hydrophobic, so drastic changes in the polarity of these amino acids as a result of fluorine substitution would be expected to negatively affect their recognition. Proline is likely to be recognized by its unique ring structure, and the aromatic amino acids by their aromatic character. These properties are not changed as a result of fluorine substitution, so these fluorinated amino acids would be expected to be recognized more readily.

In a report where TFMet was substituted into bacteriophage )c lysozyme (Duewel et al., 19971, an 18 kDa protein with three methionine residues, it was possible to achieve 70% substitution of Met by TFMet. However, this protein was expressed with 1 mM (185 mg/L) TFMet in the induction medium, and protein expression was induced for 9.5 h. So, it may be possible to increase the level of incorporation of fluorinated aliphatic amino acids such as TFMet, but it seems to require the use of rather unconventional methods. The researchers also noted the cytotodc effects of TFMet. Another possibility in the case of expression of CaM with of TFLeu and TFMet is that the fluorinated amino acids are incorporated Fluorinated ali~haticamino acids in calmodulin 154 into the protein at high levels, but the properties of these substituted proteins are different from CaM in that they cannot be purified by the traditional phenyl-Sepharose chromatography. The hydrophobic properties of Leu and, especially, Met are very important for the function of the hydrophobic properties of CaM, so it wouldn't be unexpected that the highly substituted proteins could not be purified. Assuming this, then it would be elrpected that some of the positions (e.g. Leu4 or Met76) which are not involved in the hydrophobic patches or the hydrophobic cores of the protein, would be more highly substituted in the purified fraction than other positions. This could explain why some 19F peaks are more intense than others, but it is difficult to tell for sure. Apart from sequencing of the substituted proteins, there is no way to determine if this is the case. Another problem encountered with these CaMs with fluorinated aliphatic amino acids is their stability. FPro-CaM is less stable than WT- CaM. The breakdown process is accelerated by exposure to high temperatures (Figure 4.71, which means that the cleavage is most likely to be spontaneous. It could be due to contamination with a thermally stable protease, but this is unlikely because the instability of FPro-CaM was seen in several preparations but not seen in any other CaM preparations. It is possible that the location of cleavage of the polypeptide backbone is at the FPro residues themselves, but the stability of the FPro-CaMs are Ca2+- dependent, and the cleavage pattern of Pro43Gly-Fpro-CaM is the same as FPro-CaM, so the cleavage location is most likely not at the FPro residues. Moreover, wild-type CaM also eventually breaks down with the same cleavage pattern as the FPro-CaMs, albeit at a much slower rate (not shown). More likely, the FPro-substituted proteins are globally less stable. Due to the high receptivity of the fluorine-19 nucleus, however, it Fluorinated aliphatic amino acids in calmodulin 155 was not difficult to obtain good 19F NMR spectra of the substituted proteins, even if the level of incorporation was low or the stability of the proteins were limited. As expected, however, the dispersion of the peaks in the spectra of these proteins is not as good as the spectra of proteins with fluorinated aromatic amino acids, such as CaMs with fluoro-phenylminns and fluoro-tyrosines (David, 19971, or GST with 2-fluorotyrosine (Figure 4.1). The peaks in the spectrum of TFLeu-CaM (Figure 4.9) are dispersed over -2.5 ppm and the peaks for the TFMet-CaM spectrum (Figure 4.13) are spread over -3 ppm, while the spectrum of 2-Ftyr-GST (Figure 4.1) is spread over -10 ppm. Figure 4.6 shows the 1D 'H and lgF NMR spectra and the 'T, 'H HETCOSY of Ca2+-FPro-CaM. There are two distinct lgF peaks from the two FPro residues in the protein. For simplification, the 19F spectrum was acquired with proton decoupling because of the great deal of splitting of the 'OF signal by the protons in the FPro ring (Figure 4.4). The coupling is evident in the HETCOSY of FPro-CaM (Figure 4.6) as it is in the spectrum of FPro (Figure 4.5). The protons with the most intense crosspeaks in the HETCOSY spectra (5.28 ppm in Figure 4.5 and 5.28 and 5.58 ppm in Figure 4.6) are the geminal H4 protons. The other protons were not definitively assigned but are likely to be one P (H3)and one 6 (H4) proton. Neither the and P' nor the 6 and 8' protons are equivalent in the lH spectrum of FPro (not shown). The other protons would not be expected to couple as well to the 19F nucleus due to their geometry. The 1D 19F NMR spectra of apo-TFLeu-CaM at two levels of incorporation (10% and 20%) are shown in Figure 4.8. Like the spectrum of FPro-CaM, these spectra were acquired with proton decoupling. Proton decoupling does not have as large an effect with these spectra, however, because the degree of splitting is not as large; the only significant coupling Fluorinated ali~haticamino acids in calrnodulin 156 is to the H4 proton, as is seen in the HETCOSY spectra of TFLeu-CaMs (Figures 4.10., 4.12,4.13),or the HETCOSY of TFLeu (David, 1997). What is more significant is that the level of incorporation has an effect on the 19F spectra of TFLeu-CaMs. This is because intermediate levels of incorporation cause heterogeneity in the samp!e. For example, 20% incorporation of TFLeu into a protein such as CaM that has nine leucines results in a population of proteins in which a large proportion have two TFLeus substituted (see Figure 4.3a). If two or more Leus are close together in the three-dimensional structure of the protein (they need not be close together in the primary sequence) then, in the substituted protein, some TFLeus will be neighbored by other TFLeus whereas other TFLeus will only be neighbored by Leu residues. Since the chemical shift of the fluorine atom is highly influenced by its environment, there could potentially be several peaks for one TFLeu depending on the number of other TFLeus in its environment. When the level of incorporation is lower (e.g. lo%), then the chance that a TFLeu is neighbored by another TFLeu is much lower, resulting in less heterogeneous spectra. The result is less spectral overlap with TFLeu incorporation at 10% (Figure 4.8). Even though there is less spectral overlap, there are still more than nine peaks in the spectrum of Figure 4.8a. This could be because the TFLeu used in this chapter is a mixture of stereoisomers. With the stereospecificity of the E. coli translational machinery, both (25, 4R)- TFLeu and (2S,4s)-TFLeu could be expected to be incorporated. These isomers are chemically different and have different lgF-chemical shifts (David, 1997). Another possible reason for the complexity of the spectra is conformational flexibility of the side chains. The side chain of a TFLeu residue is able to take different conformations in which the positions of the Fluorinated aliphatic amino acids in calmodulin 157 methyl group and the trifuoromethyl group swap positions. This results in different possible environments, and different chemical shifts, for one TFLeu. This could result in line broadening, or, if exchange is sufficiently slow, the presence of more than on peak for one TFLeu residue. Addition of the soluble spin labe! TEMPOL to a sample is s gseful way to estimate the extent of solvent exposure of NMR-active nuclei. It was found that the TFLeu residues of TFLeu-CaM are protected from the solvent most in the apo-state (Figure 4.10) and in the peptide-bound state (Figure 4.11) of the protein. The TFLeu residues are more solvent exposed in Ca2+-saturatedCaM, making it difficult to obtain 19FNMR spectra in the presence of TEMPOL (not shown). The 19F peaks also become more degenerate and less distinct as Ca" is titrated into apo-TFLeu-CaM (Figure 4.9) as the TFLeu residues lose specific contacts with other residues and become solvent-exposed as part of the hydrophobic patches in the Ca2+-saturatedprotein. The change in spectroscopic properties as Ca" is titrated into a sample is even more evident in spectra of TFMet-CaM (Figure 4.13). This is because the Met residues in play a much more important role in the hydrophobic patches of CaM (Vogel and Zhang, 1995; Yuan et al., 1999a). Eight of the nine Met residues are in the hydrophobic patches, and again, as with TFLeu-CaM, the 19F peaks of TFMet-CaM become less distinct as Ca2+is added. The same can be seen in 'H, 13C HMQC spectra of 13C-methyl- Met labeled CaM (Chapter 4; Yuan et al., 1998a), where the spectrum becomes more crowded as Ca" is added. Moreover, because of the intermediate level of incorporation of TFMet (Figure 4.3b) there is sample heterogeneity as there was with TFLeu-CaM. In the spectrum of apo- TF1Met-CaM (Figure 4.131, the peaks at 38.5 pprn and 39 ppm both have Fluorinated ali~haticamino acids in calrnodulin 158 smaller neighboring peaks slightly upfield. Because there are two examples of Met residues being adjacent in the primary sequence (Met71, 72 and Met144, 145) the effects of intermediate incorporation are very significant. The importance of the Met residues in the hydrophobic patches of CaM is also demonstrated in the 1D 19F NMR spectrum of Ca2+-TFMet-CaM with 1 equivalent of the CaMKI peptide (Figure 4-14), which looks very different from the spectrum of uncomplexed Ca2+-TFMet-CaM(Figure 4.13). This shows the interactions that take place between the TFMet side chains and the hydrophobic face of the CaMKI peptide (see also Yuan et al., 1999a). The CaMKI peptide has a Trp residue which inserts into the C- terminal lobe of Ca2+-CaM,as well as a Phe residue on its hydrophobic face. Both of these aromatic side chains, through ring-current shifts, significantly affect the chemical shifts of the TFMet residues. In conclusion, the fluorinated aliphatic amino acids cis-4- fluoroproline (FPro), 5,5,5-trifluoroleucine (TFLeu), and S-trifluoromethyl- homocysteine (trifluoromethionine; TFMet) have all been demonstrated as potentially useful probes in examining the properties of proteins. However, TFLeu and TFMet, because of the drastic changes in their polarity resulting From the fluorine substitution, were incorporated into CaM at low levels. Moreover, due to the mobility of the side chains of these amino acids, the 19F NMR spectra of the substituted proteins were not as disperse as spectra of proteins with fluorinated aromatic amino acids. Thus, it is not likely that these amino acids will enjoy as wide spread popularity as the fluorinated aromatic amino acids. The resolution of the spectra of FPro-CaM and TFLeu-CaM was improved, however, by the application of the I9F, 'H heteronuclear COSY (HETCOSY). Also, the Fluorinated aliphatic amino acids in calrnodulin 159 results with the substituted calmodulins have shown the importance of the Leu and Met residues in the hydrophobic patches in CaM, and in the interaction of the hydrophobic patches with CaM-binding peptides. Thrombic -fragmentsof calmodulin 160

CEZAPTER FIVE: Peptide and metal ion dependent association of thrombic fragments of calmodulin

ABSTRACT.

The ubiquitous eukaryotic calcium binding protein calmodulin (CaM)has been cleaved in vitro by the protease thrombin to create two fragments, each of which contain an isolated "EF-hand" helix-loop-helix Ca2+-bindingdomain. Fragment TM1 (1-106)contains the intact N- terminal lobe of CaM as well as the major portion of the third helix-loop- helix Ca2+-bindingdomain; and fragment TM2 (107-148)contains the last part of helix 6, from the third Ca2+-bindingdomain, as well as the entire fourth Ca2+-bindingdomain of CaM. TM1 and TM2 were purified to homogeneity by gel filtration methods. Detailed structural and biochemical studies of TM1 and TM2, and their potential to associate in a complimentary fashion to create a CaM-like complex, were undertaken by a variety of methods. Circular dichroism and 'H, I3C, and lt3CdNMR data all showed that TM1 and TM2 can associate in a metal-ion dependent fashion to form a species with secondary structure and metal ion binding properties similar to CaM. However, this interaction was weak, illustrating the importance of the location of a pair of helix-loop-helix domains on a single polypeptide chain. This was shown by the broad peaks in 'H,13C HMQC spectra of I3C-methyl-Met labeled TM1 and TM2 and the inability of TM1 and TM2 to form a complex in native-gel bandshift assays. Thrombic fragments of calmodulin 161

Additionally, native-gel bandshifi assays and with 13C and '13Cd NMR data indicate that TM1 and TM2, with metal ions, will bind to CaM-binding target molecules, such as CaM-target peptides and the CaM-binding drug trifluoperazine. The interaction with a target peptide further strengthens the association between TMI and TM2 such that the Ca2+- TM1:TMa:peptide ternary complex is able to be visualized on a native- PAGE gel. Although TM1 and TM2 were able to form a complex with CaM- binding peptides, they did not exhibit any synergistic complementarity in activating the CaM dependent enzymes calcineurin, smooth muscle myosin light-chain kinase, or 3':5'-cyclic nucleotide phosphodiesterase. This may either be due to an interaction between TM1 and TM2 and the target enzymes that is too weak, or perhaps that the proper complex was formed but disruption of the C-terminal lobe by the thrombic cleavage was sufficient to abolish any activation. The work presented in this chapter extends on previous studies of fragment complementation in other helix- loop-helix Ca2+-bindingproteins with the important distinction that thrombic cleavage of CaM is disproportionate, with the cleavage site being in the middle of a helix flanking a Ca2+-bindingloop rather than in the linker region between two helix-loop-helix Ca2+-bindingsites. This leads to the intriguing possibility that fragment complementation could possibly occur irrespective of the location of the break between the fragments.

As explained in Chapter 1 of this thesis, calmodulin (CaM)belongs to the "EF-hand" superfamily of calcium binding proteins (Strynadka and James, 1989; Marsden et al., 1990). Other important members of this Thrombic framents of calmoddin 162 family include the troponins C, which are the CaM homologs in muscle tissues, the SlOO proteins, which are involved in a variety of Ca"- dependent signaling processes, and the calbindins, which sequester Ca" ions from the intestinal lumen. A brief survey of these proteins reveals that the "EF-hand" helix-loop-helix Ca2+-bindingdomains predominantly exist in pairs. This is important because the pair of domains can bind Ca2+ ions in a co-operative manner, resulting in tighter binding of metal ions than would be possible for one site alone. Co-operative Ca2+binding occurs because metal ion binding by one site causes a conformational change in that site from a metal ion free "closed" form to a metal ion bound "open" form. A similar change then takes place in the other helix-loop-helix motif in the pair such that the second site binds metal ions much more readily. This co-operativity of Ca" ion binding occurs all across the subclasses of EF-hand proteins, including the calbindins (Akke et al., 1991, 19951, which are Ca2+-bindingbuffer proteins that do not show a very large overall conformational change upon Ca2+binding, and calrnodulin (Linse et al., 1991), which, being a Ca2+-ionbinding regulatory protein, does display a large conformational change upon Ca" binding. Key structural features of the EF-hand pair include a small two-stranded P-sheet between the two opposing loops, and contacts between conserved hydrophobic residues on opposing a-helices (Strynadka and James, 1989). Overall, these pairs of helix-loop-helix motifs associate to form a globular domain with two-fold rotational symmetry between the two units. The necessity of a pair of helix-loop-helix domains for proper CaZ+ binding was first discovered when researchers examined the metal ion binding properties of synthetic peptides comprising the sequence of isolated Ca2+binding loops and helix-loop-helix domains. In a family of Thrombic -f?amentsof calmodulin 163 peptides derived &om the third Ca2+-bindingsite of rabbit skeletal troponin C (Reid et al., 1981; Gari6py et al., 19821, researchers noted the importance of the helices flanking the Ca" binding loops, especially the N-terminal helix, in increasing the Ca2+affinity of the peptides. Importantly, all of these peptides, including the ones which had flanking helices. bound Ca2+ much more poorly than intact domains containing two sites. Later, it was found that a 34-residue peptide comprising the entire site I11 helix-loop- helix domain of chicken skeletal troponin C (TnC) associated, in the presence Ca2+,to form a head-to-tail dimer very similar in structure to the intact C-terminal domain of TnC (Shaw et al., 1990). A proteolytic fragment from site IV of TnC was also found to dimerize in the same manner (Kay et al., 1991). Importantly, it was found that this dimer structure could be induced in the %-residue peptide after binding only one Ca2+ion per two peptide molecules (Shaw et al., 1991), and that after the first ion was bound the affinity of the second site for Ca2+ increased substantially (dissociation constants were determined to be >1 mM for the first Ca2+ion and 3 pM for the second Ca" ion). This lent very significant evidence to the theory of a co-operative transition in Ca2' ion binding. More recently (Shaw and Sykes, 1996), it was shown that synthetic peptides from domain 111 and domain IV of TnC preferentially associated to form a heterodimer with a structure similar to the intact C-terminal domain of TnC. Apart from the Ca"-binding regulatory protein troponin C, helix- loop-helix domain association has also been demonstrated in the calbindins, which are involved in buffering intracellular Ca2+levels rather than in Ca2+-dependentcelldar signaling. In an elegant experiment (Finn et al., 19921, fi-agments of the two-site Ca2+binding protein calbindin D,, Thrombic fragments of calmodulin 164 were created by introduction of a unique methionine residue followed by cyanogen bromide cleavage. In contrast to the TnC studies, the structures of the cleaved and uncleaved proteins were very similar, and the two fragments associated to form the heterodimer even in the absence of Ca2+. This probably reflects calbindin's more static role as a Ca2+buffer rather than as a Ca" signaling protein. The heterodimer was stabilized by the addition of Ca" ions (Finn et a1 ., 19921, probably not only through inducing the dimer conformation but also, importantly, through neutralization of negative charges in the Ca2+-bindingloops that repelled dimer formation. The stability of EF-hand dimers of calbindin D,, were increased even further by the introduction of a disulfide bond (Linse et al., 19931, such that the disulfide-bonded heterodimer was of equal stability to intact calbindin D,,. In contrast to CaM and TnC, in which the two two-site globular lobes are largely autonomous, studies of synthetic helix-loop-helix fragments of calbindin D,,, a six-site Ca2+-bindingprotein, showed that the six fragments packed into one globular structure (Linse et al., 1997). Similarly, tryptic half-molecules of sarcoplasmic calcium-binding protein, each of which contain two helix-loop-helix domains, dimerize in a CaZ+- dependent manner to form a globular structure with four sites (Durussel et al., 19931, similar to the native structure (Vijay-Kumar and Cook, 1992). Again, this is probably a reflection of the function of these proteins as intracellular metal ion buffers, rather than in Ca2+-signaling. Studies of the association of proteolytic fragments have been undertaken for all sorts of proteins. A classic example is the ribonuclease- S system, which has been examined for quite some time by a variety of methods (Graziano et al., 1996, and references within). Proteolytic fragment reconstitution experiments that successfully created complexes Thrombic fiaments of caLmoduZin 166 similar to their native proteins include studies of barnase (Kippen et al., 19941, chymotrypsin inhibitor-2 (de Prat Gay and Ferscht, 19941, and thioredoxin (Tasayco and Chao, 1995). The work stems from limited proteolysis experiments, and their ability to identify domain boundaries (Fontana et al., 1986, Hubbard, 1998). The underlying premise is that the linker regions between protein domains are quite flexible. So, if a protein is subjected to cleavage by a digestive protease, such as trypsin, under conditions in which the native structure of the protein is maintained, proteolysis will be limited to accessible areas such as the interdomain regions with the domains themselves left relatively intact. This is how most of these fragments are generated, with the exception of creation of fragments through chemical cleavage or peptide synthesis. Figure 5.1 is a cartoon diagram of calmodulin showing the cleavage sites by two proteases, trypsin and thrombin. The apo-state of CaM is quite flexible, and tryptic digestion occurs quite rapidly at a variety of sites. By contrast, the Ca2+-formof calmodulin is cleaved by trypsin exclusively in the central linker region of the protein (Walsh et al., 1977; Andersson et al., 1983a; Thulin et al., 19841, a region which is now proven to be quite flexible (Heidorn and Trewhella, 1988; Barbato et al., 1992). The tryptic half-molecules of CaM generated by digestion in the presence of Ca2+are able to activate several CaM-dependent target enzymes to varying degrees (Newton et al., 1984; Persechini et al., 1994). From this work it seems that the target enzyme binds first to the C-terminal lobe of CaM, and then to the N-terminal lobe. The fbnction of the central helix in the activation of many target molecules is simply to increase the local concentration of the N-terminal lobe. In contrast to trypsin, thrombin is a selective protease which Thrombic fragments of calmodulin 166 Figure 5.1.

Figure 5.1. Schematic drawing of the CaM protein, with the Ca2+-binding loops shown as thin-line loops, numbered with Roman numerals, and a- helices shown as bars, numbered with Arabic numerals. Trypsin cleaves Ca2+-CaMat three sites (Arg74, Lys75, Lys771, but Lys77 is by far the major site. Thrombin cleaves apo-CaM at ArglO6 (major site) and Arg37 (minor site; in brackets). Adapted from Andersson et al. (1983).

cleaves only aRer certain arginine residues. Thrombin only cleaves apo- CaM, and not Ca2+-CaM,at a single major site, the Arg106-His107 peptide bond (Wall et al., 1981, Andersson et al., 1983a; see Figure 1.2 for the primary sequence of CaM). This generates two fragments: TM1 (1-1061, which contains the first two helix-loop-helix domains of CaM as well as the major portion of the third domain, and TM2 (107-1481, which has part of helix 6, the C-terminal helix of domain 111, as well as the entire domain IV. There is also a minor site of cleavage at the Arg37-Ser38 peptide bond (Shea et al., 1996) but with proper manipulation of the conditions cleavage at this site can largely be avoided. The fragment TM1 can activate rabbit skeletal MLCK, although only when present at high concentrations, whereas TM2 cannot (Wall et al., 1981). TMl, like intact CaM, can also bind to the hydrophobic matrix phenyl sepharose (Vogel et al., 1983), Thrombic fragments of calmodulin 167 probably through its N-terminal lobe which remains unaffected by the thrombic cleavage. Also, as one would expect, the isolated Ca2+-binding sites (site 111 of TM1 and site TV of TM2) have poor metal-ion binding properties, as can be seen by "Td NMR (Andersson et al., 1983a). Cadmium-113 is extremely useful because it is a spin-1/2 calcium substitute. Although it is a "softern metal ion than Ca2+,Cd2+ is suitable because it has a very similar ionic radius (0.92 A for Cd2+vs 0.94 A for Ca2+; Williams and Frausto da Silva, 1996). Cadmium-113 NMR has been proven useful for study of many proteins (for reviews see Summers, 1988; Coleman, 1993; Clarke and Vogel, 20001, including Ca2+-bindingproteins such as insulin (Sudmeier et al., 19811, troponin-C (Forsen et al., 1979), calmodulin (Andersson et al., 1982), calbindin D,, (Kordel et al., 1992), and a-lactalburnin and lysozyme (Aramini et al., 1995). Cadmium is also a useful substitute for zinc ions, such as in a study of histidinol dehydrogenase (Kanaori et al., 1996). One important problem to avoid with 'I3Cd NMR is the fact that Cd" has a very high affinity for halides such as chloride ions, such that l13Cd peaks are broadened beyond recognition if there is any chloride in the sample. Consequently, other counter ions such as perchlorate or sulfate should be used when preparing solutions (Summers, 1988). '13Cd NMR reports things like metal ion affinity and influences of sample conditions on metal ion binding site properties. For example, it is commonly known that CaM has two high- afEnity Ca"-binding sites. This is shown in the l13Cd NMR spectra of "3Cd2+-saturatedCaM (Andersson et al., 1982, 1983a) which only have two peaks; the peaks for '13CcP+ bound at the low aflinity sites exchanged too rapidly to be seen at room temperature. Through the use of proteolytic fragments of CaM, including the C-terminal tryptic half-molecule as well Thrombic fraPments qf calmodulin 168 as TM1 and TM2, Andersson et al. (1983a) were able to assign the '13Cd signals for the two high-affinity sites to sites I11 and IV of CaM. Moreover, addition of CaM-binding drugs (Andersson et al., 1983b, Thulin et a1 ., 1984) or peptides (Zhang et al., 1995a) increased the affinity of CaM for metal ions, as was shown by the appearance of two additional peaks in lL3Cd NMR spectra which arise from 113Cd2+bound by sites I and II (Thulin et al., 1984). Although Andersson et al. (1983a) successfully identified the "'Cd signals for the two high-affinity metal ion binding sites in CaM by using thrombic fragments, they did not examine the effects of recombining TM1 and TM2 on the metal ion affinity. Given the significance of the work on fragment complementation in EF-helix-loop-helix Ca2+-bindingproteins, it would be worthwhile to study these effects. Not only would studies of TM1:TMS association expand the work of fragment recombination to the very important Ca2+-bindingprotein calmodulin, but also TM1 and TM2 have properties differing from the fragments studied thus far. Generally, studies of synthetic peptides or proteolytic fragments of Ca2+-binding proteins have used entire helix-loop-helix domains. By virtue of its cleavage location, which is in the middle of helix 6 of CaM, thrombin creates fragments which have a disproportionate distribution of helix- loop-helix Ca2'-binding domains. TM1 contains the major portion, but not quite all, of domain I11 of CaM. TM2 actually comprises the latter part of helix 6, the second helix from domain 111, in addition to containing the entire domain IV. The studies of reconstitution and complementation of TM1 and TM2 presented in this chapter thus extend on previous work on fragment complementation in EF-hand Ca2+-bindingproteins. Moreover, no studies to date have determined the idluence of target peptides on the Thrornbic -fragments of calmodulin 169 association of EF-hand fragments. This chapter demonstrates that CaM- target peptides can promote the association of TM1 and TM2 in forming TM1:TMZ:peptide ternary complexes. The work in this chapter also examines proteolytic fragment complementation by several other methods, including gel bandshifi assays and multinuclear NMR spectrsocopy.

MATERfALS AND METHODS.

Materials. Bovine thrombin, essentially free of other clotting factors and plasminogen and plasmin, and trifluoperazine were obtained from Sigma. The CaM-binding peptides from skeletal muscle myosin light-chain kinase (skMLCK; sequence KRRWKKNFIAVSAANRFKKISS; Ikura et al., 1992), and CaM-kinase I (CaMKI; sequence AKSKWKQAFNATAVVRHMRU; Gornes et al., 20001, and the peptide corresponding to the first characterized CaM-binding domain of phosphodiesterase (PDEa;sequence Acetyl-QTEKMWQRLKGILRCLVKQL-NH?;Sonnenburg et al., 1995; Yuan et al., 1999b) were synthesized by the Peptide Synthesis Facility, Queens University, Kingston, Ont., Canada, and were judged to be >95% pure by mass spectrometry and HPLC. Escherichia coli strains MM294 and DL41 (AMet) were obtained from the E. coli genetic stock center, Yale University. Deuterium oxide (D, 99.9%) was obtained from CDIN Isotopes (Point Claire, Que., Canada). Cadmium-113 oxide (l13Cd, 95%) and carbon-

13-methyl methionine (13C, 99%) were obtained from Cambridge Isotope Laboratories (Cambridge, Mass.). Chelex-100 resin was obtained from Bio- Rad. Sephadex G-75 size exclusion chromatography matrix was obtained from Pharmacia. All other reagents were obtained Bom reputable sources. Thrombic -fragments of calmodulin 170

Preparation of thrombic fragments of calmodulin. Wild-type CaM was expressed in MM294 cells with pCaM according to the methods of Chapter 2. I3C-methyl-Met CaM was expressed in DL41 (Met)cells according to the methods of Chapter 2 with the exception that, for induction, cells were transferred from the rich medium to the MOPS- based defined medium containing all the natural amino acids except Met, which was replaced by L-13C-methyl-Metat 25 mgL. Wild-type CaM and 13C-methyl-MetCaM were purified according to the methods of Chapter 2. Typical yields from 2 L of cell culture were -80 mg for WT-CaM or -60 mg for 13C-methyl-MetCaM. The level of incorporation of 13C into 13C-methyl- Met CaM was not determined, but from the quality of NMR spectra obtained from the samples (see below) the level was quite adequate. The cleavage of CaM with thrombin and the purification of the proteolytic fragments were performed according to the methods of Wall et al. (1981);Andersson et al. (1983a); and Brokx and Vogel(2000b). Apo-CaM for thrombic cleavage was prepared by dissolving the protein in 50 mM NH,HCO,, then passing this solution through a 7 mL Chelex-100 column equilibrated with 50 mM NH,HC03. Fractions containing CaM (determined by checking A,) were pooled and lyophilized. 50-100 mg of lyophilized apo-CaM was dissolved in 5 mL thrombic cleavage buffer (prepared fresh): 50 mM Tris-HC1 (pH 8.5), 5 mM ethylene glycol-bis-2- aminoethyl ether N, N, N', N'-tetraacetic acid (EGTA), 1 mM DTT, and 50- 100 pL of thrombin stock solution (1 unit/pl; 1 unithg CaM) was added. The solution was incubated at 37 "C for -90 min. The digest was then rapidly cooled and applied to a 2.5 cm X 100 cm Sephadex (3-50 column equilibrated with Chelex-treated 50 mM NH,HC03. The column was run Thrombic -fiamnents of calmodulin 171 at 0.5 mumin at 4 "C with Chelex-treated 50 mM NH,HCO, as the buffer, and 10 min fractions were collected. Fractions were usually pooled into four separate pools: 0: undigested CaM (if any) plus TM1, 1: pure TMly 2: TM1 and TM2, 3: pure TM2. Pool 0 was saved and used in the next round of proteolysis. Pool 2 was saved and used in the next round of size- exclusion chromatography. Pools 1 and 3, containing pure TM1 and TM2 respectively, were lyophilized and stored at -20 OC. Metal ions were removed from protein samples by passing a solution of the protein through a 5 mL Chelex-100 column.

W absorption spectroscopy. Samples of CaM, TM1, and TM2 were prepared by dissolving appropriate amounts of the proteins in 50 mM Tris-HC1, pH 7.5. Single scans between 320 nm and 240 nm were collected for each sample on a Cary 1 UV/visible spectrophotometer.

Gel bandshie assays. Urea-PAGE bandshift assays were performed according to the methods of Erickson-Viitanen and Degrado (1987) and Brokx and Vogel (2000a). Samples of 50 pL were prepared in 100 mM Tris-HC1, pH 7.2, 4 M urea, 2 mM CaCl?, and 30 pM CaM, TMly and/or peptide. Samples with TM2 had 60 pM in order to easily visualize the TM2 band on the gel and to drive complex formation. Af'ter a l h incubation at room temperature, 50 pL 50% glycerol was added. Samples were loaded onto a 15 % PAGE gel containing 4 M urea, 0.1 mM CaCl,, and 0.375 M Tris-HC1, pH 8.8 as the buffer. Ten pL ( equivalent to 0.15 nmol protein except 0.30 nmol TM2) were loaded per lane. Gels were run for -3 h at a constant voltage of 100 V Thrombic -framentsof calmodulin 172 in a running buffer of 25 mM Tris, 0.192 M glycine, and 0.1 mM CaCl?, after which they were stained with Coomassie Blue according to standard protocols (Sambrook et al., 1989). Native-PAGE bandshift assays were performed exactly according to the methods above, except that urea was excluded from both the samples and the polyacrvlamide gel and appropriate volumes of water were substituted in its place.

Circular dichroism spectroscopy. Circular dichroism spectra were obtained on a Jasco 5-715 spectrapolarimeter (Department of Chemistry) according to published methods (Yuan et al., 1995; Brokx and Vogel, 2000a). Fragment concentrations were determined by qualitative amino acid analysis; from the amino acid analysis results, molar extinction coefficients of e2,, = 2360

M"*cm"for TM1 and E?,, = 1860 M"*crn-'for TM2 were determined and were used for later concentration determinations. CaM concentrations were determined by an extinction coefficient of els, = 1.8 (r,= 3000 M-'*cm"). Peptide concentrations were determined either by qualitative amino acid analysis or by an extinction coefficient of E, = 5500 M-'*crn-lfor a unique tryptophan residue within the peptide. For far-UV CD spectra, samples contained 10 pM protein and/or peptide, 10 mM Tris-HC1, pH 7.2, and either 2 mM CaC1, or 2 mM EDTA, in a total volume of 200 pL in a cylindrical cuvette of 0.1 cm path length. Scan parameters for far-UV CD spectroscopy were a spectral width from 255 nm to 185 nm with a 0.2 nm step resolution, a 50 nm/min scan rate, a 2 s response time, and 20 mdeg sensitivity. Five scans were taken for each spectrum with protein, snd ten scans were recorded for spectra of peptide or buffer alone, and spectra were smoothed with the JASCO sohare. The spectra of buffer alone served as Thrombic -framnents of calrnodulin 173 a baseline which was subtracted from all other spectra. For the far-UV CD experiments with the PDEa peptide, which has a Cys residue, oxidation of the samples was prevented by preincubating a 40 solution of the peptide in 4 mM DTT at 37 "C for 1 h prior to sample preparation and all samples contained a fmal concentration of 1 mM DTT (Yuan et al.. 1999b; Brokx and Vogel, 2000a). For near-W CD spectra, samples consisted of 50 pM protein andlor peptide, 25 mM Tris-HC1 pH 7.2, 100 mM KC1, and either 2 mM CaCl, or 5 mM EDTA. A sample volume of 2 mL and a 1 cm cylindrical cuvette was used. Twenty scans between 320 nm and 250 nm were collected for each spectrum; scan parameters were the same as for the far-UV CD spectra except that the sensitivity was decreased from 20 mdeg to 10 mdeg. Baseline correction for the near-UV CD spectra was performed as for the far-W CD spectra; near-W CD spectra were plotted without smoothing.

Enzyme assays. Calcineurin assays were performed according to the methods of Chapter 2. The activation assays for bovine brain 3':5'-cyclic nucleotide phosphodiesterase (PDE) (Sigma) and chicken gizzard smooth muscle myosin light-chain kinase (smMLCK) were performed by the laboratory of Dr. Michael P. Walsh, Department of Medical Biochemistry. Assays for activation of PDE were performed according to the method of Wang et al. (1972). Assays of activation of smMLCK were performed according to Weber et al. (1999);the purified 20 kDa light chain of myosin (10 pM) was used as a substrate and the concentration of srnMLCK was set at 0.2 pdd. Thrombic framnents of calmodulin 174 Proton and carbon-13 NMR spectroscopy. One- and two-dimensionallH-NMR spectra were acquired on a Bruker AMX500 spectrometer equipped with a 5 mm z-axis gradient shielded triple resonance probe according to the methods of Chapter 2. Protein concentrations were determined by weight; ID 'H NMR spectra, as well as TOCSYs and NOESYs for TM1 and/or TM2 were performed at 298 K both in H?O and in D,O. 'H,I3C heteronuclear multiple quantum coherence (HMQC) spectra of 13C- methyl-Met TM1 and TM2 with gradient coherence selection were acquired at 298K on a Bruker AMX5OO spectrometer using the pulse sequence of Wider and Wiithrich (19931, according to protocols established in our laboratory (Siivari et al., 1995; Yuan et al., 1999a). The I3C- methyl-Met labeled samples were dissolved in 500 pL D,O, 100 mM KCl; pH was adjusted to 7.4 with a thin-stem electrode and was not corrected for the isotope effect. The refocusing delay for the 'H, I3C HMQC spectra (= '1,) was 3.6 ms, with a 1.5 s relaxation delay between scans. Quadrature detection in the F1 dimension was obtained using the time-proportional phase incrementation (TPPI) method (Marion and Wiithrich, 1983). Spectra consisted of 1024 ('H; F2) by 128 (13C; F1) data points with sweep widths of 6 ppm in each dimension, centered at 3.32 ppm ('HI and 17.31 ppm (13C). The 'H dimension was referenced to the water peak at 4.78 ppm; the 13C dimension was referenced indirectly to the 'H frequency of internal 2,2-dimethyl-2-silapentane-5- sulfonate (DSS) by applying a conversion factor of 0.251449530 (Wishart et al., 1995). Generally 16 scans were acquired per increment, with a total acquisition time per experiment of -1 h. For 'H, 13C HMQC spectra of TM1 and TM2 with the CaMKT peptide, aliquots of a 5.44 mM stock solution of the peptide (in D,O, pH 7.4) were added to the TM1:TMZ NMR sample. In Thrombic -fi.amentsof calmodulin 175 an attempt to dissolve the precipitate, the solution was diluted 2 X in D20, 100 mM KC1 and the pH was readjusted to 7.4. Spectra of 32 scans (2 h) were acquired for these samples. All 1D 'H NMR spectra were processed with the SwaN-MR software (Balacco, 19941, operating on a Power Macintosh 6100/60 personal computer. All 2D NMR spectra were processed with the NMRPipe software (Delaglio et al., 1995) on a Silicon Graphics Indy workstation and were viewed with the accompanying NMRDraw graphics package.

Cadmium-I13 NMR spectroscopy. One-dimensional cadmium-113 NMR spectra were acquired on a Bruker AM400 wide-bore spectrometer equipped with a 10 mm broadband probe ( "=Cd v, = 88.75 MHz). To remove contaminating metal ions, acid- washed glassware was used throughout all sample preparations. Methods were similar to Aramini et al. (19951, except that sulfate, rather than perchlorate, was used as the counter ion (Sudmeier et al., 1981; Brokx and Vogel, 2000a). This was done for safety and practical reasons; perchlorate salts of polyvalent metal ions are notoriously unstable and explosive, and, moreover, the sulfate method entirely omits the potential interference from chloride ions in the initial step of dissolving the t'3Cd0 in HCI (Aramini et al., 1995). In the method used here, a stock solution of l13CdS0, was prepared by dissolving lI3CdO (32.1 mg) in a few drops of 3 M H,SO, and evaporating the solution to dryness under low heat and a steady stream of N, gas. The resulting white precipitate was dissolved in 2.5 mL 50% D,O:H?O (to 100 mM 113CdS0,),and the pH of the stock solution was adjusted to 4.0 with dilute NaOH and H2S0,. A 100 mM solution of CdSO, at natural abundance (12% '13Cd) in 50% D,O:H,O pH 4.0, prepared Thrornbic framents of calmodulin 176 similarly, was used as a chemical shift reference for the '13Cd spectra. Samples for '13Cd NMR were prepared by dissolving the lyophilized protein in 2 mL 10% D,0:H20.After adding appropriate amounts of the '13CdS0, stock solution, sample pH was adjusted to 7.4 with dilute NaOH and H,SO,. Spectra consisted of a simple 1D pulse sequence with a 20 000 Hz (225 ppm) sweep width and a 1 s relaxation delay. Spectra were processed with SwaN-MR (Balacco, 1994) on a Power Macintosh 6100/60 personal computer, and were referenced to external 100 mM CdSO, in 50 % D,0:H20, pH 4.0 at 0 ppm.

RESULTS.

W absorption spectroscopy. The UV absorption spectra for CaM, TM1, and TM2 are shown in Figure 5.2. Each of the two thrombic fragments of CaM has a tyrosine residue, which makes quantitation by UV spectroscopy quite straightforward. TM1 also has seven Phe residues whereas TM2 has only one, which makes the spectra of the two fragments quite different from each other. Thus, a qualitative assesment of the purity of a preparation of one of the fragments can be made by examination of the W spectrum of the sample. The W absorption spectrum of a 1:l mixture of TM1 and TM2 (not shown) looks quite similar to the spectrum of intact CaM.

Gel Bandshift Assays. Figure 5.3a is a picture of the gel bandsha assay in the presence of 4 M urea and 0.1 mM Ca2+. There is a clear bandshift when either the skMLCK or the CaMKI peptide is added to CaM, indicative of the Thrornbic .fiamentsof calmodulin 177 formation of a complex between CaM and the peptide that has reduced mobility relative to CaM alone. This is a result not only of the increased mass of the complex, but also the decreased negative charge relative to uncomplexed CaM. TM1 and TM2 do not associate with each other, as shown by the presence of separate bands for the two fragments in the lane containing TM1 and TM2. Moreover, there is no significant bandshift when either the skMLCK or the CaMKI peptide is added to TM1 and TM2 (Fig. 5.3a, lanes 7 and to, or when a peptide is added to TM1 or TM2 alone (not shown). Thus the interactions between TM1 and TM2 are not strong enough, even with a peptide present, to form a complex in the presence of 4 M urea. The results of the native gel bandshifi in the absence of urea are significantly different (Figure 5.3b, c). The overall quality of the gel is poorer with streaking of the bands in many of the lanes, owing to the absence of urea. There is still no complex being formed between TM1 and TM2 (Figure 5.3b, lane 6); however, when TM1 and TM2 are combined in the presence of a peptide, there is a single, new species formed with mobility very similar to that of the respective CaM:peptide complex. This finding strongly supports the claim that TM1 and TM2 can associate, in the presence of a peptide, to form a complex with CaM-like properties. There is no significant bandshift of either TM1 or TM2 with a peptide, although there is some streaking of the lane with TM1 and CaMKI (Figure 5.3c, lane 61, and especially TM1 and skMLCK (not shown). This may reflect the stronger binding constant between skMLCK and CaM (1 nM), as opposed to CaMH and CaM (3-10 nM) (O'Neil and DeGrado, 1990, and references therein). A gel bandshift assay with TM1 and TM2 in the presence of EDTA was not performed, but since no bandshift is observed with CaM and Thrombic -fragments of calmodulin 178 Figure 5.2.

Figure 5.2. W-absorption spectra of a) CaM, b) TM1,and c) TM2 in 50 mM Tris-HC1, pH 7.5. Thrombic -framentsof calmodulin 179

Figure 5.3. Figure 5.3. (previous page) a) 4 M urea-PAGE gel of: lane 1: CaM, 2: CaM + CaMKI, 3: CaM + skMLCK, 4: TM1,5: TM2,6 TM1+ TM2,7: TM1+ TM2 + CaMKI, 8: TM1 + TM2 + skMLCK. b) Native-PAGE gel with the lane designations same as in a). c) Native-PAGE gel of lane 1: CaM, 2: CaM + CaMKI, 3: TM1,4: TM2,5: TM1+ TM2,6:TMl + CaMKI, 7: TM2 + CaMKI, 8: TMl + TM2 + CaMKI. Conditions are outlined in the Methods section.

either the skMLCK or the CaMKI peptide in the presence of EDTA it should be assumed that there would be no significant interaction with TM1 and TM2 under these conditions as well.

Enzyme activation assays. Activation of calcineurin (Figure 5.4a), smMLCK (Figure 5.4b), and PDE (Figure 5.4~)by TM1 and TM2 was determined. TM1 and TM2,either alone or in combination, are very poor at activating any of the three enzymes; the activity that is seen at higher concentrations of the fragments (e.g. Figures 5.4b, c) is most likely due to contamination by small amounts of intact CaM. For example, the concentrations of maximal activation by CaM and TM1 for smMLCK and PDE differ by about two orders of magnitude, so the activity of TM1 could be explained by a 1% contamination by CaM.

Circular dichroism spectroscopy. Figure 5.5a shows the far-UV circular dichroism spectra of TM1 and TM2,both alone and in combination, in the presence of Ca2+ions. There is significant a-helical structure in TM1, as shown by the negative ellipticities at 208 nm and 222 nm. The CD spectrum of the smaller TM2 Thrombic framents of calmodulin 181 shows little secondary structure. When TM1 and TM2 are combined, the resulting spectrum (TM1:TMP) has greater negative ellipticities than the sum of the two individual spectra of TM1 and TM2. However, the CD spectrum of TMl:TM2 does not have as large negative peaks as the spectrum of intact CaM. Thus, some a-helical structure in CaM is lost as a result of cleavage at the Arg106-His107 peptide bond by thrombin. When TM1 and TM2 are combined in the absence of Ca2+(Figure 5.6a), the CD spectrum is very similar to the sum of the two individual spectra, indicating that the interaction between the two fragments is Ca2+- dependent. Moreover, there is significantly less a-helical structure in the two fragments than in intact apo-CaM. When CaM is combined, in the presence of Ca2+,with a CaM-binding target peptide, such as MLCK (Figure 5.5b), or PDEa (Figure 5.5~)there is a resultant increase in the a-helical structure, indicated by an increase in the negative ellipticity in CD spectra. It is well established that this is not the result of structural changes in CaM, but instead is primarily due to a structural change in the target peptide, from a largely unstructured form in solution to a highly a-helical form in the CaM-bound state (Zhang et al., 1994b). When TM1 and TM2 are combined with MLCK (Figure 5.5b), or PDEa (Figure 5.5~)in the presence of Ca2+,the result is a very significant increase in negative ellipticity over that of TMl:TM2, indicating some interaction between TM1, TM2, and the target peptide. Moreover, the increase in ellipticity is larger than that when the target peptides are bound to CaM. This may possibly be the result of not only stabilization of a-helical structure in the peptide by TMl:TM2, but also stabilization of helical structures within TMl and TM2 by the target peptide. When PDEa is combined with CaM in the absence of Ca2+(Figure Thrombic -+a-ments qf calmodulin 182

Figure 5.4. a)

[Protein] (nM)

---- [7--- TM1 --+- TMI:TM2 -- CaM Thrombic -fragments of calmodulin 183

Figure 5.4. b)

-12 -11 -10 -9 -8 -7 -6 -5 -4 [Fragment] (M) Thrombic -fragments of calrnodulin 184

Figure 5.4. C)

-11 -10 -9 -8 -7 -6 -5 -4 -3 [Fragment] (M)

Figure 5.4. Activation assays of a) calcineurin, b) smooth muscle myosin light-chain kinase, and c) cyclic nucleotide 3'' 5'-phosphodiesterase by TM1 and/or TM2. Sample conditions are outlined in the Methods section. Thrombic fra~mentsof calmodulin 185 Figure 5.6. a)

o.o.mem.me TMI :TM2 - CaM Thrornbic -framnentsof calrnodulin 186 Figure 5.5. b)

...... MLCK

m-m------m TM1 :TM2: MLCK CaM Thrombic fiaments of calmodulin 187

Figure 5.5. c)

... l..ll.l...... PDE - ....-.--. TM1 :TM2 + PDE CaM ----- CaM + PDE

Figure 5.5. Far-UV circular dichroism spectra of a) TM1, TM2, the summation of TM1 and TM2 (TMl+ TM2), a 1:l mixture of TM1 and TM2 (TMl:TMB), and CaM; b) TM1:TMS and MLCK compared to CaM and MLCK; c) TM1:TMS and PDEa compared to CaM and PDEa. Protein and peptide concentrations were 10 pM; spectra were acquired in the presence of 2 mM CaCI, . Thrombic fiaments of calmodulin 188

Figure 5.6. a) Thrombic -fragments of calrnodulin 189 Figure 5.6. b)

..-.-.-.- TM1 :TM2

**...... *.***....lPDE ...*...... TM1:TM2 + PDE

----- Apo-CaM + PDE

Figure 5.6. Far-W CD spectra of a) TM1, TM2, the summation of TM1 and TM2 (TM1 + TMZ), a 1:l mixture of TMl and TM2 (TMl:TMS), and CaM; b) TM1:TMB and the PDEa peptide compared to CaM and the PDEa peptide. Protein and peptide concentrations were 10 pM; spectra were acquired in the presence of 8 mM EDTA. Thrombic -fragmentsof calmod ulin 190 5.6b), there is an enhancement of negative ellipticity due to structural changes in the PDEa peptide, which adopts a structure with some helix and some turn components as it binds to the C-terminal lobe of apo-CaM (Yuan et al., 1999b). The interaction of PDEa with TM1 and TM2 in the absence of Ca2+is not as significant (Figure 5.6b), which is to be expected since it is the C-terminal lobe of CaM that is disrupted as a result of the thrombic cleavage. Near-UV CD spectra for CaM, TM1, and TM2 under various conditions are depicted in Figure 5.7. The near-W CD spectrum of CaY'- saturated CaM (Figure 5.7a) shows negative ellipticity peaks at 263 nrn and 270 nm which are the result of electronic transitions in the eight Phe residues of the protein. The broad peak at 283 nm is from the two Tyr residues in CaM. The near-W CD spectrum of TM1 has strong Phe peaks, owing to the fact that it has seven of the eight Phe residues originally contained in CaM. It also has one Tyr residue. The near-W CD spectrum of TM2 is less intense due to the fact that it has only one Phe and one Tyr residue. When TM1 and TM2 are combined in the presence of Ca2+(Figure 5.7a), the resulting spectrum is similar to that of Ca2+-CaM,although the Tyr peaks are not quite as intense. The near-UV CD spectrum of a 1:l mixture of TM1 and TM2 in the absence of Ca" is unlike that of apo-CaM (Figure 5.7~)and quite similar to the summation of the two spectra for apo- TM1 and TM2, again suggesting that there is little interaction between the two eagments in the absence of Ca2+. When Ca2+-CaMis combined with the CaMKI peptide, two positive CD peaks appear at 288 nm and 295 nm (Figure 5.7b). These are the result of positive 'L. and 'L electronic transitions (Barth et al., 1998) that occur in the Trp residue of the peptide when it is bound by CaM (Yuan et al., Thrombic -fia-ments of calmodulin 191 1999b; Gomes et al., 2000); there are no Trp residues in CaM. The near- UV CD spectnun of a 1:l:l mixture of TM1, TM2, and the CaMKI peptide in the presence of Ca2+ (Figure 5.7b) also has the same Trp peaks, suggesting that the TM1:TMS complex binds the CaMKI peptide in a manner similar to CaM.

Proton and carbon-13 NMR spectroscopy. The 1D NMR spectra of TM1,TM2, TMl:TM2, and CaM in D20in the presence of Ca2+ions are shown in Figure 5.8. When TM1 and TM2 are combined, the peaks for the tyrosines (Tyr99 and Tyr138) shift upfield significantly to positions similar to that of CaM. This is important because the two tyrosines are separated as a result of the thrombic cleavage. There are also several downfield-shifted a protons in the spectrum of Ca2+-TMl:TM2,similar to CaM. These are from residues in the small two-strand P-sheets that form between the two adjacent Ca2+- binding loops in each lobe. The spectrum of TM1 also has peaks in this region, which is to be expected because the P-sheet in the N-terminal lobe is likely to be intact. However, further peaks appear in this region when TM1 and TM2 are combined, indicative of a p-sheet interaction between the two fragments. As an aside, the spectrum of TM2 alone in D,O has two peaks at 7.7 and 8.5 ppm that are downfield from the aromatic peaks. COSY and TOCSY spectra (not shown) indicate that they are members of the same spin system, likely a phenylalanine ring. They are definitely not histidine ring protons or amide protons. As well as the P-sheet a protons in the 1D 'H NMR spectrum of TMl:TM2 in D,O, there is also evidence in 'H spectra in H,O that indicate proper conformation of Ca2+-bindingloops. Four downfield-shifted amide Thrombic fiaments of calmodulin 192

Figure 5.7. a) Thrombic -fiaments of calrnodulin 193 Figure 5.7. b) Thrombic -fragments of calmodulin 194 Figure 5.7.

C) Thrombic -fiamnentsof calmodulin 195 Figure 5.7. (previous pages) Near-UV CD spectra of a), b) TM1, TM2, the summation of TM1 and TM2 (TM1 + TM2), a 1: 1 mixture of TM1 and TM2 (TMl:TM2), and intact CaM; c) TMl:TM2, CaM, the CaMKI peptide, TM1:TMB with the CaMKI peptide, and the CaM:CaMKI peptide complex. Protein and peptide concentrations were 50 yM; samples in a) and c) contained 5 mM CaCl,, and samples in b) contained 5 mM EDTA.

Figure 5.8. Figure 5.8. 500 MHz 1D 'H NMR spectra of a) TM1 (-1.5 mM), b) TM2 (-1 mM), C) TM1:TMS (-1 mM), and d) CaM (-1 mM) in D,O, 100 mM KCl, 10 mM CaCl,, pH 7.4, at 298K. 256 scans were acquired for each spectrum, except for d) which had 128 scans. Peaks in d) are labeled with the chemical-shift assignments for wild-type CaM (Ikura et al., 1991). Experimental parameters are outlined in the Methods section.

proton peaks are present in the NOESY spectrum of Ca2+-TM1:TMPin H,O (Figure 5.91, like those in the spectrum of CaM. These are from four glycine residues (Gly25, Gly61, Gly98, and Gly134), one in each loop, that participate in an important hydrogen bond in the Ca2+-boundconformation (Ikura et al., 1987). The spectrum of TM1 has two such peaks, corresponding to the two loop glycines in the N-terminal lobe, as well as a third at significantly lower intensity, likely Gly98 from site 111. TM2 also has a weak signal in this area that is probably from Gly134. When TM1 and TM2 are combined, four strong peaks are present, indicating that Ca2+-bindingsites 111 and IV come together to form a conformation that binds Ca2+more tightly. The 'H,13C HMQC spectra of methyl-13C-Met labeled TM1, TM2, and CaM under various conditions are in Figures 5.10 and 5.11. Four resolvable peaks exist in the spectrum of apo-TM1 (Figure 5.10a), which correspond to the Met residues in the intact N-terminal lobe (Met36, Met51, Met71, Met72). The peaks for all four Met residues of apo-TM2 (Figure 5.10~)all overlap and are found at a chemical shift position close to the value of Met methyls in a random coil state (Wiithrich, 1986), indicating a lack of structure in TM2 without Ca2+. The 'H, 13C HMQC spectrum of TMl:TM2 without Ca2+(Figure 5.10e) is very similar to the Thrornbic framents of calnodulin 197

Figure 5.9.

Figure 5.9. 500 MHz 'H, lH NOESY spectra of a) TM1, b) TM2, and c) TM1:TMP in H,O:10 % D,O, 10 mM CaCl,, 100 mM KC1, pH 6.3, at 298K Fragment concentrations were -1.5 mM. NOESY crosspeaks labeled in c) are deduced from a comparison to the NOESY spectrum of intact Ca2+-CaM (Ikura et al., 1987). Experimental conditions are outlined in the Methods section. Thrombic -fiamentsof calmodulin 198 sum of the two individual spectra, demonstrating a lack of any significant interaction between the two fragments. When CaZ+is added to either TM1 or TM2 alone (Figure 5.10b, dl, the Met methyl peaks shift, but the change is incomplete even after the addition of ten equivalents (10 mM) of Ca", showing that the isolated Ca2+-bindingsites (domains I11 and IV) bind Ca" very poorly. When TM1 and TM2 are combined, the Met methyl peaks become more distinct (Figure 5.10e) and shift to values similar to those of Ca2+-CaM(Figure 5.11). However, the peaks are still quite broad compared to those of intact Ca2+-CaM,especially in the I3C dimension, and, moreover, there are some smaller peaks at lower intensity, indicating conformational exchange in the TM1:TMP complex. In fact, the peaks in all of the HMQC spectra of methyl-13C-Met labeled TM1 and/or TM2 are all quite broad, indicative of the lack of a stable conformation. Figure 5.11 shows the 'H, 13C HMQC spectra of rnetl~yl-~~C-Met labeled TM1:TMS and CaM in the absence of Ca", the presence of Ca2+,and the presence of Ca2+and a synthetic peptide corresponding to the CaM- binding domain of CaM-dependent protein kinase I (CaMKI). When the CaMKI peptide is added to Ca2+-TMl:TM2(Figure 5.119, the Met methyl peaks shift to values close to those of the Ca2+,-CaM-CaMKIcomplex, indicating that the peptide is being bound by the TMl:TM2 complex in a manner similar to CaM.

Cadmium-113 NMR spectroscopy. The 1D WdNMR spectra of TM1, TM2, and CaM are shown in Figure 5.12. The two peaks in the spectrum of CaM are of l13Cd2+bound by sites I11 and IV, the two high-affinity sites of CaM (Andersson et al., 1983a). The signals from l13Cd2+bound by sites I and I1 are too weak to be Figure 5.10. Thrombic fragments of calrnodulin 200 Figure 5.11.

I.... 18 2.5 2:0 Thrombic fi-aments of calmodulin 201 Figure 5.10. (two pages previous) 'H,13C HMQC spectra of methyl-13C- Met labeled a), b) TM1,c), d) TM2, and e), f) TMl:TM2, in D,O, 100 mM KCl, pH 7.4, at 298K Samples for spectra b), d), and f) also contained 10 mM CaCl2. Fragment concentrations were 0.9 mM. Boxes in f) are at locations where additional peaks are seen at lower contour levels. Experimental conditions are outlined in the Methods section.

Figure 5.11. (previous page) 'H, 13C HMQC spectra of methyl-13C-Met labeled A, C, E CaM (1 mM) (Yuan et al., 1999a), and B, D TM1:TMB (0.9 mM), and F TM1:TMS (0.45 mM) in D,O, 100 mM KC1, pH 7.4, at 298K, with A, B, no CaCl, added; C, D 10 mM CaCl, added; and E, F 10 mM CaCl, plus 1 equivalent of the CaMKI peptide added. Experimental conditions are outlined in the Methods section. Peak assignments in A, C, and E are @om Yuan et al. (1999a).

seen in these spectra. The peaks for "3Cd'+bound by TM1 and TM2 (Figure 5.12a, b) are quite broad compared to intact CaM, indicating weaker binding of the metal ion. The two sharp peaks in the spectrum of TM1 (Figure 5.12a) most likely result from contamination of the sample either by intact CaM or by TM2. When TM1 and TM2 are combined (spectrum c), the two lL3Cdpeaks sharpen substantially to widths similar to those of intact CaM, indicating significant enhancement of metal ion affinity when TM1 and TM2 are combined. This is most likely the result of association and complementation of the two fragments to reconstitute an intact two- site domain with high affinity for metal ions. When the CaM-binding antipsychotic drug trifluoperazine (TFP)is added to CaM, two additional peaks arise in '13Cd NMR spectra (Andersson et al., 1983b), which are from "3Cd2+bound by sites I and 11, which have their affinity for metal ions increased by binding of target molecules. When TFP is added to a lI3Cd2+- Thrombic fiaments of calmodulin 202 Figure 5.12.

Figure 6.12. Cadmium-113 NMR spectra of a) 1.3 mM TM1, 3.9 mM 113CdS0,;b) 1.3 mM TM2, 2.5 mM 1'3CdS0,;c) 1.3 mM TMl:TM2, and 5.2 mM "3CdS0,; and d) 1.1 mM CaM,4.5 mM 113CdS0,in H,O:10 % D20 at 298 K. Spectra consisted of a)-c) 32 000 scans, or d) 30 000 scans. The small, sharp peaks at -91 and -112 ppm in a) come from a contaminating amount of TM2, or possibly intact CaM. Experimental conditions are outline in the Methods section. Thrombic -fra~rnentsof calrnodulin 203 Figure 5.13.

TFP TM1 :TM2

Figure 5.13. Cadmium-113 NMR spectra of 1.3 mM TMl:TMB, 5.2 mM "3CdS0, in H,O:10 % D,O at 298 K with a) 0 equivalents, b) 1 equivalent, C) 2 equivalents, and d) 3 equivalents trifluoperazine (TFP)added. 19 200 scans were taken for each spectrum. Thrornbic fragments of calmodulin 204 TMl:TM2 (Figure 5.13) additional peaks arise in a similar fashion.

DISCUSSION.

Apo-calmodulin has been cleaved at the Arg106-His107 peptide bond by the specific protease thrombin to create two fragments: TM1 (1-106), which contains Ca2+-bindinghelix-loop-helix domains I and I1 as well as most of domain 111, and TM2 (107-148) which contains the last part of the second helix from domain I11 as well as the entire domain IV. By ensuring that the protein was completely Ca2+-freeand by limiting the proteolysis time, cleavage at Arg36-Ser37 (Shea et al., 1996) was avoided. It is interesting that, besides these two Arg residues, CaM has four other Arg residues that are not cleavage sites. Thrombic cleavage sites vary a great deal, and the specificity of thrombin is difficult to explain, but thrombin particularly cleaves peptide bonds in which a proline directly precedes the arginine (Kettner and Shaw, 1991). None of the arginines of CaM are preceded by Pro residues. It is also known that hydrophobic residues near the active site of thrombin and a basic surface, the "exosite," far removed from the active site, have also been implicated in protein binding (Bode et al., 1992, and references therein). But, neither kg106 nor kg36 are in the vicinity of any acidic residues that could potentially interact with the exosite, nor do they preceed any hydrophobic resiudes that could potentially occupy the hydrophobic binding site. In fact, given those two criteria, some of the other four arginines could be considered as better potential targets. More likely, thrornbic cleavage occurs at these two positions because of their accessibility, and perhaps also due to other interactions between thrombin and other areas on the surface of apo-CaM (recall that thrombin only cleaves apo-CaM). Regardless of how or why digestion of CaM with thrombin occurs predominantly at ArglO6, the properties of the two fragments generated are quite interesting. As expected, the isolated Ca2+-bindingsites of the two thrombic fragments of CaM (site I11 of TM1 and site IV of TM2) bind metal ions much more weakly than intact CaM. This was shown by '13Cd NMR spectroscopy (Andersson et al., 1983, and this chapter), and 'H NMR. lH and "C NMR and circular dichroism spectroscopy also showed that TM1 and TM2 have little a-helical structure with the exception of the intact N- terminal domain of TM1. The absence of secondary structure and the poor metal-ion binding properties of these fragments parallel results obtained for other isolated EF-hand helix-loop-helix motifs, such as sites I11 and IV of troponin C (Shaw et al., 1990, 1991; Kay et al., 1991). These studies also demonstrated the metal-ion dependent association of EF-hand fragments, which was again found in this study. NMR and CD spectra show that TM1 and TM2 can interact in the presence of Ca2+to form a complex with enhanced a-helical structure and enhanced metal ion binding properties. Far-W CD spectroscopy (Figure 5.5a) clearly shows enhancement of negative ellipticity at 208 nm and 222 nm, possibly indicative of formation of additional a-helix structures, upon mixing of TM1 and TM2. This interaction is also clearly dependent on the presence of Ca2+ions (c.f. Figure 5.5a and Figure 5.6a); there is no interaction in the presence of EDTA. However, the CD spectrum of Ca2+-TM1:TMZis not as intense as that of Ca2+-CaM(Figure 5.5a), probably due to some local perturbations, such as fraying of helix 6, around the thrombin cleavage site. Perhaps also complexation of TM1 and TM2 is incomplete at the 10 pM concentration required for far-W CD spectra, and so there are some Thrombic -fragments of calmodulin 206 undissociated TM1 and TM2 monomers. Quantitation of the changes in CD spectra with respect to changes in secondary structures was not attempted because it is well-known that far-W CD spectra often do not accurately reflect the relative amounts of secondary structural elements for CaM and related helix-loop-helix Ca2+-bindingproteins, and it is dificult to interpret changes in far-W CD spectra in terms of changes in secondary structures alone. For example, although there is a substantial Ca2+-dependentincrease in the negative CD ellipticity of CaM at 208 nm and 222 nm (Bayley and Martin, 1986, and this chapter), secondary structure assignments by NMR for Ca2+-saturated(Ikura et al., 1991) and apo-CaM (Zhang et al., 1995b; Kuboniwa et al., 1995) showed that the amount of various secondary structures is actually very similar for the two states of the protein. Instead, the increase in the negative CD ellipticity is thought to be due to changes in the spatial orientation of the helices upon Ca2+-binding,as is the case for the very similar helix-loop- helix Ca2+-bindingprotein troponin-C (Gagne et al., 1994). What is important here is the enhancement of the CD signal when TM1 and TM2 are combined in the presence of Ca". This shows the production of a structure, most likely the result of complexation, that is more like CaM than either of the two fragments alone, whether it be due to increased a- helicity or proper orientation of already existing a-helices. The CaZ+- dependent production of a CaM-like structure is also shown in near-UV CD spectra (Figure 5.71, and is substantiated by NMR (see below). Since complementation of TM1 and TM2 are shown at the micromolar concentrations required for CD spectroscopy, one would certainly expect changes in NMR spectra upon mixing of TM1 and TM2 and indeed, this is the case. Important peaks arise in the 1D lH-NMR Thrombic -fiagrnentsof calmodulin 207 spectrum of Ca2+-TM1:TMZin DLO(Figure 5.81, which are the result of a small two-stranded P-sheet interaction between Tyr99 and IlelOO in loop I11 of TM1 and Val136 and Asnl37 of loop IV in TM2. These $-sheets have been shown to have very important roles in stabilizing CaM and increasing the afEnity of CaM for Ca2+and target proteins (Browne et al., 1997). Also, other peaks arise in the 'H,'H NOESY spectrum of Ca2+- TM1:TMB in H,O (Figure 5.9) that are from the NH protons of Gly25, Gly61, Giy98, and Gly 134, the four glycine residues that form a very important hydrogen bond within each Ca" ion binding loop in the CaL+- bound conformation (Ikura et al., 1987). When TM1 and TM2 are combined, the intensities of Gly98 and Gly134 are greatly increased and NOES to neighboring residues become apparent. This indicates a well- defined Ca2+-boundconformation of the C-terminal lobe which is the result of complementation between the TM1 and TM2 fragments. Apart from 2D lH NMR spectra, we have also used 'H,'3C HMQC spectra of 'T-methyl-Met labeled TM1 and TM2 to determine the properties of the fragments. These spectra have been proven very useful in determining the environment of the methionine residues in CaM (Ouyang and Vogel, 1998; Yuan et al., 1999a; Brokx and Vogei, 2000a), which are very important for target peptide recognition and binding (Vogel and Zhang, 1995). The results here (Figures 5.10 and 5.11) show that the N-terminal domain of CaM largely retains its conformation after cleavage at Arg106-His107 by thrombin. However, the methionine residues in the C-terminal lobe of the molecule (MetlO9, Met124, Met144, and Met145), when they are separated from the rest of the molecule by thrombic cleavage, become degenerate at a chemical shift position that is the same as for Met methyls in random coils (Wuthrich, 1986). When TM1 Thrombic -fragments of calmodulin 208 and TM2 are combined, the conformational variability of the Met methyls increases and there seems to be more than one conformation for many of the Met methyl groups. Even after addition of excess amounts of Ca2+,the TM1:TMB complex does not seem to be fully titrated. This may be a reflection of poor Ca2'-binding properties, or perhaps an equilibrium between the TM1:TMP complex and the respective monomers, which was also suggested in the above discussion of the CD results. Cadmium-113 NMR results (Figure 5.12) indicate a very significant enhancement of metal-ion binding when TM1 and TM2 are combined. This is most likely the result of the formation of a lt3Cd2+-TMl:TM2 complex. However, some TM1 and TM2 monomers may also exist under these conditions. The peaks for "3Cd'+ bound by TM1 and TM2 alone are quite broad, with the peak for TM1 being the broader of the two. This is possibly the result of the disproportionate cleavage of CaM by thrombin, such that the third EF-helix-loop-helix domain in TM1 is incomplete. TM2, on the other hand, has an additional piece of an a-helix which is N- terminal to domain N which may enhance the metal ion affinity of this site. With regard to this, the study of this chapter is unique because of the site of thrombic cleavage. Most of the other studies of EF-hand fragments have concerned entire helix-loop-helix domains, either chemically synthesized (Shaw et al., 1990, 1991; Kay et al., 1991; Linse et al., 1997) or proteolytically produced (Finn et al., 1992). Here we have shown that fragment complementation can still occur when the cleavage is inside the C-terminal helix of one of the domains. This could be examined further by introducing cleavage locations in other areas in the CaM molecule, such as even within a Ca2'-binding loop. It would be interesting to see if fragment complementation would occur with cleavage at this location, Thrombic -fi.amentsof calrnodulin 209 although given the dependence of complementation on metal ion binding by the fragments this might be difficult. Taken as a whole, the NMR and CD results all point towards TM1 and TM2 associating in solution to form a stable complex. However, TM1 and TM2 are not able to associate under the conditions of native-PAGE, even with no urea present (Figure 5.3b). This may be because the concentrations of TM1 and TM2 are too low to associate during the electrophoresis, or perhaps because the polyacrylamide matrix disrupts the interactions between TM1 and TM2. In any event, the gel bandshift assay results are very beneficial because they conclusively show the Ca2+- dependent binding of CaM-target peptides, namely CaMKI and skMLCK, by a mixture of TM1 and TM2. A gel band shiR occurs only when all three species (TM1,TM2, and the peptide) are present, with the resulting Ca2+- TM1:TMa:peptide quaternary complex having properties very similar to Ca"-CaM:peptide complexes. This peptide-dependent association of the fragments it the first demonstration of such an interaction in fragments containing isolated helix-loop-helix domains. It is interesting given the work of other groups (e.g. Zhang et al., 1995a; Porumb et al., 1994, and references therein) in which it was found that the presence of a target peptide increases the affinity of CaM for metal ions. The presence of a peptide is not enough to induce complexation of TM1 and TM2 in 4 M urea, however. In our experience, non-denaturing urea-PAGE detects CaM:peptide complexes with dissociation constants of approximately 100 nM or less, so the association of TM1 and TM2 with a peptide is probably considerably weaker than this. In addition to gel bandshifts, peptide binding by TM1 and TM2 is also clearly shown by CD and NMR spectroscopy. When the MLCK or the Thrombic fragments qf calnodulin 210 PDEa peptide is added to a TM1:TMZ mixture, a significant increase in the amount of a-helical structure occurs, similar in fashion to when CaM binds these peptides, as shown by far-UV CD spectroscopy (Figures 5.5b and 5.5c, respectively). This is the result of a change in the structure of the peptide from an unfolded conformation to an a-helical conformation. Upon careful examination of the spectra, it seems that the apparent amount of a-helical structure increases more when a peptide is complexed by TM1:TMB than by CaM. This may be the result of stabilization of secondary structural elements in the TM1:TMB complex by the peptide, or perhaps that there are less dissociated TM1 and TM2 monomers when a peptide is added to the mixture. Not surprisingly, the interaction of the peptides with TM1 and TM2 is Ca2+-dependent. The PDEa peptide represents the first characterized CaM-binding domain of phosphodiesterase (Sonnenburg et al ., 1995) which, in addition to binding Ca2+-CaM,can also bind to apo-CaM (Yuan et al., 1999b). When the PDEa peptide is added to a 1:l mixture of TM1 and TM2 in the presence of EDTA, no significant changes to the far-UV spectrum occur (Figure 6b), despite the significant interaction of the PDEa peptide with apo-CaM (Figure 5.6b), in which the PDEa peptide forms a structure which is partially a- helical and also contains p-turns as it interacts with the C-terminal lobe of Call4 (Yuan et al., 1999b). Because the interaction of PDEa with apo-CaM is solely through the C-terminal lobe, an absence of an interaction of the PDEa peptide with TM1 and TM2 without Ca2+is not unexpected since it is the C-terminal lobe of CaM that is disrupted by thrombic cleavage. Importantly, near-UV CD spectra show two positive CD peaks that appear at 288 nm and 295 nm when the CaMKI peptide is added to Ca2+- saturated TM1:TMS (Figure 5.7b), resulting in a spectrum very similar to Thrornbic -framentsof calmodulin 211 the CaM:CaMKI complex. These positive peaks are the result of positive 'I,and 'I+ electronic transitions that occur in the lone Trp residue of the CaMKI peptide (Trp4) when it is bound by CaM (Barth et a1 ., 1998); there are no Trp residues in CaM. The CaMKI peptide binds to Ca2+-CaMin an antiparallel orientation, resulting in the Trp residue of the peptide being buried in the C-terminal lobe of CaM (Yuan et al., 1998a). The near-W CD results, then, indicate that TM1, TM2 and the peptide associate to form a Ca2+-TMl:TM2:CaMKI quaternary complex in which the proper binding environment for the Trp residue of the peptide is maintained. It should be mentioned here that the CaMKI peptide was used instead of the MLCK peptide for the near-UV CD studies simply because it was available in greater amounts; the two peptides bind to CaM in the same orientation with their lone Trp residue buried in the C-terminal lobe of CaM (Yuan et al., 1998a), and their CaM-bound structures are thought to be very similar (Gomes et al., 2000). Complexation of the CaMKI peptide by TM1 and TM2 is also evident in 'H,13C HMQC spectra (Figure 5.11). The spectrum of methyl-13C-Met labeled TMl:TM2 with CaMKI in the presence of Ca2+is very similar to the spectrum of the Ca2'-CaM-CaMKI complex (Brokx and Vogel, 2000a). This includes peaks for Met residues 109, 124, and 145, all of which shift substantially upon complexation of the CaMKT peptide by Ca2+-CaM (Figure 5.11, c and e). Since all of these residues are found in the TM2 fragment of CaM, TM2 must be involved in complexation of the CaMKI peptide. The peak for Met72 also appzrently shifts upon addition of the CaMKI peptide to TM1:TM2, indicating that TM1 interacts with the CaMKI peptide through its intact N-terminal lobe as well. Additionally, isothermal titration calorimetry results (see Chapter Thronbic -fia~mentsof caZmodulin 212 7) show that the release of heat is greater when a solution of the CaMKI peptide is titrated into a 1:l mixture of TM1 and TM2 than when CaMKI is titrated into a sample of intact CaM. This is most likely the result of induction of a stable complex of TM1 and TM2 by the CaMKI peptide. Assuming that the binding of CaMKI to TMl:TM2 produces a similar enthalpy to the binding of CaMKl to CaM, then the extra heat is a result of the enthalpy of binding of TM1 to TM2 (see discussion in Chapter 7). The stabilization of this type of a complex by a CaM-binding peptide is also hinted at by 13C NMR relaxation studies (Lee et al., 20001, in which hydrophobic amino acid side chains involved in interactions between two helix-loop-helix domains become more rigid as a peptide is bound. Although peptide binding by TM1 and TM2 is clearly evident in gel bandshifts, CD spectra, 'H, 13C HMQC spectra, and by isothermal titration calorimetry, a mixture of the fragments was unable to complement each other in activating the CaM-dependent enzymes calcineurin, smMLCK, and PDE (Figure 5.4). Most likely, the concentration of the fragments and/or Cag+(in the nM to pM range) was simply too low to result in complex formation by the fragments in order to activate any of the enzymes. An additional intriguing explanation for the inability of TM1 and TM2 to activate these target enzymes is that activation and binding of the target enzymes are separate events. The target proteins possibly bind to TM1 and/or TM2, but the cleavage of Arg106-His107 of CaM by thrombin disrupts the surface on the C-terminal lobe of the protein that is required for activation. These types of findings were reported for activation of MLCK by some CaM mutants, such as those at ThrllO, Leull2, and Lysll5 (Su et al., 19941, all of which are in helix 6 of CaM near the thrombin cleavage site. These mutant CaMs were competitive Thrornbic fragments of calrnodulin 213 inhibitors for activation of MLCK by wild-type CaM, indicating that the mutants bound but did not activate the target enzymes. Of course, this leads to the logical design of an experiment where TM1 and TM2 are tested as inhibitors of activation of calcineurin, smMLCK, and PDE by CaM. These experiments have not yet been carried out, with the one exception where TM2 was tested as an inhibitor of CaM activation of calcineurin (Newton et al., 1984) and it was found to have no effect. The fact that neither TM1 nor TM2 alone can form a stable complex with either the skMLCK peptide or the CaMKI peptide in native gel bandshift assays (Figure 5.3~)seems to indicate that these fragments probably could not act as potent CaM inhibitors. Apart from these studies, there are plenty of other opportunities for future research with these thrombic fragments of calmodulin. Although peak broadening due to exchange problems likely renders determination of the three-dimensional structure of Ca2+-TMI:TM2 or Ca2'- TM1:TMa:peptide complexes impossible by triple-resonance NMR methods (see above discussion of 'H, 13C HMQC spectra), it may be possible to learn more about the dynamic stability of these structures through NMR studies. For example heteronuclear relaxation studies of "N-labeled proteins can yield information about the dynamics of protein backbones (for examples, see Kay et al., 1989; Barbato et al., 1992; and Chapter 6 of this thesis), which could be used to determine the strength of TM1:TMB interactions. Moreover, there exists the possibility of creation of other fragments of calmodulin through a combination of thrombic cleavage and other means. For example, the cleavage of CaM by thrombin at Arg37- Ser38 has already been discussed (Shea et al., 1996). In one instance in my hands where thrombic cleavage of CaM was allowed to proceed too long, Thrornbic -fragments of calmodulin 214 the 1-37 fragment of CaM was created; amino acid sequencing (performed by Dr. Don McKay, data not shown) revealed that this fragment copurified with TM2 by gel filtration. Due to the differences in negative charge this fragment was easily separated from TM2 by DEAE-Sephadex chromatography using 50 mM Tris-HC1, pH 7.4 as the buffer and a 0-0.5 M NaCl gradient (not shown). In addition, fragment 1-37 could also be created by cleavage of recombinant TRlC, the N-terminal tryptic half- molecule of CaM, to produce two fragments (1-37 and 38-77). Studies of complementation of these fragments could prove interesting. Not only is the site of thrombic cleavage in the N-terminal domain closer to the linker region between the two helix-loop-helix domains, but also the weaker binding of Cat+by the two N-terminal sites may mean that the association equilibrium between the two fragments could be different. Finally, TRPC (78-1481, the other half-molecule of CaM, can be made by tryptic cleavage of whole CaM. TR2C can in turn be cleaved by thrombin to create two smaller fragments (78-106and 107-148). Thus, all four single EF helix- loop-helix domains can be created through different combinations of proteolytic cleavage (see Figure 5.1). However, given their very similar properties, it may be a challenge to purify these fragments. Ion-exchange chromatography methods provide the most promise for their separation. Although the study of the four single domain fragments would certainly be interesting, what is most interesting about TM1 and TM2 is that the TM1 fragment still has an intact N-terminal domain. However, it was found by native-PAGE analysis that TM1 was not able to bind CaM- target peptides alone; it needed TM2 to form such a complex. Thus, our results not only demonstrate the importance of having two interacting EF- hand domains for proper metal ion binding, but also having two EF-hand Thrombic fiapments of calrnodulin 215 pairs for proper binding of target peptides. The two pairs, located on separate lobes in CaM, act co-operatively to bind target molecules. Thus, simply through a series of gene duplication events, nature was able to create a protein with considerable internal homology that has tight but reversible Ca2+-bindingproperties and a vastly important physiological function. Llynamics of the central linker of CaM 216

CaAPTER SIX The backbone dynamic properties of the central linker region of calmodulin in 35% trifluoroethanol

ABSTRACT.

The backbone dynamic properties of uniformly ISN-labeled Ca2+- saturated calmodulin (CaM) in 35% 2,2,2-trifluoroethanol (TFE)have been examined by nitrogen-15 NMR relaxation methods. This particular solvent was chosen in order to mimic the conditions in which CaM was crystallized, which included the presence of alcohols. Special attention was paid to the central linker region of CaM, which is a long, solvent- exposed a-helix in the crystal structure but is known to be quite flexible in solution. lSN TI, T2, and 15N{'H}NOE values were determined for both CaM in H,O and CaM in 35% TFE, and the results indicated that the presence of 35% TFE did indeed induce a more ordered, stable conformation in the central linker, with order parameters for Asp78-Glu80 of 0.29, 0.17, 0.27 in H,O and 0.82, 0.66, 0.64 in 35% TFE, although LSN(lH} NOE values showed that these residues were still slightly more flexible than the rest of the molecule in 35% TFE (Asp78-Glu80 lSN(lH)NOE = 0.46, 0.46, 0.51) . However, there is still independent motion of the two lobes of CaM in 35% TFE, with motional correlation times of 10 ns and 9 ns for the N- and C-lobes, respectively, indicating that 35% TFE was not sufficient to force CaM into a rigid, dumbbell-shaped molecule, like that seen in the crystal structure. The central linker could be additionally stabilized in the crystal structure by other factors, such as crystal Dynamics of the central linker of CaM 217 packing, temperature, or pH.

INTRODUCTION.

The crystal structure of calmodu!in (CsM) caused some debete when it was first released (Babu et al., 1985, 1988). A prominent feature of the structure is the long, eight-turn a-helix in the center of the molecule, the middle portion of which is completely solvent exposed and has no contacts with the rest of the protein. Many investigators in the field had a hard time believing such a helix could exist (Kretsinger, 1992). As a matter of fact, Klee et al. (1983) had earlier concluded, through sequence comparison of CaM with other EF-hand proteins, such as the previously solved structure of parvalbumin (Kretsinger and Nockolds, 1973), that the central portion of the linker region of CaM was probably unstructured. The same differences between the predicted md the observed crystal structures were also found for the highly homologous muscle protein troponin C (TnC) (Sudaralingam et al., 1985; Satyshur et al., 1988; Herzberg and James, 1988). At the time, the unusual central helix was thought to be functionally important for these proteins, and that binding of target molecules to CaM could actually stabilize this area. In time, this proved to be incorrect. In fact, it is the flexibility of this linker that is most important for the function of CaM, as well as TnC. The structures of CaM- target peptide complexes (Ikura et al., 1992; Meador et al., 1992, 1993) reveal that the central linker of CaM expands and unwinds as CaM %rapsn around its target molecule, forming a globular complex. Although X-ray crystallographic structures of Ca2+-CaMdepicted the central linker as a-helical, significant deviations from ideal a-helical Dynamics of the central linker of CUM 218 geometry were noted even in these structures. In the structure of Babu et al. (1988), there is an distinct kink in the central linker as the $, y angles for residues Thr79-Asp81 are not those for a uniform a-helix. As well, the temperature factors for residues 75 to 86 are quite high, denoting flexibility in this sea. The stmctme of Chattotopadhyaya ct a!. (1992) has 3 straighter, more ideal a-helical central linker and the temperature factors for this region are lower, although still quite high compared to the rest of the molecule. The amino acid sequence for residues 75 to 86 of CaM is 75KMKDTDSEEEIR86.There are two helix-stabilizing salt-bridges in this sequence in the structure of Chattopadhyaya et al. (1992): one weak one between Lys75 and Asp78, and a hydrogen-bonding interaction between GluS2 and Arg86. However, when one examines the helix-forming propensity of this sequence according to the rules of Chou and Fasman (19781,Asp78 to Serdl are all weak or indifferent helix formers. Following their guidelines, having four residues of this nature in a row would break an a-helix. Other determinations of helix stability (Chakrabartty and Baldwin, 1995; see also Kretsinger, 1992) also come up with similar results. The overall shape of the CaM molecule in solution was first examined by small-angle X-ray scattering (SAXS). Initially, Seaton et al. (1985) found a radius of gyration and a maximum length for Ca2+-CaM that is consistent with the dumbbell-shaped model of the crystal structures. However, Heidorn and Trewhella ( 19881, through additional experimentation and more rigorous data analysis, determined parameters that suggested the central helix was bent. Apart from SAXS, other methods, such as NMR spectroscopy, have also been used to show that the central linker of CaM is flexible in solution. Through heteronuclear 2D D~arnicsof the central linker of CUM 219 and 3D NMR, Ikura et al. (1991) concluded that residues 78-81 adopted a "nonhelical conformation with considerable flexibility." In a landmark paper, Barbato et al. (1992) used emerging nitrogen-15 NMR relaxation studies, determining I5N Tls, T~s,and 15N{1HlNOES, to probe the backbone dynamic properties of CRM. The techniques, first employed or? the mode! protein staphylococcal nuclease (Kay et al., 1989) use uniformly nitrogen- 15 labeled proteins and give information about individual residues, including relaxation times and 15N-heteronuclear NOES. These values are then interpreted through computational methods such as the "model-freen approach (Lipari and Szabo, 1982b) to yield information such as order parameters and motional correlation times for individual residues. All parameters (Barbato et al., 1982) indicated that, for CaM, the central linker, through residues 77 to 81, was highly mobile and, moreover, the overall correlation times of the N- and C-terminal lobes of the protein indicated that they tumbled independently and were thus not linked by a rigid a-helix. In molecular dynamics simulations (van der Spoel et al., 19961, the isolated central helix of CaM quickly degenerated to a random- coil conformation, showing the intrinsic instability of the sequence. The functional significance of the flexible central linker of CaM is demonstrated in the structures of complexes between CaM and CaM- binding target peptides (Ikura et al., 1992; Meador et al., 1992,1993). Upon binding a target peptide, the central helix of CaM unwinds as CaM "wrapsn around its target molecule. Importantly, the central linker can unwind to varying degrees in order for CaM to position itself to ensure maximal interaction between the two hydrophobic patches of the protein and the hydrophobic face of the amphiphilic target sequence. For example, residues 73-83 are disordered in the crystal structure of CaM with a CaM- Dynamics of the central linker of CaM 220 dependent protein kinase I1 peptide (Meador et al., 19931, residues 73-77 are disordered in a complex of CaM with a smooth muscle myosin light- chain kinase (MLCK) peptide (Meador et al, 19921, and in a complex of CaM with a skeletal muscle MLCK peptide (Ikura et al., 1992; Clore et al., 1993). hlnide hydrogen exchange rates for residues 75 to 79 of Ca2+-CLV increase upon complexation with a MLCK peptide (Spera et al., 1991). This has led to the central helix being called an "expansion jointn (Meador et a1 ., 1993), or a "flexible tether" (Persechini and Kretsinger, 1988; Kataoka et al., 1996). The tight bending of the central helix in some target complexes is evident in studies of modifications of CaM. For example, chemical cross-linking of the N- and C-terminal lobes of CaM to create a compact, globular structure did not affect CaM's ability to activate myosin light-chain kinase (Persechini and Kretsinger, 1988), although it did affect the activation of some other enzymes (Persechini et al., 1993). Deletions of a few residues in the central helix also had little effect in activating CaM- dependent enzymes (Persechini et al., 1989; VanBerkum et al., 1990), although for some enzymes, such as phosphodiesterase, an extended conformation of the central helix may be required for activation (VanBerkum et al., 1990). Insertions and proline mutations in the central linker also had little effect on Cahrfs activity (Putkey et al., 1988). Given the fact that the central linker of CaM is flexible in solution, and that the lack of helicity in this region is in fact important for CaM's activity, it is interesting to find out why CaM has a rigid central a-helix in the X-ray crystal structure. This possibly has to do with the conditions of crystallization, which include low pH and the presence of alcohols. The original conditions in which CaM was crystallized (Cook and Sack, 1983) consisted of 50% 2-methylpentane-2,4-diol(MPD) and a pH of 5.6. Other Dvnamics sf the central linker of CaM 221 conditions (Chattopadhyaya et al., 1992) contained a combination of MPD and ethanol at a pH of 5.0. CD spectroscopy has unequivocally shown that the addition of MPD to aqueous solutions of CaM increases the a-helical content of the protein (Bayley et a1 ., 1988, Torok et al., 1992, Bayley and ?dartin, 1992). As a matter of fact, the Ca2+-CaMcrystal structure of Chattopadhyaya et al. (1992)is actually the result of a failed attempt to co-crystallize CaM with the M13 target peptide from skeletal muscle myosin light-chain kinase. Given that the crystallization conditions they used are known to stabilize the central linker, and that flexibility of the linker is required for target peptide binding, it is no surprise that the researchers were unable to crystallize the CaM:M13 complex. In crystallizing the CaM:CaMKI and CaM:smMLCK complexes, Meador et al. (1992, 1993) used polyethylene glycol as a precipitant rather than MPD. As demonstrated by circular dichroism spectroscopy (Bayley and Martin, 19921, other alcohols, such as ethanol and 1-propanol also have an effect in stabilizing helical structures within CaM, and 2,2,2-trifluoroethanol (TFE)had a greater effect than MPD, and at lower concentrations. In a wide variety of systems, alcohols, especially TFE, have been shown to stabilize secondary structures in polypeptides (Sonnichsen et al., 1992; Luo and Baldwin, 1997; Hirota et al., 1998; Buck, 1998). The mechanisms for helix stabilization by TFE are not fully understood, but are thought to be mostly due to stabilization of hydrogen bonds within the polypeptide backbone (Luo and Baldwin, 1997; Buck, 1998). Apart from the alcohols, the low pH of the crystallization media for CaM may also play a role in stabilizing the central helix. This presumably has something to do with protonation of some of the many acidic groups in the central linker (there are two aspartates and three glutamates in Dynamics of the central linker of CaM 222 residues 78 to 84 of CaM), which can reduce charge repulsion and promote helix formation. Low pH and the presence of alcohols can have a synergistic effect on increasing the helical content of CaM (Torok et al., 1992; Bayley and Martin, 1992). Moreover, the temperature at which crystals are formed may also be responsible for stabilizing the central helix. There is a significant thermal transition that occurs in CaM between 25 and 35 "C (Tsalkova and Privalov, 1985; Wintrode and Privalov, 19971, which is seen by CD spectroscopy as a decrease in the apparent helical content of the protein (Martin and Bayley, 1986; Bayley, 2000). This chapter describes a comparison, using the lSN-NMR relaxation techniques of Barbato et al. (19921, of the backbone dynamics of CaM in aqueous solution to those of CaM in a TFE:water mixture that mimics the crystallization conditions. The results indicate that, TFE does stabilize the central linker of CaM, although the presence of TFE alone is not sufficient to make the central helix completely rigid. Instead, the stability of the central helix in the crystal structure of CaM may be due to the combination of alcohols with low pH, crystal packing, or other factors.

MATERIALS AND METHODS.

Materials. The Escherichia coli strain MM294 was obtained fkom the E. coli genetic stock center at Yale University. The pCaM expression vector was a gift from Dr. T. Grundstrom (University of Umed, Sweden) and has been described elsewhere (Chapter 2; Waltersson et al., 1993; Zhang and Vogel, 1993a). 2,2,2-trifluoroethano1 (TFE) was obtained from Aldrich. Dynamics of the central linker of CaM 223

Amrnoni~rn-~~Nchloride ("N, 99%), D,O (D,99.9%), and methylene-d,-TFE (D,98%) were obtained from Cambridge Isotope Laboratories, Cambridge, Mass. All other reagents were acquired &om reputable sources.

Nitrogen-15 itnifonn labeling of CaM. Uniformly lSN-labeled CaM was expressed according to the procedure of Zhang and Vogel (1994b)with some modifications. Initially, a 2 mL LB-ampicillin culture of MM294-pCaM was inoculated from a fresh plate and allowed to grow at 30 OC for 12 h. One mL of this culture was used to inoculate 50 mL of an M9-based minimal medium (6 gfL Na,HPO,, 3 gLKH1P04, 0.5 gLNaC1, 1 mM MgSO,, 0.1 mM CaCl,, 0.01 mM FeCl,, 5 glL glucose, 2 g/L lsNH,Cl, 50 mg/L ampicillin) and this 50 mL culture was grown at 30 OC for Q1/,h. The 50 mL culture was used to inoculate 500 mL of the M9 medium, which was grown at 30 OC until the OD,,, reached 0.45 1 h. Then the temperature was increased to 37 "C for 1 h (OD,, = 0.85) to cause runaway production of the pCaM plasrnid, after which IPTG (to 0.5 mM) was added and cells were shaken for a further 7 h to express the protein. The uniformly 'SN-labeled CaM was purified according to the methods for other CaMs (Chapter 2). Determination of the percent incorporation of nitrogen-15 into the protein was performed by electrospray-ionization mass spectroscopy (Dr. Jim Chen I Dr. Gilles Lajoie, University of Waterloo).

NMR spectroscopy. For NMR spectroscopy, two lSN-labeled CaM samples (0.5 mL, 5% D,O, 1.5 mM CaM, 100 mM KC11 were prepared, one in water and one with 35% (voVvo1) TFE. The pH of each sample was adjusted to 6.3 with dilute Dynamics of the central linker of CaM 224 solutions of KOH and HC1; pH was measured with a thin-stem pH electrode and was not corrected for the isotope effect. Any precipitate or solid material was removed by microcentrifugation before placing of the sample in an NMR tube. This was especially important for the TFE sample. Acid-washed glassware md plasticware ?ves used thoughout tc remove contaminating paramagnetic species. 15N, lH heteronuclear single quantum coherence (HSQC)spectra were acquired on Bruker AMXSOO (University of Calgary) and Varian Unity 600 (University of Groningen, Netherlands) spectrometers according to the method of Wider and Wiithrich (1993). In Groningen, HSQC spectra consisted of four scans with 2048 ('H,F2) by 256 V5N,F1) data points and sweep widths of 8000 (F2) and 2500 (Fl)Hz. An HSQC spectrum was taken at 47 OC in order to match the peaks with the chemical shifts of Ikura et al. (1990). Then, a temperature titration was performed; further HSQC spectra were obtained at decreasing temperatures in order to assign the peaks at 35 OC which enabled comparison with the data of Barbato et al. (1992). The '5N{'H}NOE was measured at 600 MHz for both the CaM in water and the CaM in 35% TFE samples using the sequences of Peng and Wagner (1992) and Barbato et al. (1992). Spectra were 64 scans, and a delay of 5 s between scans was implemented to ensure complete relaxation. The data for the NOE at 600 MHz was collected as two interleaved spectra which were then separated to create two spectra with 2048 (F2) by 1024 (Fl) data points. Data collection was complex in both dimensions for the spectra acquired at 600 MHz. Nitrogen45 T1 and T2 values and lSN(lH}NOES were collected at 500 MHz in H,O and in 35% methylene-d,-TFE using published pulse sequences (Kay et al., 1989; Barbato et al., 19921, with sweep widths of Dvnamics of the central linker of CUM 225

6579 ('H, F2) by 1923 (15N,F1) HZ,a data matrix of 2048 (F2)by 256 (Fl) points, and 32 scans per increment. A T1 experiment consisted of six spectra with T1 delays of 26, 82, 138, 306, 502, and 852 rns (data collected in both water and TFE), and another T1 experiment of five spectra consisting of delays of 26, 96, 180, 404, and 922 ms, was collected in TFE only in order to have more points on the relaxation curves. T2 experiments consisted of seven spectra with T2 delays of 7.65, 22.9, 45.9, 68.8, 107.1, 160.6, and 229.4 ms. Each TI spectrum ranged between 3.5 and 6 h, and each T2 spectrum ranged between 3.5 and 4.1 h, depending on the lengths of the variable delays. Delays between scans were 2 s for both the T1 and T2 experiments. The 500 MHz 15N('H)NOES were collected in two separate spectra, one with the NOE and one without the NOE effect. Delays between scans for both spectra were 5 s to ensure complete relaxation, and 64 scans were collected per increment. Data collection for all nitrogen-15 relaxation spectra at 500 MHz was complex in the F2 dimension and used the TPPI method (Marion and Wiithrich, 1983) in the Fl dimension. The 15N and NH chemical shift values for CaM in 35% TFE at 35 "C were determined by performing a TFE titration; small amounts of the TFE sample were sequentially transferred to the water sample and vice versa (i.e. transfer a small volume fkom each to the other to create a 1%TFE and a 34% TFE sample, then another transfer to create a 2% TFE and a 33% TFE sample, and so on) and HSQC spectra at 600 MHz were acquired for each sample. TFE concentration was accurately determined by obtaining a simple 1D 'H spectrum of the sample without water presaturation, determining the ratio of the TFE methylene peak area to the water peak area, and calculating the percent TFE by multiplying a density conversion Dvnarnics of the central linker of CaM 226 factor. All 2D spectra were processed with the NMRPipe processing program (Delaglio et al., 1995) and viewed with the accompanying NMRDraw graphics package. For determination of the 600 MHz lSN{lH) NOE, peak heights were used, due to the fact that peek overlap often made integration of peak volumes of difficult. By analogy to the results of Barbato et al. (19921, the NOE values were calculated as the ratio of the intensities of the irradiated spectrum to the unirradiated spectrum, i.e. NOE = irradiated/unirradiated. In this way, NOE values for 15N range from 1 to - -3. Within a protein values generally range between 0 and 1, with 1 being the total absence of any NOE enhancement, and lower values meaning a greater effect. For the T1 and T2 series and for the 500 MHz "N{'H) NOE spectra, peaks were picked and peak volumes were calculated using the pck toolkit in the NMRDraw software. Then, the volumes of each peak in the series of spectra were fitted and compared using the nlinLS program, which is also part of the NMRPipe-Draw package. Base widths for peak fitting were 10 (F2) by 6 (Fl)points; shell scripts for the fitting routine were supplied by Peter M. Hwang (University of Toronto). Spectra were first divided into 4 or 5 manageable pieces, with 10 points of overlap between pieces, before the fitting was undertaken, and the program was run of Silicon Graphics Indigo2 and Octane computers. From the set of relative peak volumes that was generated, relaxation times and lSN('H)NOE values were calculated using the lmquick relaxation data fitting program (Farrow et al., 1994). The set of nitrogen-15 Tls, T2s, and the '5N(1H}NOE values, both at 500 MHz and 600 MHz, were then all inputted to the tm-f77 program, generously supplied by Dr. Neil A. Farrow (Farrow et at., 1994), which uses the "model £reen approach of data analysis Dynamics of the central linker of CaM 227

(Lipari and Szabo, 1982a, b) to calculate overall correlation times (7,) and order parameters (S') for each residue. Errors in T1 and T2 supplied by lmquick were used for tm-f77; the NOE values were all given errors of 0.02 (-2.5%) (Barbato et al., 1992).

RESULTS.

The expression and purification of uniformly lSN-labeled CaM from a total of 550 mL M9-based media according to the procedure outlined in the Methods section yielded 90 mg of protein. This should be viewed as an excellent yield; typical yields from this procedure are around 25 mg. The mass spectrum of the protein is in Figure 6.1; incorporation of 15N was determined to be 100%; the molecular mass is 188 a.m.u. greater than CaM at natural abundance, and there are only 188 nitrogen atoms in CaM. The degree of incorporation is also seen to be extremely good as indicated by the quality of the NMR spectra (see below). The 15N, 'H HSQC spectrum of uniformly 15N-labeledCaM in H,O at

35 OC is shown in Figure 6.2. 142 of the 144 main-chain amide resonances (the two proline residues as well as the N-terminal residue do not have any amide protons), with the exception of Asp2, Val108, and Leull2, were assigned for the protein at 35 OC, given the assignments at 47 "C (Ikura et al., 1990), and Figure 2a of Barbato et al(1992). Of these 142 amide peaks, 141 'SN{lH] NOES were determined (signal overlap made an NOE determination for Glu104 unreliable). The I5N, 'H HSQC spectrum of uniformly lSN-labeled CaM at 35 "C in 35% TFE is shown in Figure 6.3. 105 main-chain amide resonances were successfully assigned for CaM in 35% TFE. Of these 105 peaks, NOES were obtained for 92 resonances in Dynamics of the central linker of CaM 228

Figure 6.1.

Figure 6.1. Electrospray ionization mass spectrum (ESI-MS) of 'W - labeled CaM (Dr. Gilles Lajoie, University of Waterloo). Dynamics of the central linker of CUM 229

Figure 6.2. a) Dvnamics of the central linker of CaM 230

Figure 6.3. Dynamics of the central linker of' CaM 23 1

Figure 6.2. (two pages previous) 600 MHz I5N, 'H HSQC spectrum of Ca2+- saturated 15N-CaM(1.5 mM)in H,O, 100 mM KCl, pH 6.3,35 "C,a) entire spectrum, and b) window of most crowded region. Assigned peaks are numbered with their residue numbers.

Figure 6.3. (previous page) 600 MHz I5N. 'H HSQC spectrum of CaZ+- saturated 15N-CaM(1.5 mM) in 35% TFE, 100 mM KCI, pH 6.3, 35 OC. The windows a) and b) are as before.

Figure 6.4. a)

time (s) Dvnamics of the central linker of CaM 232

Figure 6.4. b)

time (ms)

Figure 6.4. a) T1 decay cwes and b) T2 decay curves of four residues of the CaM sample in 35% TFE representing the complete range of relaxation times. The curves are the result of fits to exponential decay functions which were obtained using Cricket Graph 111 running on a Power Macintosh G3 350 MHz personal computer. Dynamics of the central linker qf CaM 233 the TFE sample (63% of the protein). Most of the problems resulted from spectral overlap in the original water spectrum, which made monitoring of the migration of peaks difficult once TFE was added. In particular, residues in a-helical regions of CaM (especially helices 6 and 7) were most difficult to assign in 356 TFE. In the important central linker region (Lys75 to Arg861, NOEs were found for all but Met76, Ser81, and Ile85. The 15N-T1 decay curves for four representative residues of CaM in TFE, Glu31, Phe65, Thr79, and Leull6, are shown in Figure 6.4a, and the T2 decay cwes are shown in Figure 6.4b. Glu31 is a residue with some of the shortest relaxation times, while Thr79 (in the central helix) and Leu116 (in the linker between domains 111 and IV) are two residues with the longest relaxation times, and Phe65 has Tls and T2s of average length. The results agree well with the fitted exponential decay curves, which are also plotted. The 'W-Tls, the "N-T2s, the lSN{lH)NOEs, and the calculated motional correlation times (T,), order parameters (S2), and correlation times of internal motion (re) are shown by residue for the sample of 15N- CaM in H,O in Figure 6.5a and the values for the sample in 35% TFE are shown in Figure 6.5b. As well, the values under both conditions are superimposed as depicted in Figure 6.5~. The data in water agree well with the results of Barbato et al. (1992) in that the residues in the central linker region (Lys77-Glu83) and the residues in the domain 111-domain IV linker (Lysll5 and Glu116) have longer Tls and T2s and larger NOE enhancements, resulting in decreased order parameters in this region. The Tls are consistently longer in the TFE sample than in the water sample, and the T2s are consistently shorter. This results in the calculation of longer motional correlation times for residues in the TFE Dyzarnics of the central linker of CaM 234

Figure 6.5. a)

TI (ms) 600

T2 (ms) 100

0.75 NOE 0.5 0.25

50 75 100 125 Residue number Dynamics of the central linker of CaM 235

Figure 6.5. b)

T2 (ms) 100

0.5 NOE

25 50 75 100 125 Residue number Dynamics of the central linker of CUM 236

Figure 6.5 c)

0.5 NOE

50 75 100 125 Residue number Dynamics of the central linker qf CaM 237

Figure 6.5. Nitrogen-15 relaxation data for Ca2+-saturatedlSN-CaM a) in water, b) in 35% TFE, and c) both in water and in 35% TFE. In a) and b) the open symbols are the '5N(1H}NOE values at 600 MHz; the other data were collected at 500 MHz. In c) the filled symbols are for CaM in water and the open symbols are for CaM in 35% TFE (the '5N(1H}NOE values are at 500 MHz).

Table 6.1, a) IN WATER

1 CaM N (4-75) C (82-148)

T~ (dl) 7.7k1.8 N 85 8.3k1.8 N 43 6.9k1.3 N 38 T~ (threshold) 7.7k1.3 M 62 8.0k1.2 N 33 6.7k0.9 N 29 i b) IN 35% TFE r CaM N (4-75) C (82-148)

2, (all) 9.6i1.3 N 83 10.0*1.0 N 46 9.1k1.6 N 34 r, (threshold) 9.6k0.9 N 62 9.9i0.6 N 33 9.0k0.8 N 24

Table 6.1. Motional correlation times for CaM and the amino- and carboxy-terminal domains of CaM in water and in 35% TFE. Values were calculated from lSN-relaxationdata using tm-f77 (Farrow et al., 1994). The values for the N- and C-domains were calculated separately by using the data for the indicated residues. The designation "all" refers to all peaks used, whereas "threshold" refers to the r, calculated separately using only data whose values for their T1/T2 ratios were less than one standard deviation from the average TUT2 ratio. The number of residues used for each calculation is specified. Dvnamics of the central linker of CaM 238 sample. The global motional correlation times for the two samples are shown in Table 6.1; as well, the global motional correlation times were calculated separately for the N- and C-domains. There is also more scatter in the data for the T1 values in TFE than in water, even though there were ten delays used for these results, as opposed to six for the water sample. What is significant is that there are no large differences between the Tls and T2s for the central Iinker and the rest of the molecule for the TFE sample. There is a significant change in NOE enhancement for the central linker residues in the TFE sample, but not as large as those in the water sample (c.f. 600 MHz NOE of 0.25 for Asp78 in H,O and 0.46 for Asp78 in 35% TFE). The NOES in the linker between Ca2+-binding domains III and IV are also low (0.26 for Lysll5 and 0.40 for Leu116 at 600 MHz) for CaM in water, similar to Barbato et al. (1992). The NOE values for CaM in 35% TFE also indicate flexibility in this region (600 MHz 15N{'H}NOE = 0.25 for Lysll5 and 0.15 for Leull6). NOE values indicate that the linker between Ca2+-bindingdomains I and 11 of CaM (residues 40- 44) also seems to be slightly more flexible than the rest of the molecule, both in TFE and in H,O, but the difference is not as significant as for the other linker region. From the order parameters, it appears that the central linker is more ordered in the TFE sample than in the water sample (Asp78-Glu80 S2=0.29,0.17, 0.27 in H20,0.82,0.66, 0.64 for 35% TFE).

DISCUSSION.

The backbone dynamic properties of CaM in water and in 35% TFE were compared by nitrogen-15 relaxation methods in order to determine the effects of organic solvents on the flexibility of the central linker. TFE

C Dynamics of the central linker of CaM 239 was chosen as the solvent of choice in an attempt to mimic crystallization conditions in the context of an NMR experiment. 2-methylpentme-2,4-diol (MPD) was not used as a cosolvent because it is quite viscous, which is extremely undesirable in NiMR experiments. Also, a higher concentration of MPD relative to TFE is required to achieve the rr?aximd incre~sein cr- helicity of CaM (Bayley and Martin, 1992). It is important to note that the phenomena of protein crystallization and protein precipitation are closely linked, and so it has proved difficult to mimic the exact crystallization conditions in an NMR sample while at the same time maintaining a high enough sample concentration to make NMR experiments feasible. With TFE we were able to obtain satisfactory results at a moderate concentration of cosolvent, while still maintaining a high protein concentration. Since the effects of TFE on the secondary structure of CaM seem to be complete at 30% TFE (Bayley and Martin, 1992) we chose a 35% concentration to ensure the full effect in the NMR sample. TFE was also preferred as the cosolvent because the properties of it as a protein secondary structure stabilizer and its properties in NMR experiments are well understood (Buck, 1998) compared to MPD, which is used more generally as a precipitant in protein and nucleic acid crystallizations. The spread of the peaks in the 15N, 'H HSQC spectrum of CaZ+- saturated CaM in 35% TFE (Figure 6.3) is similar to that in the spectrum in H20(Figure 6.2). Due to spectral overlap, and the fact that the different spectra varied slightly, not all of the peaks gave reliable Tls, T2s, 500 MHz NOEs, and 600 MHz NOEs. The T1 and T2 decay curves that were obtained were quite reliable and fitted well to exponential decay functions (Figure 6.4). Thus, peaks for 85 residues in water and 83 residues in TFE were successfully inputted into the tm-f77 fitting program. The 15N Dynamics of the central linker of CaM 240 heteronuclear relaxation results of CaM in H20compared well with the data of Barbato et al. (1992). The central linker region is considerably more flexible and disordered than the rest of the molecule, and the two halves of the molecule behave as if they tumble somewhat independently. A lo~germotiond comelation time for the N-teAmhallobe of the molecule as opposed to the C-terminal lobe was also observed. Table 6.1 shows 7, values of -8.0 and -6.7 ns for the N and C-terminal lobes respectively in water while Barbato et al. (1992) report slightly shorter values of -7.1 and -6.3 ns. It is unclear why this is the case, because precautions were made to use the same conditions with respect to sample pH, ionic strength, and temperature, and the TI, T2, and NOE data themselves are quantitatively similar in the two studies. The diEerences could perhaps just be due to the different computational methods used or perhaps slight differences in temperature calibrations. The real goal of the work in this chapter was not to simply repeat the report of Barbato et al. (1992) but rather to use their methods to determine the effects of TFE on the dynamics of CaM. The lSN{lH}NOE values (Figure 6.5) seem to show that there is a significant amount of flexibility still in the central linker, but it is not to as great an extent as for CaM in H,O. There is very little difference in the T1 or the T2 values of the central linker compared to the rest of CaM. Moreover, the order parameters for the central linker region in 35% TFE are not sigdicantly different from the rest of the molecule. However, when calculated separately, the correlation times for the N- and C-lobes of CaM in 35% TFE are significantly different from each other, as they are in H20, indicating that the molecule is probably not rotating as a rigid dumbbell. The calculate motional correlation times for the TFE sample are longer than Dynamics of the central linker of CUM 241 those in water, but this could be attributed to viscosity effects (see below). Thus, TFE seems to have successfully induced a more stable, ordered structure than is present in water, although it is perhaps not as stable and well-defined as other a-helical regions. of CaM. ,Although the centrd helix is not ss different from the rest of the molecule when CaM is dissolved in 35% TFE, there are significant differences in the 15N relaxation data between CaM in 35% TFE and CaM in water. The Tls are more scattered and significantly longer, and the T2s significantly shorter, in the TFE solution. The difference is also shown in the correlation times for CaM in 35% TFE, which are significantly longer (Table 6.1). The large amount of scatter in the TI values is difficult to explain, but there is a potential explanation for the discrepancies in the Tls and T2s for CaM in 35% TFE relative to water. Mixtures of TFE and water are significantly more viscous than water alone. According to the fourth-order polynomial approximation of Shuck et al. (1998),the viscosity of 35% TFE is 1.67 times that of water. Although their experiments were carried out at 20 "C, the relative difference in viscosity between 35% TFE and water is probably similar at 35 "C. Since 2, varies with viscosity q by the relationship

(Cavanagh et al., 2000), where r, is the hydrated radius of the protein and k, is Boltzmann's constant, and assuming that all other variables remain the same (not necessarily true; if TFE does cause the central helix to stiffen then r, would most certainly increase), then r, varies directly with Dynamics of the central linker of CaM 242 q. So, a 1.67-fold increase in q would result in a 1.67-fold increase in T,. This is not what is observed; in fact, the calculated 7, for CaM in 35% TFE is only about 1.25-fold of that in water. Therefore, other factors besides the sample viscosity must be different in 35% TFE relative to water. Perhaps the redius of hydration of CLW is decreased in 359 TFE relative to that in water. This could be quite likely, since the protein could be hydrated less in a mixed solvent with less polar character than water. Given that r, varies with r,3, it would not take much of a difference in r, to result in a change to the 5,. What most certainly did not occur here, however, is an increase in r,. If 35% TFE had been able to completely stiffen the central helix, this would have resulted in an effectively larger CaM molecule (the two lobes of the protein would no longer tumble independently), resulting in a greater r,. So, although the 15N NMR relaxation data indicate that the central linker of CaM is less flexible, more ordered, and more similar to the rest of the molecule in 35% TFE than it is in H,O, this stabilization is not sufficient to cause the formation of a rigid, dumbbell-shaped CaM molecule. An TFE-dependent increase in the a-helicity of CaM has been observed previously by far-UV CD spectroscopy. In the study of Bayley and Martin (1992), TFE significantly enhances the negative ellipticity of CaM at 222 nm, indicative of additional a-helix formation, an effect which seems to be fully complete at around 30% TFE. Because far-UV CD is only an indicator of global changes in secondary structure, it is unclear if TFE does indeed stabilize the central linker of CaM. The increased ellipticity may be due to increased stability of a-helices in other parts of the protein, but the most logical conclusion of the CD results is that the central linker becomes a-helical in TFE. On the other hand, perhaps TFE induces helix Dvnamics of the central linker of CaM 243 formation in the central linker, but this helix remains flexible, accounting for the 15N('H}-NOEresults and the short correlation times for CaM in 35% TFE. It is also important to remember that, for CaM and related proteins like troponin-C (Gagne et al., 1994), increases in CD ellipticity do not necessarily indicate increases in a-helicity, but rather are dso possibly the result of changes in the relative alignment of a-helical elements. Given that TFE can often disrupt tertiary interactions, causing the formation of partially unfolded states (e.g. Buck et al., 19961, it is quite plausible that the orientation of the helices in CaM is different in 35% TFE than it is in water. There is perhaps another explanation: in another comparison of NMR and CD spectroscopy (Mufioz et al., 19951, helical populations of peptides in 30% TFE were overestimated by CD relative to NMR, whereas the opposite applied for aqueous solution. This study used homonuclear NOEs and a proton conformational shifts but not 15N{'H} NOE data in determining helical conformations by NMR, but it still might explain the NMR data for calmodulin. Other researchers (for example, Kemmink and Creighton, 1995) have found that CD can perhaps underestimate helical content. Clearly, this issue could be explored further. What would be most interesting would be to determine if there are any helical structures in the central linker of CaM in 35% TFE. With the 15N and amide proton assignments for the majority of the residues in TFE already in hand, one could use doubly I3C and '3 labeled protein to assign the rest of the nuclei for the residues in the central helix. Then it could be determined if medium-range NOEs and CaH chemical shift digereaces indeed indicate a helical structure for the central linker of CaM in 35% TFE.. Another possible explanation for the inability of 35% TFE to Dynamics of the central linker of CUM 244 completely stabilize the central a-helix of CaM is that other factors, in addition to the presence of alcohols, account for the rigidity of the central linker in the crystal structures. The other main factor is sample pH. Uncornplexed Ca2+-CaMhas traditionally been crystallized at a pH of 5.0- 5.6 (Cook and Ssck, 1983; Chattopadhyaya et al., 1992). A lo^ pH s~chas this may cause protonation of some of the many acidic residues of CaM, leading to decreased local charge repulsions and the formation of more a- helical structures. Although the pK,s of Glu and Asp residues are normally lower than this (3.9 and 4.1, respectively), with a high local concentration of them such as in the central linker of CaM (5 out of 7 residues between 78 and 84 are acidic residues), it might be expected that the pys of some of the groups could be higher than normal. Indeed, Wang (1989)found that CaM becomes more elongated upon a pH decrease from 7 to 5. Both Bayley and Martin (1992)and Torok et al. (1992) found that low pH had a synergistic effect with the presence of alcohols in increasing the negative CD intensity of CaM at 222 nm, indicative of helical structures. The NMR experiments described in this chapter were all performed at a pH of 6.3; an NMR comparison of CaM in 35% TFE at a pH of 6.3 to CaM in 35% TFE at a pH in the range of 5.0-5.5 would be warranted. However, the solubility of CaM at pH values this low is extremely poor, making NMR experiments very difficult. This is the reason why all experiments in this chapter have been at pH 6.3. Moreover, they enabled a more direct comparison to the data of Barbato et at. (1992),which were also obtained at this pH. In addition to the pH differences, temperature may also have an effect. CaM crystals are generally grown at room temperature (22 "C or

SO) whereas the NMR data here, like the data of Barbato et al. (19921, were collected at 35 "C. There is considerable experimental evidence, including Dvnamics of the central linker of CaM 245

CD spectroscopy (Martin and Bayley, 1986; Bayley, 2000) and differential scanning calorimetry (Tsalkova and Privalov, 1985; Wintrode and Privalov, 19971, that a thermal transition occurs in CaM at temperatures in this range, which is quite likely to be the result of changes in the centrd linker. The stability of the central a-helix in the crystal structure of CaM may also be accounted for by contacts between adjacent CaM molecules in the crystal lattice. These contacts would then place the two lobes of CaM a certain distance apart, and the central linker may then be held in a more unfavorable a-helical conformation. Finally, it also important to remember that, although the central linker of CaM is attractively depicted as a uniform a-helix in the crystal structures (Babu et al., 1988; Chattopadhyaya et al., 19921, in reality the crystallographic data revealed this region to be mobile, with significant deviations from an ideal a-helical structure. So, in conclusion, even though the presence of 35% TFE can cause more stability and order in the central linker of CaM, the fact that it cannot sufficiently stabilize the central linker to create a rigid dumbbell- like molecule should not be surprising. Perhaps in the future the central linker of CaM could be used as a model of a solvent-exposed a-helix, and site-directed mutants of CaM with stable central linkers could be derived and studied through the techniques described in this chapter. Energetics of ne~tidebinding to CUM 246

CHAPTER SEVEN: Energetics of peptide binding to calmodulin studied by isothermal titration calorimetry

ABSTRACT.

The energetics of calmodulin (CaM)-binding by four target peptides, the CaM-binding domains from CaM-dependent protein kinase I (CaMKI) and constitutive nitric oxide synthase (cNOS), the CaM-binding domain "A" from 3', 5'-cyclic nucleotide phosphodiesterase (PDEa), and the model peptide melittin, have been studied by isothermal titration calorimetry (ITC). Through interpretation of the results some information about the mode of binding of these peptides to CaM has been derived. No protons are gained or lost as a result of CaMKI binding to CaM, which is relevant because CaMKI and CaM both contain a histidine residue. Performing titrations of the four peptides at different temperatures yields the heat capacity change upon binding (AC,), which relates to the degree of compactness of the complex. This has helped classification of the peptides according to the relative positions of their hydrophobic anchor residues. The four peptides all bind with a greater positive enthalpy (i.e. more endothermic) than the previously studied peptide from smooth muscle myosin light-chain kinase (Wintrode and Privalov, 1997, J. Mol. Biol. 266, 1050-1062). Thus, these four peptides could be considered more "typical" in that entropic factors, such as hydrophobic interactions, play a large role in the tight binding of these peptides to CaM. ITC has been shown here to Energetics of ~e~tidebinding to CaM 247

be a very powerful technique to study CaM-peptide interactions, and it complements other techniques, such as circular dichroism, fluorescence, and NMR, in revealing information about CaM-binding by peptides in which the structure of their CaM-bound complex is not known.

INTRODUCTION,

Calorimetry is a powerful technique when examining the stability of biological macromolecules and the binding interactions between biomacrornolecules and their ligands, because it yields a direct measurement of the heat involved in folding and binding reactions. Other techniques (e.g. circular dichroism, fluorescence, and NMR), while themselves being very powerful in their own right, only enable measurement of unfolding temperatures and equilibrium constants, and do not measure the enthalpies involved. With knowledge of the enthalpy of a reaction as well as the equilibrium constant, one can then determine the relative contribution of enthalpy and entropy to the reaction in question. The two main techniques in biological calorimetry are differential scanning calorimetry (DSC)and isothermal titration calorimetry (ITC). The techniques are discussed in a number of recent reviews (Ladbury and Chowdhry, 1996; Cooper, 1999; Jelesarov and Brosshard, 1999). In DSC, the difference in power required to heat two cells (the sample cell and the reference cell) is recorded as the instrument scans across a certain temperature range. If the sample cell contains a protein, and the scan temperature approaches the unfolding temperature of the protein, then the differential power will increase as more power is required to heat the sample cell. In this way, the thermal unfolding and refolding properties of Energetics ofpentide bindine to CUM 248 biomolecules and biomolecular complexes can be studied. It is extremely useful for examining the changes to the thermal stability of proteins in response to urea, pH changes, the presence of ligands, or mutations to the protein. ITC also measures the differential power between two cells. But, as the name isothermal implies, a constant temperature is maintained, and the difference in power required to maintain this temperature is recorded. A concentrated solution of a ligand is titrated into the sample cell which contains the protein. If the binding reaction of the ligand to the protein produces or consumes heat, the differential power between the sample and reference cells will change, and by recording the differential power during a series of injections along a titration the enthalpy of binding of the ligand to the protein can be measured. As well, given a proper set of starting conditions, a binding constant for the ligand to the protein can also be determined. It is ITC of peptides binding to calmodulin that is the subject of this chapter. ITC has been successfully used in determining the enthalpies of binding of several different types of ligands to proteins (Ladbury and Chowdhry, 1996; Cooper, 1999; Jelesarov and Brosshard, 19991, including nucleic acids, peptides, other proteins, and metal ions. The properties of metal ion binding to calmodulin, CaM fragments, and other related Ca2+ binding proteins have been studied by several groups (Sellers et al., 1991; Gilli et al., 1998; Yamada, 1999). In addition to protein-ligand interactions, ITC can also be used to examine the interactions between peptides and lipids (reviewed by Seelig, 1997). This demonstrates the power of ITC in providing data on the relative role of hydrophobic effects in biomolecular interactions; hydrophobic effects, being primarily entropically driven, will Energetics of w~tidebindine to CaM 249 not produce any heat and thus if a binding interaction is tight but produces little heat or even consumes heat, then it shows the importance of hydrophobic contributions in the binding interaction. In addition to hydrophobic contributions other parameters of binding can also be examined by perfor~ingdifferect series of ITC experiments. One such series is to perform a set of titrations in which the only parameter that is varied is the buffer used (the pH in each experiment remains the same). If protons are consumed or released, i.e. if ionizable groups on the protein and/or the ligand experience a pyshift, as a result of the binding interaction, then there will be an additional contribution to the measured heat due to binding of protons by the buffer species. By plotting the apparent enthalpy of interaction versus the ionization enthalpy of the buffer, one can determine the number of protons released or consumed in the binding interaction by measuring the slope of the plot (see e.g. Wintrode and Privalov, 1997; Seelig, 1997; Jelesarov and Brosshard, 1999). One can then determine which groups experience a pK, shift by performing mutations to the protein, changing the ligand, or, in the case of ligand-lipid interactions (Seelig, 1997), choosing a different lipid. ITC can also be used to measure changes in heat capacity as a result of the binding interaction. The slope of the binding enthalpy (measured in separate experiments at different temperatures) versus temperature gives the change in heat capacity, AC,, between the complex and the two dissociated species (Ladbury and Chowdhry, 1996; Wintrode and Privalov, 1997). Knowledge of the AC, value enables an estimate of the change in solvent accessible surface area (MA)as a result of binding, since AC, is related to the amount of exposure of a molecule to a solvent (Makhatadze Energetics of ~e~tidebindine to CaM 250 and Privalov, 1995; Jelesarov and Brosshard, 1999). Since complexation of a protein and a ligand most often results in a decreased surface area as a result of the ligand being buried inside the protein, then the slope of the plot is most often negative (i.e. as temperature is increased, the binding interaction becomes more exothermic). The steepness of the slope can give a relative estimate of how much the ligand is being buried, or if perhaps the binding of the ligand induces conformational changes in the protein such that the ASA is reduced even further (the classical "induced fit" mechanism). In the case of target peptides binding to calmodulin, the induced fit mechanism is not an accurate model, since changes take place both in CaM and, more substantially, in the target peptide. This has been revealed by the structural determination of three canonical CaM-peptide complexes (Ikura et al., 1992; Meador et al., 1992; 1993; see Figure 1.2). The target peptide, which is unstructured in solution, takes on an a-helical structure as it is bound to CaM. Ca2+-saturatedCaM, although being dumbbell-shaped in the crystal structure (Babu et al., 1988; Chattopadhyaya et al., 1992), in reality is comprised of two globular domains connected by a flexible linker (Heidorn and Trewhella, 1988; Barbato et al., 1992); this central linker unwinds as CaM envelops the target peptide, forming a globular complex. Importantly, the central linker of CaM unwinds to varying degrees as CaM binds different target peptides, resulting in the central linker being called an "expansion joint" (Meador et al., 1993). For example, CaM-binding regions can be classified according to the alignment of their hydrophobic "anchor" residues (Rhoads and Friedberg, 1997; Figure 11, which form contacts with the hydrophobic clefts on each lobe of CaM. When the anchor residues are closer together, Energetics of oe~tidebinding to CaM 25 1 the central helix unwinds to a greater degree enabling the two lobes of CaM to come closer together (Meador et al., 1993). Other results (Yao and Squier, 1996) suggest that, while the overall shape of different CaM- peptide complexes may be similar, tertiary structures and solvent accessibility within the complexes can be different. If the structures of the unbound and bound forms of the protein and the ligand are known, then it is possible to empirically calculate USA as a result of the complexation. This is the case for CaM, in which the structure of the Ca2+-saturatedprotein is known (Babu et al., 1988; Chattopadhyaya et al., 19921, as well as complexes of CaM with CaM- binding peptides from skeletal muscle myosin light-chain kinase (Ikura et al., 1992),smooth muscle myosin light-chain kinase (Meador et al., 19921, CaM-dependent protein kinase I1 (Meador et al., 19931, CaM-dependent protein base kinase (Osawa et al., 19991, and a Ca" pump (Elshorst et al., 1999). Once changes in ASA are determined for various types of groups (polar groups, aliphatic side chains, aromatic side chains), then the enthalpy, entropy, and Gibbs' free energy changes of hydration as a result of complexation can be calculated (Makhatadze and Privalov, 1995; Wintrode and Privalov, 1997). These calculations can then be used to interpret the results of ITC experiments. This type of detailed investigation of a peptide binding to CaM was undertaken by Wintrode and Privalov (1997),in which the properties of smMLCKp, the peptide comprising the CaM-binding domain of smooth muscle mysosin light-chain kinase, were examined by ITC. This study represented the fist time in which the enthalpy of binding of a peptide to CaM was determined in detail. The results were surprising in that it was found that enthalpic factors, not entropic factors, were the major driving Enerrretics of ~e~tidebinding to CUM 262 force in the formation of the complex. This contradicted the popular notion that CaM binding of target peptides is driven by the hydrophobic effect, which is comprised mainly of entropic contributions. In addition to the Wintrode and Privalov work, there have been a couple of other ITC studies of CaM-binding peptides (Moorthy et a!., 1999; Tsvetkov er nl., 19991, bnt these examine how peptide binding properties change as a result of changes to CaM or to experimental conditions. This chapter describes how CaM, under what could be considered standard conditions, binds four different CaM binding peptides. The four peptides are the CaM binding domains from calmodulin-dependent protein kinase I (CaMKI), neuronal constitutive nitric oxide synthase (cNOS), and 3',5'-cyclic nucleotide phosphodiesterase (PDEa), and the model CaM-binding peptide melittin (Figure 7.1). CaMKI could be considered a canonical CaM-binding peptide, and its properties are well known (Yuan et al., 1999a; Gomes et al., 2000). Melittin was also previously considered to be a typical CaM-binding peptide, but it is now known that melittin binds to CaM with the major population binding in a parallel orientation, opposite to other CaM-binding peptides (Yuan et al., 2000b). PDEa was the first characterized CaM- binding domain from bovine brain cyclic nucleotide phosphodiesterase 1A2 (Sonnenburg et al., 1995); it is thought that this peptide is only a partial CaM-binding sequence. It is also known that PDEa can bind apo-CaM (Yuan et al., 1999b), although the binding of PDEa to apo-CaM is not investigated in this chapter. There are two major isoforms of nitric oxide synthase that differ in their regulation by calmodulin (Marletta, 1994); neuronal cNOS is a constitutively expressed isoform of nitric oxide synthase that is activated by CaM in a Ca2+-dependentfashion in the usual way. Another isoform of cNOS is found in vascular endothelium. Energetics of neptide binding to CUM 253

There is also an isoform of NOS, found in marophages, that is inducibly expressed (iNOS). iNOS has CaM as an integral subunit, both in the presence or absence of Ca2+ions, and the iNOS target peptide can bind either apo-CaM or Ca2+-CaMvery tightly (Yuan et al., 1998b). Instead of Ca2+-regulation,the activity of iWOS is regulated by its expression. In addition to an investigation of the binding of these four peptides to CaM, the potential of using mutants andlor fragments of both CaM and CaM- binding peptides to learn more about CaM-peptide interactions is also discussed.

MATl3IRIALS AND MEZTHODS.

Materials. Wild-type calmodulin was expressed and purified according to the methods of Chapter 2. The thrombic fragments of calmodulin TM1 and TM2 were generated and purified according to the methods of Chapter 5. The peptides derived from the calmodulin-binding domains of CaM- dependent protein base I (CaMKI; sequence AKSKWKQAFNATAVVRH- MRKLQ), constitutive (rat cerebellar) nitric oxide synthase (cNOS; sequence KRRAIGFKKLAEAVKFSAKLMGQ), inducible (murine macrophage) nitric oxide synthase (iNOS; sequence RRREIRFRVLVKW- FFASMLMRKVMAS), bovine brain 3': 5'-cyclic nucleotide phosphodiester ase 1A2 (PDEa; sequence Ac-QTEKMWQRLKGILRCLVK- QGNH,) were synthesized by the Queen's Peptide Synthesis Facility, Queen's Univ., Kingston, Ont. Melittin (sequence GIGAVLKWiTTGLPAL- ISWIKRKRQQ) was purchased from Sigma. Dialysis membranes (molecular weight cutoff 1000 Da and 3500 Da) were purchased from Energetics of ~eptidebinding to CaM 254 Figure 7.1.

CNOS CdMKI PDEa

melittin

Figure 7.1. The four CaM-binding peptides in this chapter, as well as three peptides for which the structure of the CaM-peptide complex is known; "Meador et al., 1993, bIkura et al., 1992, 'Meador et al., 1992; Wintrode and Privalov, 1997. The sequences are aligned according to the position of their N-terminal hydrophobic anchoring residues (which bind to the C-terminal lobe of CaM), shown in the large dark grey bar and numbered as residue 1. The C-terminal hydrophobic anchoring residues are also shown in dark grey bars. Other conserved hydrophobic residues are shown in white bars. Additionally, conserved basic residues are shown in light grey bars. All basic residues (Lys and Arg) are underlined. Only the N-terminal anchor residues of the PDEa peptide are highlighted because this sequence comprises only one of the two CaMbinding domains of PDE which are thought to act in concert to bind CaM (Sonnenburg et al., 1995; see text). Melittin is shown in the opposite orientation as the other three peptides because 80% of it binds to CaM in a parallel orientation, opposite to the other peptides (Yuan et al., 2000b). Energetics of ~e~tidebinding to CaM 255

Spectrapor. Tris(carboxyethy1)phosphine (TCEP)was purchased from Sigma. Trifluoroacetic acid (TFA) was purchased from Pierce or Fischer. Acetonitrile (OmniSolv, HPLC grade) was obtained from EM Science. All other chemicals were obtained from reputable sources. The CaM-binding peptides were purified by high-peformance liquid chromatography (HPLC) with an &TA system (Pharmacia). Solvents containing water were filtered and degassed before use. CaMKI was purified to >95% purity using a SOURCE 15RPC ST 4.61100 column (Pharmacia), employing a gradient from solvent A (H20, 0.065% TFA) to 100% solvent B (acetonitrile, 0.05% TFA). Cameluted at around 25 % B. The other peptides were purified with a Sephasil Peptide C18 (5 ym ST 4.61100) column (lab of Dr. George Makhatadze, Hershey Medical Center of Penn State University), employing a gradient from solvent A (HZO,0.1% TFA) to 100% solvent B (acetonitrile, 0.005% TFA). Peptides eluted between 30 and 50% B; PDEa eluted as two peaks with essentially identical spectra, presumably one of which was a reduced monomer and one which was a &sulfide-linked dimer; the pooled fractions from the first peak were completely reduced to the free thiol form (see below) and used in the experiments.

Isothermal titration calorimetry. Samples for isothermal titration calorimetry (ITC) were prepared by dissolving the protein and peptide (usually in lyophilized powder form) in the sample buffer and then dialyzing against at least two changes of 1-2 L of the sample buffer, for a total of at least 16 h. All sample buffers consisted of 5 mM buffering ion, 2 mM CaCI,, 100 mM NaCI, pH 7.0. Buffer pH's were adjusted with stock solutions of NaOH and HC1. With the Energetics ofwe~tidebinding to CaM 256 exception of the buffer dependence studies for the CaMKI peptide, Pipes was used as the buffer species. Calmodulin was dialyzed with the 3500 molecular weight cutoff (MWCO) membrane while peptides were dialyzed with the 1000 MWCO membrane. CaM and the peptide were always dialyzed in the same buffer. For titrations with the PDEe peptide, the peptide was preincubated in a solution of 2 % P-mercaptoethanol at room temperature for 1 h before dialysis. The peptide was then dialyzed extensively against sample buffer as above; the sample buffer also contained 1 mM TCEP which was used instead of the conventional thiol reducers because, according to the manufacturers of the ITC instrument, it does not react with the interior coating of the ITC sample cell. Isothermal titration calorimetry was performed on Microcal's VP- ITC isothermal titration calorimeter, with a sample cell volume of 1.43 mL, in the laboratory of Dr. George Makhatadze, Milton S. Hershey Medical Center of Penn State University, Hershey, PA. The sample cell was first washed extensively with sample buffer from the last dialysis change. To determine the sample concentration in the cell, the cell was filled with the sample solution, then this solution was removed with a syringe and a W- absorption spectrum of this sample was taken with a 1 cm path length (3 mL) cuvette and a UVNis spectrophotometer (Hitachi). To determine the concentration of injectant in the automatic syringe, a UV-absorption spectrum was taken with either a 0.1 cm (450 pL) or a 1 cm (100 pL) cuvette. Correction for scattering was taken by extrapolation of a linear regression of absorbance values between 370 nm and 320 nm; the logarithmic correction protocol of Winder and Gent (1971) did not give satisfactory results, especially with CaM samples. For calmodulin, a molar extinction coefficient of h, = 2900 M-'*crn-'was used. The thrombic Energetics of pe~tidebindine to CUM 257 fragments TM1 and TM2 were both quantitated with a molar extinction coefficient of e2,, = 1450 M-l*cm'l. This value provided the best ability to correct against scattering. For peptides containing tryptophan residues a molar extinction coefficient of E?,, = 5690 M"*cm-' was used. For cNOS and iNOS, a molar extinction coefficient of E,~= 193 %f".crn" for a free phenylalanine was used, with multiplication for the number of Phe residues in the peptide. Sample concentration in the cell was typically 0.01-0.04 mM, whereas the concentration of the injectant in the syringe was typically 0.4-1 mM. Injections of 3-5 pL were typically employed, with 20-30 injections per titration. For the melittin titrations, melittin was in the sample cell and CaM was in the syringe, in order to avoid the formation of tetramers and other aggregates that can occur with melittin at high concentrations (Dempsey, 1990). In all cases, titrations were performed at 25 OC or below in order to avoid problems due to a thermal transition that occurs in Ca2+-CaMthat begins in the 30-40 OC range (Tsalkova and Privalov, 1985; Wintrode and Privalov, 1997). Data from the titrations were processed with Microcal's Origin v.5.0 software, and data were fitted using Origin's least-squares fitting algorithm with a one- site binding model. In most cases, the peak areas of several injections well after saturation were averaged to determine an enthalpy of dilution of the ligand into the buffer, which was then subtracted from all values before fitting the binding isotherm; in other cases a separate experiment of dilution of the injectant into sample buffer was performed and this value was subtracted instead. Due to the small volumes injected, the enthalpy of dilution of the sample by the injected solution was neglected. Energetics of ve~tidebinding to CUM 258

A typical titration of CaM with a target peptide that yields a negative enthalpy of binding, CaM with the CaMKI peptide in imidazole buffer at 25 "C,is shown in Figure 7.2, and a typical titration of CaM with a target peptide that yields a positive enthalpy of binding, CaM with the cNOS peptide in Pipes buffer at 5.2 OC, is shown in Figure 7.3. Note that in each case there are plenty of peaks that have the same, maximum area. These result from stoichiometric addition of the peptide to CaM (i.e. all of the peptide is being bound by CaM) which give a relatively large degree of certainty in determining the enthalpy of binding. The binding constant is less certain due to the fact that it is large in the context of these experiments, and there are thus few points on the titration curve in which the addition of the peptide to CaM is not stoichiometric (e.g. in Figures 7.2 and 7.3, there are only a few peaks in the titration where the differential power is intermediate between the maximal level and the baseline level). The CaMKI peptide was titrated into CaM at a temperature of 25 "C at pH 7.0 in four different buffers: cacodylate, Mops, Pipes, and imidazole. The plot of the apparent enthalpy, AH,,, vs. the ionization enthalpy of the buffer, AH,,, is shown in Figure 7.4. The plot is linear, as expected, due to the fact that the AHap, and AH, have the following relationship:

AH,, = AH,, + N,, *a, where AH, is the red enthalpy of binding of the peptide to CaM and N,, Energetics of ne~h'debinding to CaM 259

Figure 7.2.

24 ] I I I 0 30 60 90 120

time (min)

Figure 7.2. Raw data from a typical titration that yields a negative binding enthalpy. The titration consisted of 3 pL injections of the CaMKI peptide (0.4 mM) into CaM (0.015 mM) at 25.0 "C in imidazole buffer. Energetics of ve~tidebinding to CaM 260 Figure 7.3.

time (min)

Figure 7.3. Raw data from a typical titration that yields a positive binding enthalpy. This titration consisted of injections of 3 pL of the cNOS peptide (0.409 mM) into CaM (0.0141 mM) in Pipes buffer at 5.2 OC. Energetics of ~eptidebinding to CUM 261

Figure 7.4.

cacodylate Pipes

T 7 MOPS

imidazole

Figure 7.4. Plot of measured binding enthalpy. vs. enthalpy of ionization of the buffer for titrations of the CaMKI peptide into CaM at 25.0 "C. The buffer used is labeled beside each point. The linear regression line of the data is drawn. Energetics of ne~tidebinding to CaM 262

(the slope of the line) is equal to the number of protons that are taken up by the complex as a result of binding. If N is negative then protons are released as a result of binding. The slope of the regression line in Figure 7.4 is -0.032:indicating that virtually no protons are released as a result of binding of the CaMKI peptide to Ca2+-CaM. Since both CaM and CaMKI each contain one histidine residue, which can potentially be protonated or unprototonated at pH 7.0, it is most likely that the protonation states (i.e. the p&s) of these histidines are not altered as a result of binding. The temperature dependence of binding enthalpy was examined for four peptides, CaMKT, cNOS, PDEa, and melittin over a range of 5 "C to 25 "C.The results were compared to those of Wintrode and Privalov (1997) and are depicted in Figure 7.5. Although the results of this chapter were collected in Pipes buffer and the results of Wintrode and Privalov (1997) were collected in cacodylate buffer, buffer dependence curves (Figure 7,4; Wintrode and Privalov, 1997) show the binding enthalpies for smMLCKp and CaMKI to be essentially the same for the two buffers. Unfortunately, due to solubility problems with the iNOS peptide, no reliable ITC data could be acquired, even with titrations with the iNOS in the ITC cell. Moreover, the peptide was not sufficiently soluble in 2 M urea. All the peptides shown have a negative linear relationship of binding enthalpy to temperature, with the slope equal to the change in heat capacity AC, as a result of binding (the complex has a lower heat capacity than the sum of unbound CaM and peptide). The line for CaMKI is closest to that for smMLCKp, with a negative enthalpy of binding even at moderately low temperatures. The other peptides all have more positive enthalpies of binding, although their binding constants are still very large (see, e.g. Energetics of e en tide bindine to CaM 263

Figure 7.5.

temperature ("C)

CaMKl I cNOS 0 PDEa

Figure 7.5. Plot of binding enthalpy vs. temperature for the four peptides studied, plus smMLCKp (data from Wintrode and Privalov, 1997). Linear regression lines are drawn. Energetics of ~eptidebinding to CaM 264

Figure 7.3, which shows endothermic binding of cNOS to CaM even though binding of this peptide is very tight). The AH,,, and AC, are listed for all the peptides in Table 7.1. The cNOS peptide has very similar AC, to the CaMKI peptide (-3.67 kJ-mol-'*klvs. -3.54 kJ-mol-l.K1),although its enthdpy of bhding is positive, even at relatively high temperatures. The PDEa and melittin peptides have even greater positive enthalpies of binding. This may reflect the importance of entropic factors, such as hydrophobic interactions, in driving the binding of these peptides. The AC, of binding of melittin is smaller than that of CaMKT and cNOS and is more similar to that of smMLCK (Wintrode and Privalov, 1997). The AC, of binding of the PDEa peptide is smaller still; this may reflect that binding of the PDEa peptide produces a less globular complex, due to the fact that the peptide may not comprise the entire CaM-binding domain of cyclic nucleotide phosphodiesterase (see Discussion). The ITC data for the titration of TM2, fragment 107-148 of calmodulin, into TM1, fragment 1-106 of calmodulin, in the presence of 2 mM CaC1, is shown in Figure 7.6, and the titration of the CaMKT peptide into a mixture of TM1 and TM2 in the presence of 2 mM CaCl, is shown in Figure 7.7. As already shown by a variety of methods (Chapter 5; Brokx and Vogel, 2000a), TM1 and TM2 can associate in the presence of metal ions and CaM-binding peptides to form CaM-like complexes. Due to the limited availability of the fragments, TM2 was not titrated into TM1 to complete saturation. The titration reveals a.estimated K, of around lo3 M-' (K, in mM range) for the TMl:TM2 complex, which is a surprisingly poor binding constant given the favorable results from CD and NMR spectroscopy (Brokx and Vogel, 2000a; Chapter 5). The AH of binding is also somewhat uncertain, but given the large enthalpy of dilution of TM1 Energetics of pe~tidebinding to CaM 265 Table 7.1.

Peptide AH (kJ*mof'1 AC, (kJarnol-' *Ex ) CaMKI -50.4 -3.54 cNOS -11.7 -3.67 PDE 13.8 -1.98 melittin 48.9 -2.79 smMLCKp -68.5 -2.77

Table 7.1. Table of binding enthalpies at 25 "C and heat capacity changes upon binding for the four peptides in this study, as well as smMLCKp (Wintrode and Privalov, 1997). The values are calculated from the linear regression lines in Figure 7.5.

of-4.6 kcal~rn~l*~which was determined in a separate experiment (data not shown), AH,,, for TM2 to TM1 is negative, and could be approximately 4 kcal-mol-l. The titration of CaMKI into a mixture of TM1 and TM2 revealed a K. of about 5-10' M-', which, although not as large as CaMKI for CaM, is still quite high. Moreover, the AH of binding is -19 kcal-mol-l, which is about 6 kcal-mol-l larger than the AH,, of CaMKI to CaM. This extra enthalpy could be due to the release of heat by the association of TM1 and TM2 (see Discussion). Energetics ofpe~tide binding to CaM 266

Figure 7.6.

time (min)

TM2/TM1 molar ratio Energetics of peptide binding to CaM 267

Figure 7.7.

time (min)

CaMKl I (TMI:TM2) molar ratio Energetics ofpe~tide binding to CUM 268

Figure 7.6. (two pages previous) A. Raw calorimetric trace for the titration of the TM2 fragment of CaM (0.524 mM) into the TM1 fragment (0.0228 mM). The peaks are not all uniform due to changes in the injection volume as a result of experimental error; this is corrected tar in panel A, as was the baseline jump in the ninth injection. The peaks are all positive because of the enthalpy of dilution of TM1 which is quite large in the context of the rest of the data in this chapter; it was calculated to be -4.6 kcal*mol" in a separate experiment (not shown). B. Binding isotherm calculated from the experiment in panel A; the enthalpy of dilution of TM1 was subtracted from the raw data to arrive at the AH values plotted in panel B.

Figure 7.7. (previous page) A. Raw calorimetric trace for the titration of the CaMKI (0.485 mM) peptide into a solution of TM1 and TM2 at roughly equal concentrations (0.02 mM). The first peak is from a 6 pL injection; all other injections are 4 pL. Ao Binding isotherm derived from the raw trace in panel A. The solid line is the fit to the Leveberg-Marquhardt binding equation. Energetics of pe~tidebinding to CaM 269

DISCUSSION.

The enthalpy of binding to Ca2+-saturatedCaM was determined for four CaM-binding peptides, all at pH 7.0 in 100 mM NaCl and 2 mM CaClr (saturating Ca2+conditions). Three peptides (CaMKI, cNOS, and PDEa) comprise the CaM-binding domains of CaM-dependent target enzymes, while melittin is a model peptide derived from CaM inhibitors naturally found in bee venom. Due to the experimental conditions, the results provided accurate estimates of the binding enthalpy (AHbi,,) and the binding stoichiometry but only a rough estimate of the binding constant y.In order to obtain a more reliable estimate of the binding constant, one should ideally choose experimental conditions such that product of the concentration of the protein in the sample cell and & is 100 at the most and thus, in practice, it is often best to obtain AH,,, and y in separate experiments with different concentrations (Jelesarov and Brosshard, 1999). The lowest sample concentration used in the titrations in this chapter is about 0.01 mM. With a Y of approximately lo8, this gives a product of 1000. Thus, to obtain a good estimate of Ka the sample must be diluted at least 10X, or even more. Given the results from typical ITC traces of CaM-peptide binding (Figures 7.2, 7.3), one would expect the differential power in this concentration range to be no more than 0.1 pcaYsec, which pushes the limitations of the instrument. It may be possible to use ITC to determine binding constants for CaM-binding peptides, but it would no doubt be challenging. The real advantage of ITC is the direct determination of binding enthalpy. The enthalpy of binding of the CaMKI peptide was determined in four different buffers (cacodylate, Mops, Pipes, and imidazole) in order to Energetics of veotide bindine to CUM 270 determine if any protons are bound or released as a result of the complexation. This was undertaken for CaMKI because this peptide was available in sufficient amounts to enable this detailed study; moreover, CaMKI has a histidine in its sequence, while the other peptides do not. The imidazole functional group of the His residue is able to be either protonated or deprotonated at pH 7.0 due to its p& (6.8)being very close to this value. With the possible exception of the Cys thiol (p& = 8.01, this is the only amino acid side chain which can be titrated near physiological pH values. CaM also has one His residue, HislO7. The plot in Figure 7.4 is very reliable, with an r' value of 0.994. With a slope of only -0.032, it demonstrates that virtually no protons are transferred to or from the buffer as a result of the CaMKI peptide binding to CaM. That is to say that no ionization state of any residues changes as a result of the binding interaction at pH 7.0, or possibly protons are transferred from one binding species to the other without interaction with the buffer. Since the CaMKI peptide is quite basic (there are four Arg residues and 2 Lys residues in the 22-residue peptide) one can assume that protonation of the His residue is disfavored and thus it is unprotonated at pH 7.0. Most likely then it would remain unprotonated after binding of the peptide to CaM. This may be somewhat surprising given that positive charges on the peptide can act favorably with acidic residues on CaM, especially around the central linker region (Ikura et al., 1992; Meador et al., 1992, 1993), but since there are already a large number of basic residues on the CaMKI peptide that can fill this role it seems that the His residue, through the aromatic character of its side chain, perhaps binds to CaM through hydrophobic interactions. Additionally, the absence of any pK, shift as a result of binding is also shown for the smMLCK peptide (Wintrode and Privalov, Energetics of pe~tidebinding to CaM 271

1997). However, the X-ray crystal structure of the smMLCKp-CaM complex (Meador et al., 1992), shows that the His residue of smMLCKp is involved in a salt-bridge to CaM. These crystals were grown at a pH of 4.6, conditions which definitely favor protonation; under more physiological conditions such as those used in the ITC experiments the protonation state of the His could be much different. The main focus of the experiments presented in this chapter was to utilize the methods of Wintrode and Privalov (1997) with other peptides and CaM fragments. The previous results with the smMLCK peptide were surprising in that they revealed that binding of the peptide to CaM is enthalpically driven and is not due to entropic factors. This is surprising because the dogma was that hydrophobic interactions drive peptide binding to CaM. In order to examine if binding of all peptides to CaM are enthalpically driven in the same way as smMLCKp, titrations were performed with the four peptides at a series of temperatures. For these titrations, 5 mM Pipes was chosen as the buffer because it gave the most consistent results with the CaMKI peptide. The standard conditions of 100 mM NaCI, 2 mM CaCl,, and a pH of 7.0 were kept. Buffer dependence studies for smMLCKp (Wintrode and Privalov, 1997) and CaMKI (Figure 7.4) showed that the binding enthalpies are virtually identical in cacodylate and Pipes, which enabled direct comparison of the results for the peptides in this chapter to the results for the smMLCK peptide (Wintrode and Privalov, 1997). The temperatures chosen varied from 5 "C to 25 "C;temperatures higher than this were not used in order to avoid a thermal transition of CaM that occurs at temperatures above this range (Tsalkova and Privalov, 1985; Wintrode and Privalov, 1997). The results (Figure 7.5) show that the four peptides studied all have Energetics of we~tidebinding to CaM 272 higher enthalpies of binding than smMLCK. In fact, cNOS, PDEa, and melittin all have positive enthalpies of binding below about 25 "C or so. The binding of all of the peptides is still very tight at these temperatures, so there must be a substantial positive entropy contribution to the binding, unlike that concluded for smMLCK. The entropy need not be solely due to hydrophobic interactions but could in part be due to other entropic factors, such as the induction of flexible regions in CaM or the more unhindered rotation of amino acid side chains as the peptides bind. However, at the level of protein backbone and amino acid side chains, the degrees of freedom of CaM and the peptide are most likely reduced as a result of binding. The peptide, which is unstructured in solution, takes on an ordered a-helical structure in the bound state. Moreover, binding of the peptide by CaM takes place solely through interactions of amino acid side chains, so it is likely that the side chains are less ordered in the bound state as rigid binding interactions take place. With this in mind, classical hydrophobic effects (the entropically favorable release of bulk solvent) are then most likely a major reason for the tight binding. A detailed study of side chain entropy changes as CaM binds the smMLCK peptide was undertaken by 13C NMR relaxation methods (Lee et al., 2000). The results demonstrate that CaM "possesses greater than average side chain mobility, which is considerably reduced upon binding to smMLCKp," although some loss in entropy is compensated by an increase in motional eeedom in side chains in other parts of CaM. There is also compensation by enthalpic factors, suggesting that binding of the smMLCK peptide is enthalpically driven as was found by Wintrode and Privalov (1997). These enthalpic contributions can still be called hydrophobic effects, under the term "non-classical" hydrophobic effects Energetics of peptide binding to CaM 273

(Seelig, 1997, and references therein; Wieprecht et al., 1999). These are the result of van der Wads interactions between hydrophobic groups of the ligand and the receptor (protein, lipid, etc) such that it is possible to have tight hydrophobic binding that is predominantly due to enthalpic factors. This could most certainly be the case in binding of peptides such as smMLCKp to CaM, where bulky hydrophobic side chains on the peptide, such as the Trp, bury in the hydrophobic patches and interact with the hydrophobic residues of CaM. This side chain penetration is also seen to be important in the binding of some peptides to lipid bilayers (Wieprecht et al., 1999). Also, unlike the more static lipid groups, the malleable surfaces on the hydrophobic patches of CaM, with the high number of polarizeable Met side chains (Vogel and Zhang, 1995; Yuan et al., 1999a), are able to adjust to give optimal van der Wads interactions with the peptides. SO, the binding of smMLCKp to CaM could still be predominantly hydrophobic in nature, albeit in a non-classical way. Undoubtedly, some of the enthalpy of binding can also be explained by the formation of an a-helix in smMLCKp (c.f. Wieprecht et al., 19991, but this should be expected to be relatively similar for all the CaM-binding peptides in this study. Perhaps an examination of the CaMKK peptide, which does completely form an a- helix in the canonical way upon binding to CaM (Osawa et al., 19991, could help explain how much the formation of the a-helix actually contributes to the enthalpy of binding. The ITC results of this chapter indicate that this "non-classical" hydrophobic mode of binding to CaM as occurred with smMLCKp might not be the case for all CaM-binding peptides. Since the peptides in this study all bind to CaM with a more positive enthalpy than smMLCKp at moderate temperatures, the binding here is more likely of a classical Energetics of ~evtidebinding to CUM 274 hydrophobic nature. It would be most interesting to complete the 13C relaxation studies of Lee et al. (2000)for other CaM:peptide complexes, such as those with cNOS or CaMKI, and see if there are other entropic contributions to binding, such as a greater relative freedom in the backbone and/or side chains of the peptide iuld CAI in the complex. Besides the use of NMR relaxation methods to study internal dynamics upon ligand binding, a straightforward way to analyze various contributions to the AG, m, AS, and AC, upon binding is through analysis of three-dimensional structures. As already mentioned, the crystal structure of Ca2+-saturatedCaM has been solved by a variety of groups (Babu et al., 1988; Chattopadhyaya et al., 19921, and the structures of three "canonical" CaM-peptide complexes, that is where the peptide forms an a- helix upon binding and the complex is globular as CaM wraps around its target, have also been solved. These are namely complexes of Ca2+- saturated CaM with CaM binding peptides from skMLCK (Ikura et al., 19921, CaMKII (Meador et al., 19931, and smMLCK (Meador et al., 19921, the same peptide which was studied by ITC by Wintrode and Privalov (1997). With the crystal structures in hand, Wintrode and Privalov (1997) were able to calculate the change in water accessible surface area (AASA) for four classes of surfaces (backbone, polar side chains, aromatic side chains, and aromatic side chains) as a result of smMLCK binding by CaM. From this, the changes in the various energy parameters of hydration (hGhyd, AH^^^^ AS^^^), can be calculated, and AC, can be estimated. The calculations give a AC, of -2467 J~rn01-'-~~,which compares well with their experimental result of -2.7 kJ.mol"*kl. Overall, AH^^^ is very unfavorable, which leads to an unfavorable AG'". The favorable binding of smMLCK, then, must come from enthalpic factors such as van der Waals Energetics of pe~tidebindinn to CaM 275 interactions (including the non-classical hydrophobic effect), hydrogen bonds, and salt bridges. Enthalpic factors may be less important in binding of the CaMKI, cNOS, PDEa, and melittin peptides, as the results of Figure 7.5 suggest. However, crystal structures of the CiM-bound state of these peptides are not available, nor are any results from heteronuclear NMR relaxation studies, so a direct explanation of the results for these four peptides is not as simple. One method of deriving information may be to fit the sequences for these peptides into already existing CaM-peptide structures. However, this would most likely give similar calculated MA,AG~~~, AHhyd,and dShd values and thus not solve the apparent conflict in the ITC results. As already demonstrated in the existing structures of CsM-peptide complexes, the overall shape of the complex is also different for various CaM-binding peptides as a result of differential unwinding of the central linker of CaM (Clore et al., 1993; Meador et al., 1993). Thus, simple fitting of a new peptide into existing structures may not be necessarily valid. However, plenty of information about the binding of CaMKI, cNOS, PDEa, and melittin to CaM is already known through other spectroscopic and biophysical methods. The CaMKI peptide probably binds to CaM in a fashion more similar to smMLCKp than the other three peptides, due to the fact that its behavior in CaM-binding is very similar to smMLCKp with respect to methods such as fluorescence and circular dichroism spectroscopy, and it, like smMLCKp, has a Trp residue in a similar location which is bound to the C-terminal lobe of CaM ( Yuan et al., 1998a; Brokx and Vogel, 2000a; Gomes et al., 2000). The similar mode in binding to CaM is supported by the ITC results (Figure 7.5, Table 7.11, in which the enthalpy of binding is similar to the smMLCK peptide. However, the AC, of Energetics of ~eotidebinding to CUM 276 binding is somewhat different (-3.54 kJ*mol-'-K'for CaMKI compared to - 2.77 kJ*rn~l-~.~'for smMLCK); this may be because it has a different motif for CaM binding (Rhoads and Friedberg, 1997). A sequence alignment of the peptides (Figure 7.1) shows that position 14 of the smMLCK peptide is an anchoring ieucine residue whereas there is a Met at the corresponding position of CaMKI; the anchoring residue in CaMKI is thought to be a Val at position 10. Thus, with the anchor residues closer together by approximately one turn of an a-helix in CaMKI versus smMLCK, perhaps the mode of binding of this peptide is somewhat different: the lobes of CaM may move closer together to accommodate the different sequence, resulting in a more compact complex. The formation of a tight complex such as this is actually what happens with CaM and a peptide from CaM- kinase I1 (CaMKII) (Meador et al., 19931, a peptide which is more closely related to CaMKI (Figure 7.1). This formation of a tight complex, resulting in a larger USA, might explain the larger AC, of binding for CaMKI. In another study (Yuan, 19981, however, spin labeling studies demonstrated that the Met residue at position 14 of the CaMKI peptide (Figure 7.1) did indeed interact with the N-terminal lobe of CaM and could perhaps play an anchoring role. We await the crystal structure of the CaM:CaMKI complex in order to determine which residues are primarily involved in the binding of CaMKI to CaM. One could also investigate the binding of CaMKI peptides with altered sequences to CaM by ITC. Moreover, one could evaluate if there is any correlation between positioning of anchor residues and AC, by determining the AC, of binding of the CaMKII peptide to CaM, and seeing if it is similar to CaMKI. The cNOS peptide has hydrophobic anchoring residues in the same relative positions as the smMLCK peptide although it has no Trp residue. Energetics of oe~tidebinding to CUM 277

This is especially intriguing because cNOS binds tightly to CaM even without the bulky Trp residue to be inserted into the C-terminal hydrophobic cleft of CaM; the K, for the cNOS peptide to CaM is in the order of 2.2 nM (Zhang and Vogel, 1993b). This may be reflected in the enthalpy of binding of cNOS to CaM (Table :.I), which is more positive than that of CaMKI or smMLCK; burial of a bulky Trp residue would be expected to be enthalpically favorable since it results in a potentially large number of van der Waals interactions. In other words, the binding of cNOS to CaM could be more due to the classical hydrophobic effect. Interestingly the AC, of binding for cNOS is very similar to CaMKI (-3.67 kJ*rn~l*~*K"for cNOS and -3.54 kJ-mol"+K1for CaMKI). This contradicts prediction from sequence alignment (Figure 7.1; Zhang and Vogel, 1993b; Rhoads and Friedberg, 1997), in which cNOS seems to align more closely with the smMLCK peptide and thus would be expected to give a complex of a similar shape, resulting in a AC, of binding similar to smMLCK. Perhaps the real C-terminal anchoring residue of cNOS is not Leul4, as it is in the corresponding position in the smMLCK peptide, but rather PhelO, as it is in the CaMKI peptide (position 10 in CaMKI is a Val residue, whereas in smMLCK it is an alanine). Apart from solving the three- dimensional structure of the CaM:cNOS complex, examination of the binding enthalpy of cNOS peptides with altered sequences could be a possible way to test this hypothesis. The PDEa peptide has the lowest AC, of binding for any of the peptides studied (-1.98 kJ-mol-'=K1;Table 7.1). This is not surprising because PDEa has some unique properties among the CaM-binding peptides in this study. Although, like CaMKI and smMLCK, it has a Trp residue which is buried in the C-terminal lobe of CaM (Yuan et al., 1999b), Energetics of ~e~tidebinding to CaM 278 its sequence and mode of binding is somewhat different. The sequence of the PDEa peptide does not align well with the other peptides near its C- terminal end (Figure 7.11, which could mean that binding to the N-lobe of CaM is different, resulting in a less globular complex. Small-angle X-ray scattering dntn (H. Yoshino, personal communication), showed that although the Ca2+-CaM:PDEacomplex moved toward a globular structure, it remained larger than other Ca2+-CaM:peptidecomplexes such as that with skMLCK. Although the CaM:PDEa complex may be less globular, the ITC data seem to indicate that the PDEa peptide binds Ca2+-CaMwith a high affinity (Table 7.11, similar to the other peptides studied. This differs from the results of Yuan et al. (1999b3, who determined a considerably weaker K, of PDEa for Ca2+-CaMof 224 nM. However, these results were obtained by a myosin light-chain kinase inhibition assay which uses different conditions than the ITC work. The assays were run in the presence of 5 mM MgZ+, and it is now known that Mg2+ions can decrease the affinity of CaM for some peptides, including PDEa (Ohki et al., 1997). Moreover, the ITC data were collected under conditions that only provide a rough estimate of the association constant for PDEa, and for all other peptides for that matter. Perhaps the ITC experiments could be repeated at lower concentrations in order to determine a more accurate K, although the heat output changes may become immeasurably low (see discussion above). Importantly, the PDEa peptide may not comprise the entire CaM- binding sequence of bovine 3':5'-cyclic nucleotide phosphodiesterase 1A2; another potential CaM-binding sequence, about 50 amino acids distant from the one which the PDEa peptide comprises, has also been identified (Sonnenburg et al., 1995). In fact, that is why in this thesis the PDE Energetics of oe~tidebinding to CUM 279 peptide has been appended with the "a" suffix and called PDEa. Synthetic peptides from the other CaM-binding domain, domain B, have the ability to competitively inhibit CaM in enzyme activation assays (Somenburg et al., 1995). It seems likely that the CaM-binding domains A and B of PDE act in tandem, binding ~irnult~eciualy,resulting in an active TDE-CaM complex. Interestingly, Charbonneau et al. (1991) found that the PDEa peptide bound to CaM in a 2:l ratio, although the ITC results here strongly suggest a 1:l stoichiometry. Perhaps, under some conditions, the PDEa peptide can occupy the site on CaM normally occupied by domain B of PDE, or perhaps PDEa can bind to CaM as a dimer. At any result, it is also known that phosphorylation of PDE renders the enzyme less sensitive to Ca2+-CaMactivation, but of the two putative CaM-binding domains only domain B can be phosphorylated (Sonnenburg et al., 1995, and references therein). Clearly, ITC of both CaM-binding peptides from PDE, both alone and in tandem, is necessary for a more complete uaderstanding of CaM activation of PDE. Our lab, in collaboration with Dr. Makhatadze's lab, intends to undertake these studies in the future. Moreover, the PDEa peptide can also bind to apo-CaM, and the CaM-bound form of the peptide in this state appears to have some helix and some turn structures (Yuan et al., 1999b). Without calcium, the PDEa peptide binds exclusively to the C- terminal lobe of CaM, and the affinity is considerably weaker than that for Ca2+-saturatedCaM. It would be interesting to characterize CaM-PDE interactions in the absence of calcium by examining the binding of both PDE peptides to CaM by ITC. Melittin has the most positive enthalpy of binding to Ca2+-CaMfor any of the peptides studied (Table 7.11, although its AC, is similar to that of smMLCK (-2.79 and -2.77 kJ-mol-'-K1, respectively). A detailed Energetics of ~ewtidebinding to CaM 280 characterization of CaM-melittin interactions (Yuan et al., 2000b) has shown that the peptide binds CaM in an unusual manner. Fluorescence data indicate that the Trp residue of melittin is not as buried upon CaM- binding as it is with other CaM-binding peptides (Yuan et al., 2000b). This means a tower potential for enthalpic stabilization by van der Waals interactions (the non-classical hydrophobic effect). Classical hydrophobic interactions cculd play an important role for melittin binding to CaM. A detailed structural determination of the Ca2+-CaM:melittincomplex has been elusive up until this point, however, so it may be difficult to determine why melittin binds with such a large positive enthalpy. It appears that melittin actually binds to CaM in two distinct orientations (Yuan et al., 2000b): approximately 208 of the peptide binds in an antiparallel fashion, like the other CaM-binding peptides, whereas the other 80% binds in a parallel orientation, which enables the Trp residue to interact with the hydrophobic cleft of the C-terminal lobe. The study of Yuan et al. (2000) demonstrated the facility of using truncated melittin peptides (MelN and MelC) and tryptic half-molecules of CaM (TRlC and TR2C) to determine this result. It would be very interesting to use ITC to determine the binding enthalpies of these fragments in various combinations; these studies are ongoing. Although this chapter has demonstrated that isothermal titration calorimetry studies of calmodulin-peptide interactions can be very fruitful, the studies so far have only just scratched the surface, as the reader will no doubt agree. The study of the smMLCK-CaM interaction Wintrode and Privalov (1997) was interesting in that it, along with the complete structural determination of the complex by Meador et al. (1992) and the carbon-13 relaxation study of the complex by Lee et al. (2000), represents Energetics of we~tidebinding to CaM 281 the only thorough investigation of a CaM-peptide interaction. Given that the enthalpies of binding of the peptides in this chapter to CaM are significantly different than the smMLCK peptide, it would be very interesting to apply this "three-pronged" approach of calorimetry, structural determination, and carbon-13 relaation studies to another peptide such as Cam, CaMKII, or cNOS. This would then enable a complete understanding of the contribution of enthalpic factors, hydrophobic interactions, and other entropic factors to the binding of the peptide to CaM. It seems likely that these peptides could bind CaM quite differently than smMLCR In addition to structural studies, additional ITC work could also provide more information about CaM:peptide interactions. For example, all of the results in this chapter have been obtained in the presence of saturating Ca" levels. It is well-known, however, that many target proteins and peptides can bind to apo-CaM. The PDEa peptide (Yuan et al., 1999b), and iNOS (Yuan et al., 1998b), along with the neurornodulins (Zhang et al., 1994a; Gerendasy et al., 19951, can bind apo-CaM with relatively high affinity (K,s generally in the pM range). Unfortunately, solubility problems have thus fa. precluded any ITC work with the iNOS peptide, although perhaps with manipulation of the sample conditions characterization of the Ca2+-dependentand independent binding of NOS to CaM could be possible. The interactions of other peptides with Ca2+-CaMcould also be hrther examined by the use of CaM fragments. As with the melittin peptide (Yuan et al., 2000b), TRlC and TR2C, the tryptic half molecules of CaM, could be used to study CaM-peptide interactions. Dynamic light scattering studies have already demonstrated that CaM-binding peptides Energetics of peptide binding to CaM 282 such as CaMKI can interact with TRlC and TRPC simultaneously and induce their association (Papish et al., 2000). Calorimetry studies with TRlC and TRPC could provide information on the role of the central linker in stabilizing the interactions between peptides and the two lobes of CaM, md deterxine which part of the peptide binds to xhich lobe of CaM. TRlC and TRPC could also be used to determine the binding properties of CaM-target proteins like PDE, in which there are two CaM-binding domains. It could be that each peptide interacts separately with one lobe of CaM. In addition to PDE, other CaM-target proteins with two CaM- binding domains include caldesmon (Zhou et al., 1997, and references therein), and the y subunit of phosphorylase kinase (Dasgupta et al., 1989). Another potentially interesting CaM-binding peptide is the CaM-binding domain from petunia glutamate decarboxylase (Yuan and Vogel, 19981, which actually binds C3'-CaM in a ratio of 2 peptides per CaM molecule. As well as tryptic fragments of CaM, the thrombic fragments described in Chapter 5 could also be used to further characterize CaM:peptide interactions. As well, the interactions between the thrombic fragments themselves are interesting. Preliminary results with TM1 and TM2 alone (Figure 7.6) show that the fragments do not bind as tightly in the presence of Ca" as CD results have suggested. More experiments at higher sample concentrations may provide more reliable answers. Choice of a different temperature for the titration could also help and would also provide some data about the AC, of binding of TM1 and TM2. Additionally, as a result of the thrombic cleavage, the only histidine of CaM ends up at the N-terminus of TM2;by repeating the titration in different buffers one could determine if there are any pK, shifts in TM1 and TM2 as a result of binding. This would provide strong evidence as to whether or not the Energetics of pentide binding to CaM 283 histidine residue, and thus the N-terminus, of TM2 is involved in the interaction. A titration of the CaMKI peptide into a mixture of TM1 and TM2 is shown in Figure 7.7. The stoichiometry of binding (not shown) is not equal to 1, but this is probably only because the original TM1 and TM2 minicure did not contain exact equimolar amounts of the two fragments, and thus the concentration of the TM1:TMP complex would be lower than calculated. What is important is AHbin,,which is about 6 kcal~mol"larger than that for CaMKI binding to CaM (this number is calculated relative to the concentration of the CaMKI peptide, and is thus not subject to any errors in the TM1 or TM2 concentrations). This extra heat released is probably due to the association of TM1 and TM2 being induced by the addition of the CaMKI peptide. If one considers the following binding equilibrium:

CaMKIt

then it seems logical that, by le Chatelier's principle, the addition of CaMKI would favor the formation of the TM1:TMZ complex, thus resulting in the extra heat. If one assumes that AH,, for CaMKI to TMl:TM2 is the same as that for CaMKI to CaM, then the AH,, for TM1 to TM2 would be Energetics of oe~tidebinding to CUM 284

-6 kcal-mot1. This is close to the -4 kcal-mot' estimated from the direct TM1:TMB titration. In order to fully understand the interaction of TM1, TM2, and CaMKI, the titration should be repeated under different temperature and buffer conditions, as with titrations of TM1 into TM2. As nel!, it might be nsefiil to titrate Calm into TM1 or T312 alone. It xouid also be interesting to try the experiment with other peptides. For example, would the cNOS peptide, without a tryptophan residue, still provide a sufficient scaffolding to induce the formation of a TM1:TMa:peptide complex? In conclusion, this chapter has expanded the use of isothermal titration calorimetry in studying the binding of several different target peptides to calmodulin. The results of this chapter show that, although the binding of a smMLCK target peptide to CaM may be enthalpically driven, this might not be the case for all target peptides. In other words, the binding of the peptides in this chapter to CaM take place through the classical hydrophobic effect, whereas non-classical hydrophobic effects appear to be more important in the binding of smMLCKp to CaM. The greatest asset of ITC in this context is the ability to give direct information about the enthalpies of binding of peptides to CaM. However, binding enthalpies provide only a starting point for a complete thermodynamic understanding; structural and dynamic information also provide crucial pieces of the puzzle. In addition, a more thorough investigation of the interactions of mutants and fragments of both CaM and peptides by ITC would provide a more microscopic view of the interaction of CaM with its target molecules. Clearly, ITC has the potential to be a standard technique in providing a vast amount of information to researchers in this field. Conclusions 285

CHAPTER EIGHT: Conclusions

The studies described in this thesis, as well as many previous theses in the Vogel laboratory, have revolved around calmodulin (CaM),the ubiquitous and important eukaryotic calcium binding messenger protein. Previous work in our lab used CaM from bovine testes, which was then subjected to chemical modifications, proteolytic cleavage, and other reactions in order to explain and understand its structure and function. More recently (about ten years ago), a CaM expression plasmid was generously supplied to us which enabled bacterial expression of the protein. This made expression and purification of large quantities of the protein easy. Also, it enabled researchers to incorporate non-natural amino acids into CaM, producing CaM variants, or alloproteins. My first project at the Vogel lab extended on this work by incorporating proline analogs into CaM. The incorporation of proline analogs was considered to be an important project because, unlike other amino acids which could be chosen through conventional site-specific mutagenesis, one could chose analogs which maintained a ring structure similar to proline. It was hoped that the analogs could be developed as new, more appropriate probes to probe and study prolyl cis-trans isomerization in proteins. The incorporation into CaM of 3,4-dehydroproline (Dhp) and azetidine-2- carboxylic acid (Azc), were studied in detail. Neither compound seemed to have any significant effect on the structure or function of the protein when incorporated into CaM, but Dhp did have very unique NMR Conclusions 286 properties, owing to the double bond function in its ring structure. This made assignment of the two Dhp residues in CaM quite straightforward. Nthough the two proline residues in CaM are not that unique or functionally important, the facility of biosynthetic incorporation of Dhp and AZCirrto the protein was in itself so important result. It r3i3ed the potential of incorporating Dhp or Azc into proteins in which prolines do play an important role and using the analogs as probes of the function of the proline residues in these proteins. Indeed, cis-trans isomerization of Xm-Pro peptide bonds is very important in the folding and function of many proteins. In an effort to fulfill this potential, Dhp was biosynthetically incorporated into bovine calbindin D,,, a protein which is related to CaM and has a proline, Pro43, which can be cis or trans in the native state. However, no peaks for minor conformations could be seen for Dhp43 in lH NMR spectra of Dhp-substituted calbindin D,,, either because the minor peaks overlapped with other peaks in the spectrum, such as the other three Dhp residues in the protein, or because Dhp substitution stabilized the trans conformation to such an extent that the cis conformation was no longer visible. If Dhp did indeed stabilize the trans conformation of the Gly42-Pro43 peptide bond of calbindin, which is most likely reason for the observations in the NMR spectra, then Dhp could potentially be generally applicable in stabilizing trans prolyl peptide bonds in proteins, thereby reducing structural heterogeneity in some proteins. Stabilization of the trans conformation by Dhp was evident in a comparison of Dhp and Azc to Pro in the tripeptide system Acetyl-Tyr-Pro- Ser. Azc had the opposite effect in that the trans conformation was less favored (although it was still favored more than the cis). By inversion transfer-NMR methods it was possible to determine the rates of cis-to- Conclusions 287 trans isomerization for the three tripeptides. It was found that Pro and Dhp were fairly similar in their rates, but Azc had a significantly faster rate owing to a greater entropy of activation. It was hoped through the NMR studies of these tripeptides that it might be possible to choose a Pro malog that "locks" the prolyl peptide exclusively in either the cis or the trans conformation; while this did not happen with the peptides it was found that by choice of the proper analog plus manipulation of the temperature it was possible to favor one conformation more than the other. Additionally, given the results of Dhp-substituted calbindin D,,, it might be possible to use Dhp to favor the trans conformation in proteins. Additionally, Azc has a significant effect on increasing the rate of cis-trans isomerization, so it would be very interesting to incorporate this analog into a protein in which prolyt cis-trans isomerization is functionally important. Perhaps incorporation of Azc into calbindin D,, could be an interesting future project. In addition to Azc and Dhp, another proline analog which was successfully incorporated into CaM was cis-4-fluoroproline (FPro). FPro has a fluorine substitution, and fluorine has properties which make it very amenable to study by NMR. Although biosynthetic incorporation of fluorinated aromatic amino acids into proteins for study by "F NMR is quite common, there aren't many studies of fluorinated aliphatic amino acids, like FPro, in the literature. To this end, 5,5,5-trifluoroleucine (TFLeu) and trifluoromethionine (TFMet) were also incorporated into CaM using the bacterial expression system, and the potential for the use of fluorinated aliphatic amino acids in "F NMR of proteins was evaluated. It was observed that the incorporation level of these fluorinated amino acids was not to as high a level as some other amino acid analogs, and some of Conclusions 288 these substituted proteins were not as stable as unsubstituted CaMs, and the lgF NMR spectra of the substituted proteins were not as disperse as the spectra of proteins with fluorinated aromatic amino acids. However, it was still possible to obtain useful information on these fluorinated proteins by s relatively now experiment, the 19F,'3 heteronuclear COSY, which was used to correlate the fluorine atoms to other protons in the amino acid side chains. Studies of TFLeu- and TFMet-substituted CaMs also demonstrated the change in the environment of both of these types of amino acids as a result of the Ca2'-dependent conformational change in CaM. When Ca2+was added, the peaks for both the TFLeu and the TFMet residues became less distinct as a result of exposure of these residues on the hydrophobic patches of CaM. Binding of CaM-target peptides such as CaMKI, to TFLeu-CaM or TFMet-CaM caused the 19F peaks for the fluorinated amino acids to change once again, showing the importance of these residues in peptide binding. Moreover, the TFLeu residues of TFLeu- CaM are protected from the soluble spin label TEMPOL by the CaMKI peptide, proving that the Leu residues become less solvent exposed when they interact with target peptides. Overall, studies of fluorinated aromatic amino acids have produced good results by 19F NMR because of the excellent chemical shift dispersion. The results of this thesis show that fluorinated aliphatic amino acids are not as useful for study of proteins by simple one-dimensional 19F-NMR spectroscopy because of chemical shift anisotropy problems and the relatively low levels of incorporation, but some of these problems can be alleviated by the use of two-dimensional experiments such as the HETCOSY in amino acids which have protons that couple to the fluorine atoms. The changes in the environment of Met residues of CaM can also be Conclusions 289 seen in 'H, 13C HMQC spectra of 13C-methyl-Met labeled proteins, which were exploited in the studies of thrombic fragments in Chapter 5. Thrombic cleavage of CaM creates two fragments, TM1 and TM2, both of which contain one isolated Ca2'-binding domain. It was found, not only by

13C *ZR but dso by 2 vzri,-;,et;.cf other methods, thst T?A1 and TM2 can interact with each other and enhance their metal ion binding affinity. The interaction between TM1 and TM2 was metal ion dependent, as was shown by NMR and CD spectroscopy, and the interaction increased the metal ion affinity of both fragments, as was shown by cadmium-113 NMR. The interaction of TM1 and TM2 was interesting because, up to this point, all studies of isolated helix-loop-helix Ca2+-bindingdomains have examined fragments of entire domains. In this system the site of thrombic cleavage was not exactly in between the two Ca2+-bindingdomains, but rather in the middle of an a-helix flanking one of the sites, yet it was shown that these fragments can still associate. Moreover, CaM-binding target peptides were also examined for their effect on the strength of the association of TM1 and TM2. It was found that their association is enhanced by the presence of the peptides, forming a TM1:TMS:peptide complex that has properties very similar to CaM:peptide complexes, as was shown by 'T CRand CD spectroscopy. This is important because it is the C-terminal lobe of CaM, which is disrupted by the thrombic cleavage, that plays an important role in the binding of hydrophobic anchor residues from target peptides. This is the first study in which the peptide- dependent association of isolated EF-hand fragments has been examined. Further studies in this area could include the study of other CaM fragments, taking advantage of other cleavage points such as cleavage in the central part of the molecule by trypsin and cleavage between the first Conclusions 290 two Ca2+-bindingdomains by thrombin. In Chapter 7 the binding of several target peptides to CaM was studied by isothermal titration calorimetry (ITC) in order to determine the contributions of enthalpic and entropic factors to the high affinity binding of several peptides to CEM. ITC is 3 ~sefidtechnique because it eoebles direct determination of the enthalpies of protein-ligand interactions. The high quality of the results demonstrated the potential of ITC as a standard tool in characterizing the binding interactions of CaM and target peptides. The results obtained for four peptides showed that entropic effects, such as hydrophobic interactions, are indeed important in the binding of peptides to CaM, unlike what was previously concluded with another CaM-binding peptide. Thus, it is important to determine the binding enthalpies of CaM binding by each peptide individually, rather than making generalizations. Moreover, ITC also enables the researcher to make some conclusions about the degree of compactness of the complex when a peptide binds to CaM, thereby developing hypotheses about the positions of hydrophobic anchor residues, the orientation of the two lobes of CaM in the complex, and the degree of unwinding of the central a-helix of CaM. There is a great potential for fbture work by ITC in this area, including the comparison of the ITC results to known structures of CaM:peptide complexes and to dynamic NMR studies. ITC is also potentially very useful for the investigation of peptide binding by CaM mutants or CaM fragments, or conversely the investigation of binding of altered peptides or fragments of peptides to CaM. The central linker of CaM was itself the focus of another chapter of this thesis. In crystal structures of the protein, the central linker appears to be a rigid a-helix, and indeed it is still widely called this in the Conclusions 291 vernacular. However, it is now commonly understood that this region of CaM is in fact quite flexible, and this flexibility is functionally important for the protein. It is thought that a main reason for the stability of the central linker in the crystal structure of CaM is the presence of alcohols as precipitsnts in the crystallization media, so thc dynamic properties of CaM were investigated in 35% trifluoroethanol (TFE),paying special attention to the central linker. The results showed that 35% TFE could induce a stable, ordered structure in the central linker region of CaM, although the dynamic properties of CaM as a whole seemed to indicate that the presence of 35% TFE was not sufficient to produce a solid, dumbbell-shaped CaM with a rigid central a-helix. Still, the properties of TFE as a helix stabilizer were proven in yet another system. Additional stabilization for the central linker in crystal structures of CaM could come from the low pHs used, the low temperatures of crystallization, or other factors. It would be interesting in the future to try and make mutants of CaM in which the central linker is indeed a stable a-helix, such as ones with alanines substituted into the region. Then, not only the structure but also the function of these mutants could be investigated. Carbon-13 NMR of selectively 13C-Ala labeled CaMs could be useful in this case. In conclusion, calmodulin is a well-known and well-characterized protein such that it can be used as a model system in a variety of studies about proteins in general. However, still much is yet to be known about this very important protein, including its metal-ion binding properties, how it is able to activate so many different targets, and its true functions in the cell. Additionally, there is an entire class of related proteins and calmodulins in other organisms that are yet to be well understood. I look forward to future studies on calmodulin and other members of this Conclusions 292 important class of regulatory Ca2+-bindingproteins. References 293

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