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Structural constraints for the folding, stability and function of cytochrome C Murphy, Michael E. P. 1993

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STRUCTURAL CONSTRAINTS FOR THE FOLDING, STABILITY AND FUNCTION OF CYTOCHROME C By Michael Edward Patrick Murphy B.Sc., The University of Alberta, 1987  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF BIOCHEMISTRY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January 1993 © Michael Edward Patrick Murphy, 1993  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, ence and study.  I agree that the Library shall make it freely available for refer-  I further agree that permission for extensive copying of this thesis for scholarly  purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  The Department of Biochemistry The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1Z3  Date:  Abstract  The relationship between structure, stability and function in yeast (Saccharomyces cerevisiae) cytochrome c was studied through the investigation of mutant proteins. Two isozymes of cytochrome c can be isolated from yeast, iso-1 and iso-2-cytochrome c. The structure of iso-l-cytochrome c had been previously determined in this laboratory using X-ray diffraction analyses. In the first part of this study, the structures of wild-type yeast iso-2-cytochrome c and a composite mutant protein (B-2036) composed of segments derived from the genes of both isozymes were structurally characterized by X-ray analyses. The overall fold of the composite is similar to the two native isozymes, however, specific intra-molecular atomic interactions are altered by the presence of amino acid substitutions. These alterations are proposed to explain the loss of thermodynamic stability of the composite relative to the wild-type proteins. In a second study, a-Loop A of iso-l-cytochrome c was replaced with the corresponding loop from cytochrome c2 of Rhodospirillum rubrum to produce a mutant protein called RepA2 cytochrome c. Structural analysis of the RepA2 protein revealed that a-Loop A had folded in a conformation more similar to the original loop it replaced than the conformation of a-Loop A in cytochrome c2. Two substitutions, Va120Phe and His26Asn, that result from the a-Loop A replacement were shown to cause structural perturbations. Phe20 was back substituted to Va120 to produce RepA2(Va120) cytochrome c. In vivo functional studies and reduction potential measurements of these S2Loop A replacements demonstrate that Phe20 lowers the reduction potential by 19 mV at 25 °C and diminishes cytochrome c function at higher temperatures. A third aspect of this work was to investigate the role of Pro71. The structures of four partially functional Pro71 substitutions were determined: Pro7lAla, Pro7lIle, Pro7lSer and Pro71Val cytochromes c. Analysis of these structures revealed that loss of function could be explained by the disruption of residues 82 and 83 which have been shown to be important in the binding and transfer of electrons to  ii  complexed electron transfer partners. Larger side chain replacements, Pro71Val and Pro7lIle, result in additional disruptions at the site of mutation which is part of a highly conserved segment of polypeptide chain. A comparison of all the mutant structures investigated in this work revealed that the overall fold of the protein was preserved at the expense of disrupting the packing of the hydrophobic core and highly mobile surface loops. Amino acid substitutions that cannot be incorporated into the cytochrome c fold without structural perturbations occur in conformationally restricted regions. In addition, the displacement of groups by these replacements indicates the limits present in specific regions in terms of conformational flexibility. The structural perturbations that result from the replacement of conformationally restricted residues have been shown to diminish both the reduction potential and in vivo function of cytochrome c.  iii  Table of Contents  Abstract^  ii  Table of Contents^  iv  List of Tables^  vii  List of Figures^  ix  List of Abbreviations and Structure Nomenclature ^  xi  Acknowledgements^  xiii  1^Introduction  1  1.1^The Mitochondria' Cytochromes c ^  1  1.1.1  Structure ^  1.1.2  Stability ^  10  1.1.3  Folding ^  13  1.1.4  Function ^  14  2  1.2  The Yeast Cytochromes c ^  17  1.3  Yeast Cytochrome c Variants ^  18  1.4  1.3.1  Pro71 Replacements ^  22  1.3.2  Composite Proteins  ^  23  1.3.3  Q-loop Replacements ^  24  Thesis Objectives ^  iv  25  2  General Experimental Methods  27  2.1^Structure Determination ^  27  2.2  2.3  3  2.1.1^Crystal Growth and Characterization ^  27  2.1.2^Data Collection ^  29  2.1.3^Data Processing  31  ^  2.1.4^Structure Solution and Refinement ^  36  Structure Analysis ^  37  2.2.1^Estimation of Coordinate Error ^  37  2.2.2^Comparison of Structure Coordinates  39  ^  2.2.3^Comparison of Derived Structural Properties ^  40  Electrochemical Properties ^  41  2.3.1^Direct Electrochemistry ^  41  2.3.2^Derivation of Electrochemical Thermodynamic Properties ^  42  Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein  46  3.1  Experimental Procedures  46  3.2  Polypeptide Chain Conformations  3.3  Comparison of Iso-2, B-2036 and Iso-1-Cytochromes c ^  55  3.4  The Buried Cavity in B-2036 and Iso-l-Cytochromes c ^  61  3.5  Herne Geometry and Environment ^  64  3.6  Conserved Water Molecules ^  66  3.7  Comparison with Other Cytochromes c  66  3.8  Electrochemical Properties ^  67  3.9  Stability of Yeast Cytochromes c ^  70  ^ ^  ^  51  4 Replacements of Q-Loop A in Iso-1-Cytochrome c ^  72  4.1 Experimental Procedures ^  72  4.2 Results ^  77  4.2.1 Comparison of the Structures of RepA2, RepA2(Va120) and Iso-l-Cytochromes c ^ 4.2.2 Electrochemistry of RepA2 and RepA2(Va120) Cytochromes c ^ 4.3 Discussion ^  77 82 83  4.3.1 Comparison of the Wild Type Yeast Iso-1 and R. rubrum Structures^83 4.3.2 Structural Effects of 52-Loop A Replacements ^  86  4.3.3 In Vivo Functional Consequences of C2-Loop A Replacements ^ 88 4.3.4 The Relationship Between Herne Reduction Potential and Temperature Sensitivity ^  92  5 Replacements of an Invariant Proline in Iso-1-Cytochrome c ^94 5.1 Experimental Procedures ^  94  5.2 Results ^  96  5.2.1 The Environment of Pro71 in Yeast Iso-1-Cytochrome c ^  96  5.2.2 Structural Differences of Pro71 Mutant Cytochromes c ^ 100 5.3 Discussion ^  105  6 Summary^  113  Bibliography^  118  vi  List of Tables  1.1 Primary sequence alignment for yeast iso-1, yeast iso-2, tuna, horse and rice cytochromes c ^  4  1.2 X-ray structure determinations of mitochondrial cytochromes c . ^ 5 1.3 Structural equivalence and primary sequence identity of yeast iso-1, tuna, horse and rice cytochromes c ^ 1.4 Secondary structural elements in iso-l-cytochrome c ^ 1.5 Stability of oxidized cytochromes c ^  6 8 12  1.6 Electrochemical properties of cytochromes c from different sources ^ 15 1.7 Electrochemical properties of yeast cytochrome c and mutants ^ 16 1.8 Minimal unique residue positional boundaries of iso-2 polypeptide chain segments in each composite iso-1-cytochrome c class ^  23  2.9 The effect of neighboring background measurements on data quality ^ 35 2.10 Comparison of overall observed main chain r.m.s. deviations and the expected deviation errors ^  39  3.11 Data collection statistics of iso-2 and B-2036 cytochromes c ^  47  3.12 Sequence alignment of iso-1, B-2036 and iso-2-cytochromes c ^  48  3.13 Final stereochemistry of iso-2 and B-2036 cytochromes c ^  49  3.14 Hydrogen bond analysis of iso-2, B-2036 and iso-l-cytochromes c ^  52  3.15 Secondary structural elements in iso-2-cytochrome c ^  55  3.16 Heme solvent accessibility of mitochondrial cytochromes c ^  64  3.17 Heme conformation and ligand geometry in yeast iso-2 and B-2036 cytochromes c 65 3.18 Conserved water molecules in yeast iso-2, B-2036 and iso-l-cytochromes c . . .^67  vii  3.19 Electrochemical properties of the yeast iso-2, B-2036 and iso-l-cytochromes c . . 69 4.20 Sequence alignment of the yeast iso-1. RepA2 and RepA2(Va120) cytochromes c with cytochrome c2 from Rhodospirillum rubrum ^  73  4.21 Data collection statistics of RepA2 and RepA2(Va120) cytochromes c ^  74  4.22 Final stereochemistry of RepA2 and RepA2(Va120) cytochromes c ^  75  4.23 Electrochemical properties of the yeast iso-1, RepA2, and RepA2(Va120) cytochromes c ^  83  4.24 Hydrogen bond interactions of Q-Loop A in yeast iso-1, RepA2, RepA2(Va120) cytochromes c and R. rubrum cytochrome c2 ^  85  5.25 Data collection statistics for Pro71 mutant cytochromes c ^  95  5.26 Final refinement statistics for Pro71 mutant cytochromes c ^  97  5.27 Final stereochemistry for Pro71 mutant cytochromes c ^  97  5.28 Positional deviations of groups in the vicinity of residue 71 ^ 102 5.29 Average thermal factors of groups in the region of residue 71 ^ 106 5.30 Functional and stability studies of Pro71 mutant cytochromes c ^ 108 6.31 Amino acid additions and replacements in the eight cytochrome c structures studied in this work ^  viii  113  List of Figures  1.1 The cytochrome c fold ^ 1.2 A space-filled representation of yeast iso-l-cytochrome c 1.3 A stereographic representation of iso-1-cytochrome c ^  7  ^9 10  2.4 X-ray diffraction data collection geometry ^  30  2.5 The 0 and 8 dependence of background measurements ^  34  2.6 A sample SDBase information retrieval ^  42  2.7 Schematic of the direct electrochemistry experiment used to measure reduction potentials ^  43  2.8 A sample cyclic voltammogram of iso-2-cytochrome c ^  44  3.9 A Luzzati plot of the iso-2 and B-2036 cytochrome c structure determinations . . 50 3.10 Ramachandran plots of the iso-2 and B-2036 cytochrome c structures ^ 54 3.11 Stereo a-carbon backbone of iso-2-cytochrome c ^  56  3.12 Stereographic representation of iso-2-cytochrome c ^  56  3.13 The average main chain positional deviations between iso-2, B-2036 and iso-1cytochromes c ^  57  3.14 The region near G1y37 in iso-2, B-2036 and iso-l-cytochromes c ^  58  3.15 The region about residue 26 in iso-2, B-2036 and iso-l-cytochromes c ^  59  3.16 The average main chain thermal factors of iso-2, B-2036 and iso-l-cytochromes c 60 3.17 The region about residues 20 and 102 in iso-2, B-2036 and iso-l-cytochromes c . 62 3.18 The buried cavity in B-2036 and iso-l-cytochromes c  63  3.19 The overall average deviations of main chain atoms of mitochondrial cytochromes c 68  ix  3.20 The midpoint reduction potential versus temperature of iso-2, B-2036 and iso-lcytochromes c ^  69  4.21 A Luzzati plot of the RepA2 and RepA2(Va120) cytochrome c structure determinations ^  76  ^  78  4.23 A stereo representation of S2-Loop A of yeast iso-l-cytochrome c ^  79  4.22 A space-filled representation of a-Loop A of yeast iso-l-cytochrome c  4.24 The average main chain positional deviations between RepA2, RepA2(Va120) and iso-l-cytochromes c ^  80  4.25 The region near residue 26 in RepA2. RepA2(Va120) and iso-l-cytochromes c 4.26 The packing of residue 20 in RepA2, RepA2(Va120) and iso-l-cytochromes c  .  •  81  •  81  4.27 The midpoint reduction potential of the RepA2. RepA2(Va120) and iso-l-cytochromes c as a function of temperature. ^  82  4.28 A stereographic plot of a-Loop A from yeast iso-1-cytochrome c superimposed on that of cytochrome c2 from R. rubrum ^  84  4.29 Growth curves of experimental and control yeast strains in liquid lactate media  89  4.30 Spectra of intact yeast cells of experimental and control strains at —196 °C^. .  90  4.31 Average main chain thermal factors of 1-Loop A in RepA2, RepA2(Va120) and iso-l-cytochromes c ^ 5.32 A Luzzati plot of the Pro71 mutant cytochrome c structure determinations 5.33 The region near Pro71 in yeast iso-l-cytochrome c ^  91 .  98 99  5.34 Positional and thermal factor difference matrices for Pro71 mutant cytochromes c 101 5.35 Structural changes associated with replacing Pro71 with a smaller side chain . . 103 5.36 Structural changes associated with replacing Pro71 with a larger side chain . . . 104 5.37 Space-filled representations of wild-type and Pro71 mutant cytochromes c . . . . 109 6.38 A plot of the overall average deviation of main and side chain atoms of the structures examined in this work from iso-1-cytochrome c ^ 115  List of Abbreviations and Structure Nomenclature  A^hydrogen bond acceptor atom A.S.^saturated ammonium sulfate Angstrom unit; 1 B  A= 10 -1 ° m  crystallographic thermal factor 0  midpoint reduction potential under standard conditions  F^the Faraday constant: 23.06 cal mol -1 mV -1 calculated structure factor magnitude  F0^observed structure factor magnitude D  hydrogen bond donor atom  DTT^dithiothreitol I^observed reflection intensity K  absolute scale factor  ND^not determined NMR^nuclear magnetic resonance spectroscopy PDB^Brookhaven Protein Data Bank SHE^standard hydrogen electrode Tml^e-N-trimethyl lysine UV^ultraviolet light a, b, c^crystallographic unit cell axes h, k, 1^Miller indices e.u.^entropy unit; 1 e.u. = cal mol r.m.s.^root-mean-squared  xi  —1  K -1 = 4.184 J mo1 -1 K -1  A^wavelength of the radiation used in the X-ray diffraction experiment (CuK,=1.5418 A) ionic strength Q, X, q^Eulerian angular position of the crystal  9^Bragg diffraction angle (Tx^  estimated standard deviation of the quantity x  1. The conventions of the IUPAC-IUB Combined Commissions on Biochemical Nomenclature are followed for both three letter and one letter abbreviations for amino acids [J.  Biol. Chem. 241, 527-533 (1966); J. Biol. Chem. 243, 3557-3559 (1968)]; for designating atoms and for describing the conformational torsional angles of the polypeptide chain  [J. Biol. Chem. 245, 6489-6497 (1970)]. 2. Designations for the atoms of the protoporphyrin IX heme group are according to the Brookhaven National Laboratory Protein Data Bank (Bernstein et al., 1977). 3. The numbering scheme used to designate amino acid residues along the polypeptide chain of cytochrome c are according to those of the vertebrate cytochromes c. To conform to this scheme, the amino terminal extensions of yeast iso-1 and iso-2-cytochromes c are numbered —5 to —1 and —9 to —1, respectively. 4. The designation, yeast, used herein, refers to only the Saccharomyces cerevisiae species. All other yeast species will be referred to by their taxonomic names.  xi i  Acknowledgements  The many people who assisted in this work deserve my deepest appreciation. The protein samples were generously supplied by Profs. Barry Nall, Jacque Fetrow and Fred Sherman. Crystals and conditions for crystal growth were provided by Connie Leung, Theresa Yang and Theresa Hii. One of the Pro7lIle data sets was collected in the laboratory of Prof. Louis Delbaere. The electrochemical studies were performed in the laboratory of Prof. Grant Mauk with the assistance of Paul Barker and Steve Rafferty. Past and present members of the laboratory of Prof. Gary Brayer contributed stimulating discussions and some of the computer software used in the determination and analysis of the structures. In addition, Yaoguang Luo collected the RepA2 data set with equipment provided by Prof. Daniel Yang. Over the course of these studies, Albert Berghuis became a close friend and collaborator and critically reviewed much of the thesis. I would like to thank David Burk for giving me a place to stay in the final months of my graduate studies. I acknowledge Gary Brayer, my research advisor, for his guidance, encouragement and the freedom to pursue my own projects. Also, I recognize the other members of my committee, Profs. Bob Molday and Steve Withers, for suggesting that the electrochemistry experiments would compliment the structural studies. The Medical Research Council of Canada is recognized for financial support in the form of a Studentship. I appreciate the support of my parents and family throughout my education. I am grateful to my spouse, Gail, for constant encouragement and support as well as for reviewing the thesis.  xm  Chapter 1  Introduction  A central question in the study of proteins is how a given amino acid sequence specifies the precise three-dimensional structure of a protein and how structure determines a specific biochemical function. One approach to this question is to examine the structure and function of variants of a well characterized protein. The comparison of available mutants with wild-type forms of a protein allows better definition of those amino acid sequences that result in a folded, stable and functional protein. One system which readily lends itself to these types of studies is that of cytochrome c.  1.1 The Mitochondrial Cytochromes c Mitochondrial cytochrome c is a small soluble electron transport protein that is readily isolated from eukaryotic organisms and has, as a result, been extensively studied (Dickerson & Timkovich, 1975; Ferguson-Miller et al., 1979; Timkovich, 1979; Poulos & Finzel, 1984; Mathews, 1985; Pettigrew & Moore, 1987; Moore & Pettigrew, 1990; Sherman, 1990). Cytochrome c is differentiated from other cytochromes by the nature of attachment of the heme prosthetic group. In this protein, the heme is attached to the polypeptide chain via two thioether linkages to Cysl4 and Cys17. The central iron atom is coordinated to four pyrrole nitrogen atoms of the heme group in a square planar arrangement. Two amino acids, His18 and Met80, provide the remaining axial ligands to form an octahedral coordination geometry. The polypeptide chain component, composed of between 102 and 112 amino acids, is synthesized in the cytoplasm and is transported into the inter-membrane space of the mitochondria by heme lyase. The heme lyase also catalyzes the covalent attachment of the heme group (Dumont et al., 1988).  1  2  Chapter 1. Introduction^  As a component of the mitochondria' electron transport chain, cytochrome c transfers single electrons from Complex  III to Complex IV (Pettigrew & Moore, 1987). Also known as cyto-  chrome c reductase or cytochromes b ci, Complex  III is a large membrane bound enzyme  complex involved in energy transduction. Cytochrome c oxidase (Complex IV) is also an integral membrane protein and functions to conduct electrons to the terminal electron acceptor,  02.  Cytochrome c oxidase is also a point of energy transduction in oxidative phosphorylation. Unlike these two membrane bound complexes, cytochrome c is an isopotential electron carrier in that the electrochemical potential of the electron is conserved during transport and no biological energy transduction occurs (Salemme, 1977). Historically, the cytochromes of the electron transport chain have been defined by their unique spectroscopic properties which arise from changes in the aromatic character of the heme chromophore. The visible spectrum of cytochrome c is characterized in the reduced state by three intense absorption Soret bands at 554 nm (a), 524 nm (13) and 416 nm (7). The a and 13 bands give this protein a red color. In the oxidized state, the y Soret band shifts to 410 nm and a new absorption maxima appears at 695 nm. This band has been proposed to be linked to the iron sulfur bond of the Met80 ligand (Schechter & Saludjian, 1967). These spectral properties are used to assess the structural integrity of the protein.  1.1.1 Structure The first amino acid sequence determined for a cytochrome c was that from horse (Margoliash  et al., 1961). Subsequently, the amino acid sequences of more than 90 cytochromes c from a wide variety of eukaryotic sources have been determined by chemical sequencing of their polypeptide chains. Several compilations and analyses of these sequences have been published (Dickerson Timkovich, 1975; Dayhoff & Barker, 1976; Moore & Pettigrew, 1990). Variability plots as a function of residue position have been computed based on these compilations (Louie et al., 1988a; Moore & Pettigrew, 1990). Typically, variability is defined as the number of amino acids at a given position divided by the frequency of the most common amino acid at that position  Chapter 1. Introduction^  3  (Wu Kabat, 1970). Another approach used to present the range of amino acid residues at a given position has been to create a diagram of the sequence set where the complete sequence of yeast cytochrome c is presented along with the other observed amino acids at each position (Hampsey et al., 1986; Hampsey et al., 1988). Inspection of a compilation of 94 sequences (Moore & Pettigrew, 1990) reveals that 27 out of 102 common amino acid positions are invariant and a further 16 are found in 90 of the sequences and are highly conserved. The sequences of yeast iso-1, yeast iso-2, tuna, horse and rice cytochromes c which represent a broad range of species are presented in Table 1.1. One region of the cytochrome c sequence, residues 70 to 80, is exceptionally conserved with only a total of 4 alternative amino acids observed in 4 out of 94 sequences in this region. In addition to the large number of amino acid sequences determined for mitochondrial cytochromes c, the detailed folds of five cytochromes c have been resolved by X-ray diffraction techniques (see reviews: Salemme (1977), Matthews (1985) and Brayer & Murphy (1993)). The first mitochondrial cytochrome c structure solved was that of oxidized horse heart (Dickerson  et al., 1971). Later, the same group improved the resolution of the cytochrome c fold by determining the structure of tuna cytochrome c in both the oxidized and reduced state (Takano Dickerson, 1981a,b). In the meantime, a low resolution X-ray study of bonito cytochrome c was undertaken (Tanaka et al., 1975; Matsuura et al., 1979). Since then the structures of rice cytochrome c (Ochi et al., 1983) and yeast iso-l-cytochrome c (Louie et al., 1988a; Louie & Brayer, 1990) have been reported. The yeast structure has also been refined in the oxidized state using isomorphous crystals (Berghuis & Brayer, 1992). Recently, a higher resolution redetermination of the horse structure has also been completed (Bushnell et al., 1990). Experimental details for all the structural studies completed to date are listed in Table 1.2. Of these, the yeast iso-1 structure has been completed to the highest resolution. As has been observed in other systems, a high degree of sequence homology is exhibited in the conservation of the three-dimensional structures of the cytochromes c (Chothia Lesk, 1986). The overall main chain structural and sequence homologies of the four available high  Chapter 1. Introduction  ^  4  Table 1.1: Primary sequence alignment for yeast iso-1, yeast iso-2, tuna, horse and rice cytochromes c  -9 Iso-1^- - - - TEFKA Iso-2 AKESTGFKP Tuna ^ Horse ^ Rice^- ASFSEAPP  Iso-1 Iso-2 Tuna Horse Rice  Iso-1 Iso-2 Tuna Horse Rice  30 PNL PNL PNL PNL PNL  YL YL YL YL YL  H G I H G I GL H GL N GL  1 67 SAKK G SAKK G DVAK G DVEK G NPKA  10  G ATL 17 KTR G ATL F KTR K K T F VQK G KKI F VQK G EKI F KTK .  40^ 50 FGR H S G Q A E G YS Y TD Q V K G YS Y TD FGR HS FGR KT G QAE G YS Y TD FGR KT G QAP FT Y TD FGR QS TTP G YS Y ST  80 70 T NP J KYIPGTKM T NP J KYIPGTKM E NPKKYIPGTKM E NPKKYIPGTKM L NP J KYIPGTKM  AFG AFA I FA I FA  G G G G VFP G  L KK L KK I KK I KK L KJ  C C C C C  AN AN AN AN AN  L Q A A A  20 QCHT VE K G GPH K V G QCHT TEE GPN K V G QCHT VEN G GKH K V G QCHT VEK GKH K T G QCHT V D K G AGH K Q G 60 DENNM S E DEDSMS E NNDTL ME KEET L ME EENTL YD  IKKNVL INKNVK KSKGIV KNKGIT KDMAV I  90 EKD EKD KGE KTE PQE  RN RN RQ RE RA  DL DL DL DL DL  IT IT VA IA IS  Y Y Y Y Y  100 LKK MTK LKS LKK LKE  ACEA A A A  AKTSTNE TS-  The primary sequences of yeast iso-1 (Smith et al., 1979), yeast iso-2 (Montgomery et al., 1980), tuna (Kreil, 1965), horse (Margoliash et al., 1961) and rice (Mori & Morita, 1980) cytochromes c have been aligned so as to maximize the structural homology present. The single letter code is used to identify amino acids and the residue numbering is based on the primary sequence of tuna cytochrome c. The amidation states of residues 52 and 54 of the rice protein have been modified as suggested by Moore and Pettigrew (1990). Note the single-letter code J is used to denote E-N-trimethyl lysine. Those amino acid residues identical in all five protein sequences are enclosed by boxes (residues denoted J and K are considered equivalent).  5  Chapter 1. Introduction^  Table 1.2: X-ray structure determinations of mitochondrial cytochromes c. Crystallization Conditions  Space Group  Unit Cell  Resolution  Referencest  Yeast Iso-1 (reduced) Yeast Iso-1 (oxidized) Tuna (reduced)  92% A.S., 0.1 M phosphate, pH 6.2, 40 mM DTT 92% A.S., 0.1 M phosphate, pH 6.2, 30 mM NaNO3 85% A.S., excess ascorbate, pH 7.5  P4 3 212  1.2  (a,b)  1.9  (c)  1.5  (d)  Tuna (oxidized)  50% A.S., 15% NaNO3, 1.0 M ammonium phosphate, pH 7.0 94% A.S., 0.1 M phosphate, pH 7.5 3.6 M A.S., pH 6.0  a=b=36.46, c=137.86 a=b=36.47, c=137.24 a=34.44 b=87.10, c=37.33 a=b=74.42, c=36.30  1.8  (e,f)  1.9  (g)  1.5  (h)  Cytochrome c  Horse (oxidized) Rice (oxidized)  P43212 P21212 P43 P43 P61  (A)  a=b=53.58, c=41.83 a=b=43.78, c=110.5  (A)  tReferences: a, Sherwood & Brayer (1985); b, Louie et al. (1988); c, Berghuis & Brayer (1992); d, Takano & Dickerson (1981a); e, Swanson et al. (1977); f, Takano & Dickerson (1981b); g, Bushnell et al. (1990); h, Ochi et al. (1983).  resolution cytochrome c structures are given in Table 1.3. The sequence identity varies from 57% between the yeast iso-1 and rice cytochromes c to 83% for the horse and tuna proteins. The r.m.s. main chain deviations range from 0.51 to 0.61  A.  The high structural homology between the cytochromes c of known three-dimensional structure allows the use of the yeast iso-l-cytochrome c structure to represent the general cytochrome c fold. This fold is schematically illustrated in Figure 1.1. In total, this protein can be separated into four classes of secondary structure elements (Table 1.4). The a-helix and two 13-turn classes are well known (for review: Richardson, 1981); however, Q-loops have only relatively recently been recognized by Leszczynski & Rose (1986). An C2 loop consists of 6 -  to 16 residues which form a loop such that the terminal C c, atoms are less than 10  A apart.  6  Chapter 1. Introduction^  Table 1.3: Structural equivalence and primary sequence identity of yeast iso-1, tuna, horse and rice cytochromes c  Yeast iso-1^Tuna^Horse^Rice (reduced)^(reduced)^(oxidized) (oxidized) (108)^(103)^(104)^(111)  Yeast iso-1 Tuna Horse Rice  63 (61%) 0.40 (0.54) 0.48 (0.54) 0.40 (0.51)  0.43 (0.51) 0.45 (0.61)  60 (58%) 85 (83%) 0.49 (0.60)  62 (57%) 61 (59%) 64 (62%) —  The total number of amino acids and the oxidation state of each protein is given below its name in the heading. The upper triangular half of the matrix contains the number of identical residues in common between each pair of proteins aligned in Table 1.1. The percentage identity of the smaller protein is given in parentheses. The common 412 main chain atoms (residues 1 to 103) of each cytochrorne c were overlayed in a pairwise fashion by a least-squares method. The lower half of the matrix contains the average main chain distance deviations. The values in parentheses are the r.m.s. deviations of main chain atoms for each pair. These loops do not contain a-helices or /3-sheets as defined by Kabsch & Sander (1983). An examination of 270 Q-loops in 67 proteins reveals that these secondary structure elements form globular structures as compact as complete proteins (Leszczynski & Rose, 1986). Of the 108 amino acid residues of iso-l-cytochrome c, 52 (48%) are found to be in an a-helical conformation (Louie & Brayer, 1990). A further 51 amino acids (47%) have been defined as belonging to a-loops (Fetrow et al., 1989). The loops defined include part of Helix II (residues 49 to 54), Helix IV, all of the type II 0-turns and the 7-turn (Table 1.4). Helices II and IV which are shorter and more poorly formed than Helices I, III and V, are not considered helices according to the primary Q-loop definition. After accounting for overlapping definitions, 13 amino acid residues (12%) are not assigned to a secondary structure element. These include the 6 amino terminal residues (residues —5 to 1, Table 1.1), residues 56 to 59 that form part of a distorted /3-sheet (Louie & Brayer, 1990), a short segment (residues 85 and 86) that links  Chapter 1. Introduction^  7  Figure 1.1: The polypeptide chain of yeast iso-l-cytochrome c is represented as a ribbon. The heme group, the two thioether linkages to Cys14 and Cys17, as well as the two axial heme ligands to His18 and Met80, are also depicted.  Chapter 1. Introduction^  8  Table 1.4: Secondary structural elements in iso-l-cytochrome c  Element class Structural element Residues involved a-helix  C2-loop  0-turn  7-turn  Helix I^ 2-14 Helix II^49-55 Helix III^60-70 Helix IV^70-75 Helix V^87-102 Loop A^ 18-32 Loop B^ 34-43 Loop C^ 40-54 Loop D^ 70-84 Turn 1 (type I)^14-17 Turn 2 (type II)^21-24 Turn 3 (type II)^32-35 Turn 4 (type II)^35-38 Turn 5 (type II)t^43-46 Turn 6 (type II)^75-78 27-29  The a-helices, type II /3-turns and 7-turn are as defined in Louie & Brayer (1990). The St-loops are as defined by Fetrow et al. (1989). t Mediated through a water molecule. two secondary structure elements and the carboxy terminal residue, 103. As shown in Figures 1.2 and 1.3, the polypeptide chain of cytochrome c is folded around the heme group such that only one edge of the porphyrin ring of the heme is exposed to solvent. To a large extent, the porphyrin ring is surrounded by hydrophobic residues and forms an integral part of the hydrophobic core of the protein (Louie et al., 1988a). The total heme solvent exposure is less than 10% of its total surface area (Bushnell et al., 1990). The two negatively charged heme propionate groups are also buried in the protein matrix and are completely inaccessible to solvent. These charged groups form numerous hydrogen bonds as well as a salt bridge to the polypeptide component (Dickerson et al., 1971). These studies also show that the heme group is not planar, but is distorted into a saddle shape (Louie & Brayer, 1990).  Chapter 1. Introduction^  9  Figure 1.2: A space-filled representation of yeast iso-l-cytochrome c. The atoms of the central heme group are shown as colored black spheres. All other atoms are colored white.  Chapter 1. Introduction^  10  Figure 1.3: A stereographic representation of iso-1-cytochrome c with side chains drawn in thin lines and the polypeptide backbone and heme group in thick lines. Every fifth residue and the two termini are labeled according to the sequence alignment in Table 1.1. Four water molecules form an essential part of the structure of yeast cytochrome c. The first two, Wat121 and Wat168, are hydrogen bonded to the heme propionate A and the guanidinium group of Arg38. In the tuna and horse structures, the side chain of Arg38 interacts directly with the propionate group and Wat168 is not present. A third distinct conformation of the guanidinium group in rice cytochrome c results in a hydrogen bond network that includes two water molecules in the same position as Wat121 and Wat168 of the yeast iso-1 protein. The other two essential waters in the yeast structure are Wat110 and Wat166. Wat110 is located near the His18 ligand and Wat166 is hydrogen bonded to three residues near the Met80 ligand: Asn52, Tyr67 and Thr78. Wat166 is conserved in all four known cytochrome c structures and has been suggested to be important in the mechanism of electron transfer (Takano & Dickerson, 1981b; Berghuis & Brayer, 1992).  1.1.2 Stability The thermodynamic stability of cytochrome c has been quantitatively characterized by reversible unfolding equilibrium studies using the denaturant guanidine hydrochloride (Knapp &  Chapter 1. Introduction^  11  Pace, 1974). The presence of the folded versus unfolded protein can be monitored by tryptophan fluoresence which is quenched by the heme in the folded state or alternatively by optical rotation at 220 nm. The guanidine hydrochloride data may be extrapolated to zero denaturant to give the free energy of unfolding under standard conditions (Schellman, 1978). A study of the stability of cytochrome c from horse, donkey, dog, cow, rabbit, chicken and tuna at 25 °C, pH 7, revealed a similar unfolding free energy of 7 ± 1 kcal/mol (McLendon & Smith, 1978). The results from this study for horse (7.38 kcal/mol) agreed well with earlier experiments which measured a value of 7.27 kcal/mol under similar conditions (Knapp & Pace, 1974). In contrast, the free energy of unfolding of four cytochromes c from three yeast species, genus  Saccharomyces and Candida, is found to be 3 to 5 kcal/rnol less stable than horse cytochrome c under similar experimental conditions (Table 1.5). The number of buried hydrophobic groups has been suggested to account for the difference in stability between the yeast iso-1 and iso-2 proteins and horse cytochrome c (Nall & Landers, 1981). Global thermodynamic stability has been suggested to be involved in the regulation of protein turnover in some organisms (Knapp  & Pace, 1974). The lower stability of the yeast proteins may be correlated with the induction of their expression whereas organisms such as mammals requiring a constant level of cytochrome c correspondingly produce a globally more stable protein. The structure of ferricytochrome c is known to be pH dependent from UV visible spectroscopic studies (Theorell Akesson, 1941). The native form of cytochrome c is observed in a pH range of about 5 to 8 depending on the species and oxidation state. However, oxidized cytochrome c undergoes an alkaline transition that is characterized by the loss of the 695 nm absorbance band, having an apparent pK a of approximately 9. The pK a values of the alkaline transition for 5 different wild-type cytochromes c and some mutant forms of the yeast iso-2 protein are presented in Table 1.5. The structure of the alkaline form has remained elusive, however, the loss of the 695 nm absorbance band has been associated with the loss of the Met80 ligand (Schechter & Saludjian, 1967). Further spectroscopic studies have suggested that a deprotonated lysine &amino group is the replacement ligand (Davis et al., 1974; Brautigan et al.,  Chapter 1. Introduction^  12  Table 1.5: Stability of oxidized cytochromes c  Cytochrome c^  AG of unfolding (kcal/mol)^pK a tkt Reference 20 °C, 0.1 M sodium phosphate pH 7.0 — 7.2  pH 6.0  A. Native yeast (S. cerevisiae) iso-l-MSt  3.6  8.5  (a,b)  yeast (S. cerevisiae) iso-2  3.8  8.5  (c,d)  horse  8.5 5.9 4.6  9.2 8.9 8.6  (e,e) (e,e) (e,e)  Candida krusei Saccharomyces oviformis B. Yeast iso-1 mutants Pro71Val-MSt  2.6  (a)  Pro71Thr-MSt  1.9  (a)  Pro71Ile-MSt Asn52A1a/Cys102Ala  1.9 5.7  (a) (h)  Asn52Gly/Cys102Ala  3.8  (h)  Asn52I1e/Cys102Ala  7.0  (h)  Cys102Ala  4.7  (h)  C. Yeast iso-2 mutants Pro71Thr Pro76Gly  3.0 2.6  6.6 6.7  (d) (f,g)  References: a, Ramdas et al. (1986); b, Pearce et al. (1989); c, Osterhout et al. (1985); d, White et al. (1987); e, Saigo (1981); f, Wood et al. (1988b); g, Nall et al. (1989); h, Hickey et al. (1991) tpK a ik is the apparent pl-C a of the alkaline transition. t Residue Cys102 of these cytochromes c was modified with methyl methanethiosulfonate to prevent dimerization.  Chapter 1. Introduction^  13  1977). Acetimidylation of lysine residues has suggested that Lys72 or Lys79 may be the heme iron ligand in the alkaline form (Wallace, 1984), however, mutation of these residues to alanines did not prevent alkaline isomerization (S. Inglis, unpublished results). Inspection of the X-ray structure of cytochrome c reveals that a drastic conformational change would be required to position an c-amino group of a lysine residue near the heme iron. A NMR study of the alkaline form provides evidence for the presence of at least two conformations of cytochrome c at high pH (Hong & Dixon, 1989). The kinetics of the transition involve a rapid deprotonation step followed by a slow conformational change (Davis et al., 1974). Folding kinetic studies of iso-2cytochrome c at high pH have shown that the alkaline form is preceded by an intermediate with spectroscopic characteristics similar to those of the native protein (Nall, 1986). These results suggest that the alkaline form does possess some of the native structure observed at neutral pH.  1.1.3 Folding The kinetic properties of unfolding and refolding of horse cytochrome c by rapid changes in guanidine hydrochloride concentration were initially characterized by monitoring absorbance (Ikai et al., 1973), and later by following tryptophan fluoresence quenching (Tsong, 1976). The structures of folding intermediates have also been characterized by two-dimensional NMR spectroscopy of pulse labeled proteins (Roder et al., 1988). These studies suggest that folding is initiated by the formation of Helices I and V (Table 1.4) followed by contact between these two helices in a manner similar to the native structure. Detailed studies of the kinetic properties of folding of yeast iso-2-cytochrome c have revealed the presence of four kinetic phases. The fastest of these phases (T3) with a time constant of the order of 1 ms is poorly characterized (Nall Si Landers, 1981). Two of the phases err and T2) are observed to yield a functional product as determined by absorbance at 695 nm and ascorbic acid reduction. The differences in rate between the fast (T2) and slow (Tf) absorbance detected phases appears to arise from differences in proline isomerization in the unfolded protein. The  Chapter I. Introduction^  14  product of another phase OP detected by tryptophan fluoresence may either be a folding dead end or an intermediate on a different path to the native form (Nall, 1983). Further studies of the temperature dependence of phases rf and  rt yield activation enthalpy values in the  range expected for proline isomerization. Furthermore, the amplitudes of phases Ti and rt are independent of initial pH, suggesting the involvement of proline isomerization rather than heme ligation in these kinetic phases (Osterhout et al., 1985; Osterhout & Nall, 1985). The presence of a free sulfhydryl group in yeast iso-1-cytochrome c has interfered with the accurate characterization of folding intermediates of this protein. Cys102 of the iso-1 protein can be blocked by treatment with iodoacetamide (Zuniga & Nall, 1983) to prevent dimer formation. An examination of the folding kinetics of iodoacetamide blocked iso-l-cytochrome c revealed similar kinetic phases as observed for the iso-2 protein (Zuniga & Nall, 1983).  1.1.4 Function The functional requirements of cytochrome c are to accept and donate single electrons with biological redox partners. In this role, cytochrome c must be able to bind to a given redox partner and provide a low energy path for the electron to travel between the redox centers located within each protein. This last requirement also implies that the midpoint potential of cytochrome c must be at a level that favors electron transfer thermodynamically between the redox partners (Salemme, 1972). The midpoint reduction potential is a physical property of cytochrome c that has been measured both spectroscopically (Kreishman et al., 1978) and by direct electrochemistry (Armstrong et al., 1988). Measurement of the reduction potential of several cytochromes c from different sources reveals that this physical property is highly conserved (Table 1.6). By measuring the temperature dependence of the midpoint potential, the enthalpic and entropic contributions to the free energy of reduction may be determined. These thermodynamic parameters are also conserved in the five species studied as described in Table 1.6. Further studies have shown that  15  Chapter I. Introduction^  Table 1.6: Electrochemical properties of cytochromes c from different sources Cytochrome c  Yeast iso-1 Horse heart Tuna heart Turkey heart Candida species  ° Em (mV vs SHE)  0 AH (kcal/mol)  0 AS e.u.  261 261 265 260 260  -17.1 -16.8 -17.5 -16.8 -16.8  -37 -36 -39 -36 -36  Experimental conditions were 25 °C, pH 7.0, p, = 0.01 and SHE reference. The data was taken from Table 3 of Margalit Schejter (1973a). the reduction potential is sensitive to ionic strength and to the presence of ions that bind preferentially to one oxidation state such as chloride and phosphate (Margalit Schejter, 1973a; Margalit Schejter, 1973b). Recently, direct electrochemistry has been used to measure the midpoint reduction potential of yeast iso-l-cytochrome c (Rafferty et al., 1990). The higher potential given in Table 1.7 is due to the difference in ionic strength, the presence of different ions and the presence of small amounts of the alkaline form. An initial explanation for the high midpoint potential of cytochrome c relative to model compounds with the same axial ligands was given by Kassner (1972,1973). Using a simple electrostatic model of an ion in a sphere of constant dielectric, Kassner showed that hydrophobic groups surrounding the heme displaced solvent molecules which stabilize the positive charge on the heme in the oxidized state. Destabilization of this positive charge increases the reduction potential of cytochrome c. A related structural parameter, heme solvent exposure, has also been proposed to affect the midpoint reduction potential (Stellwagen, 1978). Schejter et al. (1982) extended the work of Kassner by separating the free energy of reduction into an electrostatic and non-electrostatic component and showed that the electrostatic term can model changes in molecular surface charge. Another electrostatic model that uses dipoles to model both the protein and the surrounding solvent has been used to demonstrate their individual roles in  16  Chapter 1. Introduction^  Table 1.7: Electrochemical properties of yeast cytochrome c and mutants  Cytochrome c  Decreased AH  0  0  290 ± 2  -14.0 ± 0.2  -9.1 ± 0.4  -6.7 ± 0.1  (a)  -14.6 ± 0.2 -14.9 ± 0.1 -14.2 -14.6 ± 0.1 -14.2  -11.6 ± 0.7 -12.6 ± 0.4 -10.7 -12.3 ± 0.3 -12.0  -6.5 -6.5 -6.3 -6.3 -6.0  ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1  (a) (b) (c) (a) (c)  -13.6 -13.0 -12.9 -12.3 -12.7 -12.6 -12.3 -12.4  -8.3 -8.1 -8.0 -6.3 -8.0 -12.5 -7.5 -8.2  -6.5 -6.0 -5.9 -5.7 -5.7 -5.4 -5.4 -5.4  ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1  (a) (a) (d) (d) (a) (e) (d) (e)  -5.9 ± 0.1 -5.7 ± 0.1 -5.5 -5.4  (a) (d) (c) (c)  0 and AScyt  0  ±2 ±2 ±2 ±2 ±2  0 and AScyt  280 260 257 249 247 234 234 232  F82Y F82A N52A T78G F82G N52IY67F Y67F N52I  F82S I75M Y48FW59F R38A  AG° (kcal/mol)  286 280 274 273 262  F82L L94S W59F F82I Y48F  Increased AH  ASc0 yt (e.u.)  E.m  Iso-1  Increased AH  AH0 (kcal/mol)  Referencet  (mV)  ±2 ±2 ±2 ±2 ±2 ±2 ±2 ±2  ± 0.1 ± 0.2 ± 0.1 ± 0.2 ± 0.2 ± 0.1 ± 0.1  ± 0.3 ± 0.5 ± 0.3 ± 0.2 ± 0.4 ± 0.6 ± 0.4  0 and decreased AS cyt  255 249 240 236  ±2 ±2 ±2 ±2  -13.8 ± 0.2 -13.8 ± 0.2 -13.0 -13.5  -11.0 ± 0.5 -11.7 ± 0.3 -9.4 -11.7  Experimental conditions were 25 °C, pH 6.0, it 0.1 M and SHE reference. tReferences: a, Rafferty et al. (1990); b, Lo et al. (1993); c, Tong et al. (1993); d, Rafferty, 1992; e, Berghuis (1993) .  Chapter 1. Introduction^  17  modulating reduction potential (Churg^Warshel, 1983; Langen et al., 1992). The charged heme propionates and their interaction with the solvent and polypeptide chain has also been proposed to be important in stabilizing the heme iron in the oxidized state (Moore, 1983). Taniguchi et al. (1980) showed that the entropy of reduction of cytochrome c is more negative than that measured for inorganic iron complexes. This decreased entropy was proposed to be due to an ordering of the polypeptide chain and the surrounding solvent upon reduction. The entropy and enthalpy of cytochrome c reduction has been measured in differing halide solutions (Kreishman et al., 1978) with the temperature dependence of the reduction potential observed to be biphasic at 42 °C in 0.1 M NaC1 solutions. These results indicate that solvent structure may play an important role in determining the entropy of reduction of cytochrome c. Furthermore, the volume of cytochrome c has been shown to decrease upon reduction (Trewhella et al., 1988) which may result in a reorganization of the surface solvent structure (Margalit Schejter, 1973a). Changes in the hydrogen bonding of surface solvent water molecules have been shown to be associated with compensating changes in enthalpy and entropy (Lumry Rajender, 1970).  1.2 The Yeast Cytochromes c Unlike other eukaryotic organisms, two isozymes of cytochrome c (iso-1 and iso-2) have been isolated from the yeast Saccharomyces cerevisiae. These two proteins are believed to have equivalent physiological roles (Mattoon & Sherman, 1966; Dethmers et al., 1979). The amount of each of these isozymes produced is controlled by differential regulation of the transcription of their respective genes in response to the presence of glucose, heme, and molecular oxygen (Laz et al., 1984). Of the cytochrome c in yeast, the relative amount of the iso-2 form present is dependent on environmental conditions, varying from 5 to 25% (Sels et al., 1965). The additional stability of apoiso-2-cytochrome c to cellular degradation relative to apoiso-1 provides another mechanism for a greater proportion of holoiso-2-cytochrome c under some conditions (Dumont et al., 1990). However, the rate of conjugation of ubiquitin to iso-2-cytochrome c in  Chapter I. Introduction^  18  vitro in a rabbit reticulocyte extract occurs at a rate five fold greater than for the iso-1 protein (Sokolik & Cohen, 1991; Sokolik & Cohen, 1992), suggesting that the iso-2 form may also be degraded at a different rate in yeast cells. Early mutational mapping led to the discovery of several nuclear genes required for production of yeast cytochrome c (Sherman et al., 1965). The structural genes for iso-1 and iso-2-cytochromes c were identified and named CYC1 and CYC7, respectively. The CYC3 gene has been identified as coding for heme lyase (Dumont et al., 1987) which is required for heme attachment and mitochondrial import of both yeast cytochromes c. Other genes were found to be involved in heme synthesis. Recently, another mitochondrial protein involved in cytochrome c import was identified as the product of the CYC2 gene (Sherman, 1990). The  CYC1 gene was cloned and sequenced by several groups (Montgomery et al., 1978; Smith et al., 1979). Subsequently, the iso-2-cytochrome c gene (Montgomery et al., 1980) was cloned and sequenced which confirmed that the iso-2-cytochrome c amino acid sequence (Dayhoff & Barker, 1976) differs from that of iso-1 by 17 substitutions and a four residue extension at the amino terminus (Table 1.1). The iso-1 and iso-2-cytochromes c have an overall 84% amino acid sequence identity.  1.3 Yeast Cytochrorne c Variants The first cytochrome c mutants were produced by treatment of yeast cells with UV radiation or chemical mutagens followed by the isolation of mutants using genetic techniques (Sherman et al., 1965). Non-functional cytochrome c mutants were screened by growth on media containing chlorolactate which inhibits those yeast cells capable of metabolizing lactate (Sherman et al., 1975). The amount of cytochrome c in intact yeast cells was measured by low temperature spectroscopy at the 550 nm a band (Sherman & Slonimski, 1964). The functional capacity and temperature sensitivity of the mutants were measured by growth of yeast cells on lactate media at varying temperatures (Schweingruber et al., 1977; Schellman, 1978). These mutants were further analyzed by genetic deletion mapping (Sherman et al.. 1975) and amino acid or DNA  Chapter 1. Introduction^  19  sequencing (Hampsey et al., 1986). Using these techniques, 44 cycl non-functional missense mutants have been identified and mapped as 30 different substitutions at 16 sites (Schweingruber et al., 1979; Hampsey et al., 1988). No mutations have been isolated that abolish function without reducing the quantity of protein in intact cells. These results suggest that all of the mutant proteins are temperature sensitive (Hampsey et al., 1988). Mutations at three residues, Cysl4, Cys17 and His18, resulted in a complete lack of protein in intact cells. These residues form either thioether or iron ligand linkages to the heme and are essential either for heme attachment or mitochondrial import. Mutations at the other heme ligand, Met80, also resulted in poor production of holo-enzyme. Other mutations resulting in less than half the normal in vivo level of cytochrome c are Gly6Asp, Leu9Pro, Gly29Asp and Gly29Asn. Gly6 is part of a section of Helix I which packs against Helix V (Table 1.4) such that a larger side chain cannot be tolerated at this position. Leu9 is also part of Helix I and a proline at this position prevents the main chain at position 9 from forming the hydrogen bonds necessary for adopting an a-helical conformation. Other non-functional mutations did in fact lead to the production of greater than 50% of the normal level of cytochrome c, however, the functional capacity of these cytochromes c were less than 5% of the wild-type protein as judged by growth on lactate. These include mutations of the hydrophobic residues that line the heme pocket: Leu32, Trp59, Tyr67, Leu68, Leu94 and Leu98. Mutation of either Pro30 or Pro7l to a leucine also resulted in a folded but completely non-functional protein. The His33Pro mutant is also non-functional, however, the amount of His33Pro cytochrome c observed in intact yeast cells was more than twice that of the Leu9Pro mutant. Residue 33 appears to be less critical for folding, but directly disrupts the electron transfer function of cytochrome c. In addition to non-functional mutants, a large number of partially to fully functional mutants, as measured by growth on lactate media, have been isolated (Sherman et al., 1974). For example, 85 amino acid replacements within the first 9 amino acid residues, as well as the complete deletion of this region, appears to be tolerated in yeast iso-1-cytochrome c (Hampsey  Chapter 1. Introduction^  20  et al., 1988). Some of the other functional replacements are of highly conserved or invariant residues, such as Tyr98Leu, Pro76Leu and Lys72Glu. These observations suggest that invariance does not necessarily imply an absolute functional requirement for these residues. However, all of the non-functional mutations with the exception of mutations to proline residues, occur at highly conserved or invariant residues (Hampsey et al., 1988). The semi-synthesis technique has been used to create mutants of horse cytochrome c (Offord, 1987). In this technique, cytochrome c is cleaved into fragments with cyanogen bromide and limited tryptic digestion. The fragments are separated, modified or replaced with synthetic substitutes, reassembled and the modified protein is then characterized. A comprehensive overview of semi-synthetic studies of cytochrome c is presented in Louie (1990). The variants produced by some semi-synthetic methods were difficult to accurately characterize due to the loose structure resulting from the non-covalent interaction of multiple peptide fragments. However, when a single site cyanogen bromide cleavage between residues 65 and 66 in the horse protein was employed, the homoserine lactone fragment (residues 1 to 65) could be religated to the carboxy terminal fragment (residues 66 to 104) to produce a molecule with properties similar to the wild-type protein (Corradin Harbury, 1974). Using this technique, chemically synthesized peptides may be substituted for the carboxy terminal fragment allowing the introduction of any amino acid, including non-natural amino acids, into the protein fold at positions 66 through 104. This technique has been exploited to replace the Met80 ligand with other functional groups to produce cytochromes c with unusual electrochemical properties (Wallace & Clark-Lewis, 1992). Recently, the mutations Met64Leu and Ser65Met have been made in the yeast protein to make its sequence in this region similar to that of the horse (Table 1.1) and thereby allow use of the semi-synthetic technique in conjunction with site-directed mutagenesis (Wallace et al., 1991). The cloning and expression of the genes of the two isozymes of yeast cytochrome c and the development of site-directed mutagenesis methods provides a means of producing specific mutants at any desired residue position (Inglis et al., 1991; Kunkel et al., 1987; Zoller & Smith, 1983). Initial structure-function studies using the technique of site-directed mutagenesis were  Chapter I. Introduction^  21  aimed at Phe82 which has been proposed to be critical in the mechanism of electron transfer (Pielak et al., 1985). Site-directed mutagenesis was required to produce mutants of this invariant residue since no mutants had been isolated by classical genetics. Replacements at this position give proteins with significantly altered electrochemical properties (Rafferty et al., 1990) and inter-protein electron transfer kinetics (Liang et al., 1988). Two Phe82 mutants, Phe82Ser and Phe82Gly cytochromes c. have been characterized structurally by X-ray crystallography (Louie et al., 1988b; Louie & Brayer, 1989). The Phe82Ser mutant structure revealed that a solvent channel had been created which increased the solvent exposed surface area of the heme. The increased heme solvent accessibility was suggested to account for the 45 mV drop in reduction potential of this mutant (Louie et al., 1988b). Structural analysis of the second mutant, Phe82Gly, revealed a refolding of the polypeptide chain in the region of residue 82 such that polar peptide groups packed against the heme. As in the Phe82Ser mutant, the increased heme polarity is proposed to account for a drop in reduction potential (Louie & Brayer, 1989). Mutants isolated by classical genetic techniques have also been used as a guide for generating site-directed mutants. For example, the Asn52Ile mutant of yeast cytochrome c was initially discovered as a second site revertant to a number of temperature sensitive mutants (Hickey  et al., 1988). Site-directed mutagenesis was then used to create the Asn52Ile mutant of iso-lcytochrome c and to characterize its structure and thermostability (Hickey et al., 1991). The thermostability of Asn52Ile was measured with the additional mutation Cys102Ala to prevent dimerization and a loss of reversibility upon unfolding of yeast iso-1-cytochrome c. Structural studies revealed that the greatly enhanced thermostability of the Asn52Ile mutation (Table 1.5) may be due to the displacement of a buried water molecule, Wat166 (Hickey et al., 1991). The normally resident residue Asn52 forms a hydrogen bond with Wat166 which has been proposed to be important in mediating oxidation state dependent conformational changes (Takano Dickerson, 1981b; Berghuis & Brayer, 1992). Several mutants of yeast cytochrome c have been analyzed to determine how the amino acid sequence influences the thermodynamics of reduction (Table 1.7). These mutants could  Chapter 1. Introduction^  22  potentially be divided into four categories based on whether each of the enthalpic and entropic contributions to the free energy of the reaction has been increased or decreased. However, only mutants in three categories are presented in Table 1.7. None of the mutants studied thus far possesses a reduction potential greater than that of native yeast iso-l-cytochrome c at 25 °C, and thus, no mutations have increased the enthalpy while decreasing the entropy of reduction. Mutants with lower reduction potentials are observed in the other three categories. The mutant with the lowest reduction potential is Asn52Ile cytochrome c (Table 1.7). The decrease in reduction potential of 58 mV resulted from a large increase in the enthalpy and a small increase in entropy of reduction. A similar 54 mV decrease in reduction potential was observed as a result of the Arg38Ala mutation, however, the thermodynamic contributions differed. In this mutant, the enthalpy of reduction increased while the reaction entropy decreased. The mutants in the third category, those in which both the enthalpy and entropy of reduction decreased, result in more modest changes in reduction potential. The Tyr48Phe cytochrome c represents the lowest potential, a decrease of 28 mV.  1.3.1 Pro71 Replacements Genetic fine structure mapping of four non-functional cycl mutants revealed a common lesion site (Sherman et al., 1975). DNA sequencing of these mutants indicates that proline was replaced by a leucine at position 71 (Ernst et al., 1985). These mutants were further treated with mutagens and partially functional revertants were selected by growth on lactate at 30 °C (Sherman et al., 1983). DNA and protein sequence analyses have identified four different revertants: Pro7lIle, Pro7lSer, Pro7lThr and Pro71Val cytochromes c (Ernst et al., 1985). From growth rates of yeast cells on lactate media, the function of these mutant cytochromes c have been estimated to be: Pro7lVal, 90%; Pro7lThr, 60%; Pro7lSer, 30%; and Pro7lIle, 20%. The lack of growth of the original Pro7lLeu mutant suggests that this mutant is completely nonfunctional. Other revertants that could have been generated by single base pair substitutions included: phenylalanine, tyrosine, glutamate, glutamine, lysine and arginine. Since none of  Chapter I. Introduction^  23  these amino acid residues were observed out of the 29 analyzed, they are also suggested to be non-functional replacements. These results suggest that structural or functional requirements constrain the amino acid at position 71 to small nonpolar residues (Ernst et al., 1985). 1.3.2 Composite Proteins  A composite protein is defined as a protein with segments of polypeptide chain derived from more than one wild-type protein. Sequence analysis of a series of revertants of a nonsense mutation of iso-l-cytochrome c at position 71 revealed that some reversions resulted from a non-allelic recombination with the CY C7 gene for iso-2-cytochrome c (Ernst et al., 1981). The resulting composite cytochromes c are composed of segments of the iso-1 and iso-2 proteins. In each of 14 composite iso-l-cytochromes c identified by peptide mapping and amino acid sequencing, a single iso-2 polypeptide chain segment representing 13% to 61% of the total amino acid sequence was present (Ernst et al., 1982). These composites were grouped into five classes based on their amino acid sequences. The minimum unique amino acid segment derived from the iso-2 gene for each class of composite is detailed in Table 1.8. The composites of Class V were most frequently observed (9 of the 14 composites isolated). This class also contains the shortest segment of the iso-2 protein, 10 residues. Table 1.8: Minimal unique residue positional boundaries of iso-2 polypeptide chain segments in each composite iso-l-cytochrome c class Class I II III  IV V  Positional Boundaries —1, 26 15, 63 26, 63 26, 83 54, 63  Yeast Strains B-2125 B-1155, B-2036 B-1585 B-2080 B-1904, B-2037, B-2038. B-2039, B-2047 B-2203, B-2204, B-2205, B-2200  Chapter 1. Introduction^  24  The composite cytochromes c from three yeast strains (B-1904, B-2080, B-2036) representing Classes II, IV and V have been further characterized (Dumont et al., 1990). All of the composites are less stable to thermal denaturation than either of the native isozymes from which they are derived. The thermal transition temperatures of iso-1 and iso-2-cytochromes c are 52.3 and 54.2 °C, respectively. The transition temperatures of the composites range from 48.9 to 49.3 °C. To achieve reversible conditions, the composites and iso-1-cytochromes c were treated with methyl methanethiosulfonate to specifically block the sulfhydryl group of Cys102 to prevent dimerization in the unfolded state. The thermodynamics of unfolding of these composite proteins, as well as the two native isozymes of yeast, are being further investigated by differential scanning micro-calorimetry (J. Liggins and B. Nall, unpublished results). Like wildtype iso-2-cytochrome c, all composite proteins with the iso-2 sequence from positions 54 to 63 are more stable to cellular degradation in their apo forms than the iso-1 protein (Dumont et al., 1990).  1.3.3 Q-loop Replacements a-loop replacements are a special class of composite protein where the replaced segment of polypeptide chain is an Q-loop. As described previously, four Q-loops comprising 47% of the total structure have been defined for the yeast iso-l-cytochrome c (Table 1.4). Oligonucleotidedirected mutagenesis has been used to delete and replace segments of these four Q-loops to study their role in cytochrome c biosynthesis and activity (Fetrow et al., 1989). Deletions within Q-Loop A. residues 22 to 28, and Q-loop D, residues 74 to 78, resulted in a complete deficiency of cytochrome c in intact yeast cells (Fetrow et al., 1989). In contrast, the deletions of either residues 37 to 40 in a-loop B or 47 to 54 in Q-loop C produced partially functional cytochrome c molecules. In the loop swap studies, Q-Loop A was replaced with the corresponding loops of four cytochromes c from tuna, Rhodospirillum rubrum c2, Pseudomonas denitrificans c550, and Pseudomonas aeruginosa c551, respectively (Fetrow et al., 1989). The tuna, R. rubrum and  Chapter 1. Introduction^  25  P. denitrificans loop replacements are of the same residue length or longer and result in cytochromes c that are partially functional. However, when Q-Loop A is replaced with the equivalent but shorter loop from P. aeruginosa cytochrome c551, the composite protein did not support growth on liquid lactate media although adequate levels of protein are present in intact cells (Fetrow et al., 1989). A fifth a-Loop A replacement was constructed using a larger loop unrelated in amino acid sequence which had been derived from porcine pancreatic esterase. This a-Loop A replacement was shown to lead to a partially functional cytochrome c (Fetrow et al., 1989).  1.4 Thesis Objectives Yeast cytochrome c is a biochemically and genetically well characterized protein ideally suited for study of the relationship between protein structure, stability and function. The genes of the two isozymes of cytochrome c from yeast have been cloned, sequenced and are amenable to site-directed mutagenesis. Also, a large number of random functional and non-functional mutants have been isolated by genetic techniques. Furthermore, the heme prosthetic group is a strong chromophore which allows for spectroscopic studies of this protein. In addition, a high resolution structure of yeast iso-1-cytochrome c is available as well as those of horse, tuna, and rice. In this work, three classes of mutants were selected for detailed investigation in order to gain insight into the functional and folding properties of cytochrome c: composite proteins, 1-loop replacements and single site mutations of the invariant proline at position 71. The B-2036 cytochrome c studied is a Class II yeast composite cytochrome c (Table 1.8). The wild-type yeast iso-2-cytochrome c was also investigated to allow comparison of this composite with both parent wild-type proteins. Both, the three-dimensional structures and electrochemical properties were determined for iso-2 and B-2036 cytochromes c. The results of these studies were combined with the data available for iso-1-cytochrome c allowing for a detailed comparison of the structure and function of all these proteins. Further analyses were initiated to  Chapter 1. Introduction^  26  compare these structures to the available in vivo and in vitro stability studies so that the relationship between the structure, stability and function could be better understood. The B-2036 and iso-2-cytochrome c proteins used for these studies were provided by Dr. B. Nall (University of Texas). The electrochemical properties were measured in the laboratory of Dr. G. Mauk (University of British Columbia). The second class of mutants investigated were a-Loop A replacements of yeast iso-1-cytochrome c. The first, RepA2 cytochrome c, contains the equivalent loop from Rhodospirillum rubrum cytochrome c2. The aim of this study was to investigate the functional role of Q-loops in cytochrome c and to evaluate if these loops are interchangeable between related proteins. A mutation of this loop replacement, RepA2(Va120) cytochrome c, was investigated to determine the role of residue 20 in stabilizing the loop-protein interface and in the overall function of yeast cytochrome c. The protein samples of both a-Loop A replacements were provided by Dr. J. Fetrow (State University of New York at Albany). The electrochemical properties of these two mutant proteins were measured in the laboratory of Dr. G. Mauk (University of British Columbia). The third class of mutants was of an invariant proline residue at position 71. The structures of four mutants, Pro7lAla, Pro7lIle, Pro7lSer and Pro7lVal cytochromes c were elucidated. The mutants were investigated to determine the role of this proline residue in directing the fold of cytochrome c and to examine the mechanism by which these amino acid replacements alter the stability and function of cytochrome c. Protein for these studies was provided by Dr. F. Sherman (University of Rochester).  Chapter 2  General Experimental Methods  An overview of the techniques used to determine structures by X-ray diffraction and to measure midpoint reduction potentials are described in this chapter. The details of each structural determination are given in the following chapters.  2.1 Structure Determination 2.1.1 Crystal Growth and Characterization The protein used in crystallization experiments was purified by established procedures (Sherman  et al., 1968; Nall & Landers, 1981) and was provided by collaborators. All of the crystals used in these studies were produced using the technique of hair seeding. The original seed crystals were derived from crystals of yeast iso-l-cytochrome  c (Sherwood & Brayer, 1985). This technique,  as it is applied to the hanging drop method (McPherson, 1982), is described in Leung  et al.  (1989). For the current work, this hair seeding technique was also applied to the liquid-liquid diffusion capillary crystallization method which was first described by Salemme (1972). In this method, a capillary tube (about 1.5 x 100 mm) is sealed at one end with a Bunsen burner. Solutions are then added to the tube with a syringe and forced to the bottom by hand centrifugationl. In a typical yeast cytochrome  c crystallization experiment, 30 pl of saturated  (NH4)•SO4 buffered to pH 6.0 to 6.5 with 0.1 M sodium phosphate and 10 to 80 mM reducing agent (dithiothreitol or sodium dithionite) is placed at the bottom of the capillary tube. A second less dense solution (10  id.) containing 80 mg/ml cytochrome c protein, 60% saturated  (NH4)2SO4 with the same buffer and reducing agent is placed near the tube opening. Seed 'The capillary tube is placed into a disposable centrifuge tube attached to a string and swung.  27  Chapter 2. General Experimental Methods^  28  crystals are then introduced by drawing a hair through a solution containing a crushed crystal of a wild-type or variant yeast cytochrome c and then by dipping the hair into the protein solution layered at the top of the capillary tube. This solution is then forced down the tube by hand centrifugation. To prevent mixing during centrifugation, the solution of highest density must always be at the bottom of the tube. Restricted by the small diameter of the capillary tube the two layered solutions mix over a period of days. Crystals of cytochrome c first appear after 12 to 36 hours and reach maximal size in 10 days. Crystals used in diffraction analyses were first transferred into a freshly prepared solution of the same buffer to maintain the reduced state of the heme group. This solution consisted of a slightly higher concentration of ammonium sulfate than the calculated final concentration used in the crystallization experiment. Dithiothreitol was substituted for Na2S2O4 in mounting solutions used for crystals grown with this reducing agent since Na2S2O4 forms a SO2 radical by a monomerization reaction which may damage the crystalline protein (Ferguson-Miller et al., 1979). The crystals were then mounted for X-ray diffraction analyses in thin wall glass capillaries with diameters of 0.7 to 1.0 mm depending on the size of the crystal. Precession photography was used to initially characterize cytochrome c crystals (Buerger, 1964). In this way, the unit cell dimensions of iso-2-cytochrome c were determined to be a = b = 36.43  A, c = 137.84 A and to belong to either of the two enantiomorphic space groups  P43212 or P41212 (Leung et al., 1989). Earlier studies had shown crystals of yeast iso-l-cytochrome c to be of the space group P43212 and to have unit cell parameters of a = b = 36.46  A,  c = 136.86 A (Louie et al., 1988a). Based on a comparison of precession photographs and unit cell parameters of the two isozymic crystals, it was assumed that these crystals were isomorphous. This assumption was subsequently confirmed by successful refinement of the iso-2 structure. The mutant crystals used were assumed to be isomorphous based on unit cell dimensions and diffraction symmetry. The assumption of isomorphism was verified by the successful refinement of these mutant structures. The volume per unit mass, V ii,, for space  Chapter 2. General Experimental Methods^  29  group P43212 assuming a single molecule per asymmetric unit, was computed from: abc V =In 8M  (2.1)  where a, b and c are the unit cell lengths and M is molecular weight of cytochrome c. The factor of 8 is the number of molecules in the unit cell. The V ii, for these yeast cytochrome c crystals is approximately 1.7 A 3 mol/g, indicating a solvent content of about 31% (Matthews, 1968).  2.1.2 Data Collection Data sets were collected for six of the eight forms of cytochrome c studied using an Enraf Nonius CAD4-F11 diffractometer. The CuK, radiation used was Ni filtered and generated from an Xray tube operated at 26 mA and 40 kV. The initial orientation of each crystal was found by small angle precession photographs. Crystals were then transferred to the diffractometer and an initial orientation matrix was derived from the angular position of one reflection and the alignment of a crystallographic axis parallel to the instrument cb axis (see Figure 2.4 for a description of data collection geometry). The orientation matrix and the cell parameters were then refined against a minimum of 16 strong centered reflections. The maximal resolution and peak scan width were selected based on the diffraction quality and size of the crystal. During data collection, each reflection was measured by a continuous SZ peak scan of 0.4 to 0.6° at 0.55°/min at an ambient temperature of 15 °C. In addition, a selection of 3 to 18 reflections were measured every 2 to 8 hours to monitor crystal decay and slippage. At least one cb independent reflection (x = 90°) was measured at 5° intervals in for use in absorption correction. Approximately 700 reflections were collected in a 24 hour period and a complete data set to 1.9  A was collected  in about 12 days. Each data set was complete to the chosen resolution and was obtained from a single crystal. Only small crystals with volumes less than 0.01 mrn 3 were obtained for the RepA2 and Pro7lIle mutants. These crystals were too small to collect diffractometer data with a standard sealed tube X-ray source. However, the combination of greater flux X-ray sources and area  Chapter 2. General Experimental Methods^  30  Diffracted beam  Direct beam  Figure 2.4: A schematic of the three circles which determine the crystal position in three-dimensional Eulerian space is presented. The crystal position is determined by progressive rotation about three angles, fl, x and 0. The diffraction angle, 20, is also indicated. detector technology allowed the collection of good quality data sets from these crystals. The two types of area detectors used to collect data were the Enraf Nonius FAST system and the Rigaku R-AXIS II imaging plate system. The oscillation method was employed in both instruments (Arndt & Wonacott, 1977). In this method, the crystal is oscillated through an angle 0 and the diffracted X-rays are collected on a two-dimensional detector in a fashion similar to using X-ray film. The rotating anode X-ray sources were operated at between 70 to 100 mA and 50 to 60 kV. In both systems, the crystal orientation and diffraction spot indexing was found by analysis of a minimum of two still frames collected at widely different 0 angles.  Hundreds of frames are typically collected on the FAST system with a small oscillation angle of 0.10 to 0.15° and a X-ray exposure time of 20 to 40 seconds per frame. The goniometer of the FAST system allows repositioning of the crystal to collect a complete data set. An oscillation  Chapter 2. General Experimental Methods^  31  angle of 0.7 to 1.5° and an exposure of 10 to 20 minutes per frame is used with the R-AXIS detector. The crystal must be manually repositioned and a new orientation found to collect the missing data cusp.  2.1.3 Data Processing A diffractometer reflection scan was divided and stored in three parts. The first 1/6 of a scan was taken to be one background intensity measurement, the middle 2/3 of the scan as the peak intensity (4), and the last 1/6 of the scan as the second background measurement. In the simplest case, the two background measurements are assumed to be representative of the background radiation of the peak and are used as the background correction. The expected statistical error in these measurements is the square root of the number of counts measured. For weak reflections with relatively few counts, this error is large. This is especially true for background measurements which are only 1/3 of the total scan. This statistical error may be decreased if one assumes that the background measurements of neighboring reflections are also representative of the current peak background. In the present study, this approach was taken and a list of candidate background measurements was made from reflections found within a sphere of reciprocal space surrounding the current reflection. An average background was then computed. The list of candidates (Ii) was then compared to the standard deviation of the average, and those measurements that differed by more than two deviations from the average were discarded. The final average background intensity (4) and the variance based on counting statistics (0 1) was calculated as follows: -  T  =^  (2.2)  2 4 at) = 77 1  (2.3)  --  where n is the number of background measurements included in the average after discarding those more than two standard deviations from the initial average. Note that the variance decreases as the number of background measurements increases, however, the assumption that  Chapter 2. General Experimental Methods ^  32  the neighboring background measurements are representative of the peak is weakened. If only the background measurements made adjacent to the peak are included, as in the individual background case, the variance is a special case of Equations 2.2 and 2.3 with n = 2. This individual variance was compared to the variance of the averaging calculation to determine if background averaging was used instead of the original individual backgrounds. Finally, the corrected peak intensity (io ) and error estimate (cri o ) was calculated by: /0 = /p  .1  0  0  —  4/b  (2.4)  420.b2  (2.5)  The factor of four arises since only a fourth of the time was spent to measure a single background compared to the peak. For non-spherical crystals, the average path of the incident and diffracted X-ray beams varies for each reflection measured. This average path difference results in a change in intensity due to absorption. This effect is strongly dependent on the crystal morphology and the data collection geometry. The method used to correct for absorption was that of North et al. (1968). The intensity of 0 independent reflections were measured at 5° rotation intervals of 0. The resulting 0 curve was then normalized and interpolated by a Fourier function to give an absorption curve  (P 4 ). This curve was then used to estimate the relative absorption (A) of a general reflection: 1 A = — [PA(cb -I- 0 cos(x)) PA(0 — 0 cos(X))1 2  (2.6)  The intensity and sigma of the reflection were corrected by dividing by the absorption factor. The above method does not account for the 0 dependence of absorbance. A linear interpolation on 0 between absorption curves computed from several / independent reflections was used to account for the 0 dependence. The absorption curves were normalized by assuming that total absorption over 0 is constant. The decay of diffracted intensities due to radiation damage was monitored periodically throughout data collection by a set of intensity control reflections. To account for the 8 dependence of crystal decay, intensity controls were collected in groups of similar resolution. A  Chapter 2. General Experimental Methods ^  33  polynomial was derived from a group of intensity controls of similar resolution by a least-squares fit. The B dependence was then calculated by normalizing the polynomials to the first intensity control group measured. The Lorentz effect arises from the different angles through which a reflection passes through the Ewald sphere during a scan. The equation used for both the Lorentz and polarization correction for diffractometer geometry is:  LP =  sin(20)  (1 — sin 2 (26)/2)  (2.7)  The intensity and sigma were multiplied by this factor. Symmetry related reflections and repeat measurements were merged by averaging to improve the accuracy of the data set. The estimated standard deviation in the averaged intensity (as) was computed by:  '2=1 = n3/2 E 2  2  (2.8)  The final intensity was converted to a structure factor amplitude by taking the square root. The final estimate of the error in the structure factor F was calculated by: QI^  aF = — =  0I -  (2.9)  A computer program, ICP 2 , was constructed to implement the data correction techniques outlined. In Figure 2.5, the 0 and  0 dependence of the background measurements obtained from a  data set collected from the Pro7lSer mutant are plotted. The greatest dependence is on 0, however there is also a correlation with 0, particularly at low resolution. In the past, individual backgrounds or a 0 dependent background function have been used to correct protein data sets for background radiation (Wyckoff, 1985). In these methods, the statistical quality of the data set is improved, however, systematic errors were introduced due to the dependence of the background radiation level on angle 'Intensity Correction Program  0 (Figure 2.5). The background averaging method described in  34  Chapter 2. General Experimental Methods^  80.0  C  z 0  60.0 0 L0) 0  a) 40.0-  v o  20.0  t^I^[^1^I^1^[^1^[ 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0  100.0  Phi Figure 2.5: A plot of average background measurements as a function of the angles 0 and 0 for data collected from the Pro7lSer mutant. Each curve represents one value of sin(e)/A: ^, 0.073; A, 0.128; 0, 0.180; 7, 0.227; 0, 0.264. The background radiation level is strongly 0 dependent and 0 dependent at lower 0 values. this work provides the needed improvement in the statistical precision of the background measurements and minimizes the introduction of systematic errors since only neighboring reflections are included in the averaging. In Table 2.9, the merging R-factor, the percentage of negative intensity measurements after background correction and the percentage of intensities that are greater than their estimated standard deviations based on counting statistics are given for data corrections including a varying number of neighboring background measurements. Clearly, including a modest number of neighboring background measurements significantly decreases the merging R-factor and reduces the number of negative intensities, indicating an improvement in the statistical consistency of the data.  35  Chapter 2. General Experimental Methods ^  Table 2.9: The effect of neighboring background measurements on data quality Leu85Phet  Pro7lSer No. of  4  2 6 10 14 22 38 74 102  n•  Pf -merget  T^n  (%)  (%)  (%)  (%)  43.7 34.1 31.8 30.7 29.4 28.6 27.8 27.8  20.0 18.8 18.0 17.8 17.3 17.0 17.1 17.2  54.1 58.8 60.4 60.9 61.5 61.8 62.4 62.6  34.7 28.8 27.4 26.8 25.7 24.8  T^T  -merge  .10 <  (%)  24.7 23.3 23.1 23.3 22.7 23.0  0  °V()) (%)  -10^  45.5 50.3 52.1 52.6 53.0 53.4  The number of reflections included in the Rni „ge calculation was 12087 and 9241 for the Pro7lSer and Leu85Phe data sets, respectively. tData for the Leu85Phe mutant was provided by T.P. Lo. t The merging R-factor over all intensities is defined as: ^Ehkl^ Rmerge  o jIz (hkl)  =^Ehki  E7-0 i ( hko i  The software used for the processing of data frames collected on the FAST instrument, MADNES, was written by Messerschmidt Pflugrath (1987). Due to the small oscillation angle, no single frame collected on the FAST instrument contains a complete diffraction spot. Adjacent frames are grouped together and a three-dimensional integration box is centered on an expected peak position. The R-AXIS data processing software is based on techniques described by Rossman et al. (1979). This method uses a two-dimensional integration box and combines partial peaks spread over two frames after intensity integration. In both methods, two scaling factors, K and B, are computed for each frame using symmetry related and duplicate intensity measurements by a least-squares fit of the expression: Is = Im x e B(sV- ) 2 -  -  ^  (2.10)  Chapter 2. General Experimental Methods ^  36  where Is and 'm are the scaled and measured intensities, respectively. These scaling factors also correct for crystal decay and absorption effects. The scaled intensities are merged, corrected for Lorentz and polarization effects, and are then converted to structure factor amplitudes. 2.1.4 Structure Solution and Refinement As discussed above, all crystal forms studied in this work were isomorphous. This isomorphism allowed the creation of an initial phase model for each data set through the construction of a model for the new form of cytochrome c based on the previous structural results obtained for iso-1-cytochrome c (Louie & Brayer, 1990). In the case of structures with mutations at only a few sites, the model was constructed by removing the side chain atoms at the sites of mutation except for the /3-carbon, to form alanine residues. The amino acid substitutions in the iso-2 and B-2036 proteins were modeled explicitly as described in Chapter 3. In the case of iso-2cytochrome c, no solvent was initially included in the refinement model, however, for the other mutant proteins, the sulfate ion and a selection of water molecules from the iso-1 structure were included in the initial refinement model. The solvent molecules were chosen based on how well they were conserved in the mutant structures previously determined and their thermal factors in the refined iso-1 structure. Difference maps were computed using the coefficients, Foil"' — Fiov, where Fio vi and Faiv are the observed structure factor magnitudes of the mutant and native structures, respectively. The resulting electron density maps were inspected with the program FRODO (Jones, 1978) as modified by S. Oatley and S. Evans. For some mutations, this type of map was used to determine the initial orientation of newly modified side chains. If the map did not suggest an unambiguous side chain placement, the side chain involved was modeled as an alanine residue and placed in the unit cell in the same orientation and position as the refined iso-1 structure. Structure refinement was then carried out by a restrained parameter least-squares procedure with the program PROLSQ (Hendrickson & Konnert, 1981) using data with F > 2o- F. A  Fo — F, electron density map was then computed to determine the conformation of the side  Chapter 2. General Experimental Methods^  37  chain of the mutant residue. Omit, Fo — Fc , 2F0 — F, and 3F0 — 211, maps were also examined at intervals during the course of refinement as a guide for the manual adjustment of side chains in the structure. Water molecules, modeled as oxygen atoms, were added to the model by searching for peaks in Fo — F, maps. A water molecule was included in the refinement if at least one hydrogen bond was formed to the existing structure and the oxygen atom refined to a reasonable thermal factor (less than 55 A 2 ).  2.2 Structure Analysis 2.2.1 Estimation of Coordinate Error In the analysis of structures of mutant proteins, atomic coordinates are compared to those of the native enzyme and other available mutant structures. A prerequisite to such a comparison is some estimate of the accuracy and reliability of the coordinate information. Three methods were used to estimate the coordinate accuracy of structure determinations. The first method, as described by Luzzati (1952), assumes that a complete data set was used in the structure determination and that the only error is the coordinate error. In this method, the conventional R-factor as a function of resolution is plotted, along with theoretical curves calculated by assuming various r.m.s. coordinate errors. Inspection of these plots for the structures described in this work suggests radial coordinate errors in the range of 0.15 to 0.20  A. This value is a  statistical estimate of the overall r.m.s. coordinate error, whereas the actual positional error of an individual atomic coordinate will depend on the location of the atom in the structure. However, neither of the intrinsic assumptions of this method was fully met. The data used for refinement was selected by the criteria that F > 2o F resulting in the use of 50 to 80% of -  the theoretically accessible data. In addition, the refinement method includes only an isotropic thermal factor term for each atom. The effect of this term on the coordinate accuracy is unknown. The second method uses an individual atom approach assuming a complete model and only coordinate error (Cruickshank. 1949; Cruickshank, 1954; Chambers & Stroud, 1979). The error  38  Chapter 2. General Experimental Methods ^  in the coordinates of a given atomic position are dependent on the fit between the observed and computed structure factors, the type of atom and the thermal factor of the atom. The overall r.m.s. error of the protein atoms (no solvent atoms included) of the proteins studied ranges from 0.15 to 0.27  A. As in the Luzzati method, the relatively incomplete solvent structure  models and the presence of disordered side chains in these structures violates the assumptions of the method. Nonetheless, the Cruickshank and Luzzati methods do give comparable global coordinate error estimates. A third indication of error in structure determinations may be derived by a comparison of the mutant structures to the wild-type yeast iso-1 structure. As a guideline, the expected overall r.m.s. deviation may be assumed to be equal to the overall r.m.s. deviation error computed from the error estimates provided by the Cruickshank method and the equation:  CA-B 7-7 1I Cr A2 +  4  (2.11)  where A and B are equivalent atoms in two different structures being compared, o A_B is the -  error in the positional deviation between atoms A and B, and uA and 5B are the coordinate errors of atoms A and B from the Cruickshank method. The actual observed overall r.m.s. main chain deviations and the calculated overall r.m.s. deviation errors using the equation above for the Pro71Ala, Pro71Ile, Pro7lSer and Pro71Val mutants are presented in Table 2.10. As expected, the computed r.m.s. deviation errors are smaller than the observed r.m.s. deviations because the calculations do not account for actual structural perturbations between the mutant and native structures. The formation of crystal contacts is another potential source of coordinate variability. A list of crystal contacts in the yeast iso-1-cytochrome c structure is presented in Louie and Brayer (1990). Since the structures studied in this work form crystals which are isomorphous with those of wild type yeast iso-l-cytochrome c, the regions of these proteins that form contacts in the crystal are very similar. Therefore, differential crystal contacts are unlikely to be a factor in the observed differences in comparisons between wild type yeast iso-l-cytochrome c and the cytochrome c variant structures studied herein.  Chapter 2. General Experimental Methods ^  39  Table 2.10: Comparison of overall observed main chain r.m.s. deviations and the expected deviation errors Mutant Structure^ Pro7lAla Pro7lIle Pro71Ser Pro71Val observed r.m.s. deviation (A) calculated r.m.s. deviation error (A)  0.18 0.14  0.16 0.10  0.19 0.13  0.22 0.14  The observed and calculated r.m.s. deviation of each mutant structure was calculated for the main chain atoms of residues 1 to 103. 2.2.2 Comparison of Structure Coordinates In the past, when a new protein structure became available the basis of protein structural analysis consisted of a detailed description of the observed structure. More recently, the availability of many closely related three-dimensional structures has required the development of new techniques for their analysis. This is particularly evident in the case of multiple structures of mutant proteins where a direct comparison to the native structure is desired. Direct visual comparison of related protein structures with computer graphic representations requires that the structures be superimposed. The structures depicted in all of the figures in this thesis were superimposed as follows. An orientation matrix was computed by a least-squares fit of the target structure to the parent structure of the common main chain atoms of residues —3 to 103 and the atoms of the heme group (43 atoms). The residues from the amino terminus to residue —4 were excluded since they are positionally disordered. The orientation matrix was then applied to the entire coordinate set of the target structure to produce a superimposed coordinate set. The iso-2 structure was used as the parent structure in the analysis described in Chapter 3, whereas the iso-1 structure is the parent structure in Chapters 4 and 5. An alternative to direct visual inspection of the atomic coordinates is the construction of difference matrices. A C a positional difference matrix is constructed by computing a set of intra-molecular C a — C a vectors for each structure. The matrix element m ii contains the difference in the length between the equivalent C a — C a, vectors in the two structures for residues  Chapter 2. General Experimental Methods^  40  i and j. A similar matrix may be constructed by comparing the difference in the average residue main chain thermal factors of two structures (Berghuis & Brayer, 1992). The matrices are symmetrical about the diagonal and thus the two types of matrices may be combined to eliminate the redundant information. In this work, the C, and thermal factor plots were combined by computing the absolute value of the matrix elements. The matrix was then contoured and plotted to provide a direct view of the main chain positional and thermal factor changes between two structures. One advantage of the difference matrix method of structural comparison is that it does not require previous superposition of the structures which may bias the comparison.  2.2.3 Comparison of Derived Structural Properties In addition to directly comparing the coordinates of two structures, a comparison of derived structural properties such as hydrogen bonds is useful in determining how structure is related to stability and function. In this study, hydrogen bonds were defined by the following criteria: a H...A distance < 2.60  A, 2.70 A or 3.05 A where A is an oxygen, nitrogen or sulfur atom  respectively; a D-H...A angle > 120°; and, a C-A...H angle > 90°. The hydrogen atom was positioned as close to the hydrogen bond acceptor as possible within the constraints of known hydrogen-heteroatom bond lengths and angles. The program Hbond created by S. Evans was used to define hydrogen bonds according to this scheme. The solvent exposed surface area is another frequently examined structural property of protein structures (Lee & Richards, 1971). In this study, the surface area exposed to solvent was computed by rolling a probe with a 1.4  A  radius over the molecular surface as implemented by Connolly (1983). All of the internal cavity volume measurements were computed by the algorithm described in Connolly (1985) using a probe radius of 1.1  A. Other derived structural properties of interest include average main chain  and side chain thermal factors, residue torsional angles and lists of residue neighbors.  Chapter 2. General Experimental Methods^  41  To date, few methods have been described that combine and compare these derived properties between many closely related structures. To address this issue for the cytochrome c structures discussed in this work, an information retrieval system was developed named SDBase. Filter programs were constructed to convert the results of several programs that produced lists of hydrogen bonds, torsional angles, solvent exposed surface area, neighboring contacts and average thermal factors to the format used by SDBase. In SDBase, both structurally derived and other related information such as literature references are stored and keyed by the name of the structure, the residue number and the type of information. The search program allows retrieval of specific combinations of information by the creation of regular expressions (Denning et al., 1978) to match the information keys. For example, a regular expression consisting of a string of ordinary characters would match any key which contained the same string. A sample SDBase search using this type of regular expression is described in Figure 2.6. The SDBase system was used in conjunction with molecular graphics programs to examine unusual structural features observed in mutant structures. In addition, this system was instrumental in preparing the tables of conserved and altered hydrogen bond interactions found in this thesis. Derived parameters from other native structures of mitochondrial cytochrome c computed from coordinates obtained from the Protein Data Bank (Bernstein et al., 1977) were also added to the database, allowing a broader comparison of the structural features of cytochrome c. A database containing more than 10000 separately keyed pieces of information on over 20 structures has been constructed and is routinely queried.  2.3 Electrochemical Properties 2.3.1 Direct Electrochemistry A schematic diagram of the direct electrochemistry experiment is presented in Figure 2.7. The experimental methods follow those described by Barker et al. (1989), Rafferty et al. (1990) and Rafferty (1992). A 25 mm diameter gold disk electrode was polished with increasingly fine grades of alumina from 0.3 pan to 0.05 pm and sonicated in deionized water. The electrode  Chapter 2. General Experimental Methods^  42  SDBase> position 26 SDBase> field bond name SDBase> structure isol iso2 b2036 SDBase> search 26 hbond B2036,iso2 ASN 31 N . ASN 26 OD1 26 hbond isol HIS 26 NE2 . GLU 44 0 26 name B2036,iso2 ASN 26 name isol HIS SDBase> Figure 2.6: In this SDBase information search, the key word "position" is used to limit the search to residue 26. The next two lines further limit the search by the use of regular expressions to information concerning hydrogen bonds, residue names and to the structures of iso-1, iso-2 and B-2036 cytochrome c. The keyword "search" initiates the search, the results of which are printed below. was electrochemically cleaned in a solution of 100 mM NaC104 and 20 mM phosphate buffer, pH 6, by cycling through a potential range of --1 to 1.2 V. The gold surface was then modified by immersion into a saturated solution of 4,4'-dithiodipyridine to sufficiently increase the rate of electron transfer to cytochrome c. A stream of water saturated argon was passed over the protein solutions to removed dissolved oxygen. A potential range of —44 to 544 mV (versus SHE) was scanned at sweep rates of 10 to 100 mV s -1 . All protein samples were oxidized with NH4[Co(dipicolinate)] and excess oxidant was removed with a Biorad P6-D6 desalting gel filtration column (1 x 20 cm). The oxidation state and concentrations of the samples were determined by UV-visible spectroscopy, assuming an extinction coefficient of 106.1 mM —l cm -1 at 409.5 nm. Experiments were performed on approximately 0.5 ml of a 0.4 mM protein solution in f.t = 0.1 M buffer (50 mM KC1 with the remaining ionic strength provided by sodium phosphate), at pH 6.0.  2.3.2 Derivation of Electrochemical Thermodynamic Properties A cyclic voltammogram of iso-2-cytochrome c indicating the peak oxidizing and reducing currents is presented in Figure 2.8. The midpoint reduction potential was computed as the average  43  Chapter 2. General Experimental Methods^  Reference electrode  Potentiostat  IWVV ■11111■1• 1■11■11.111.  •• Side arm  Auxiliary electrode  Protein solution  Capillary Working electrode (Gold) Plotter Figure 2.7: Schematic of the direct electrochemistry experimental setup used to measure reduction potentials. The three electrodes of the electrochemical cell are attached to a potentiostat. The buffer solution in the side arm is prevented from bulk mixing with the protein solution by a capillary junction. The potentiostat cycles a selected ramp voltage relative to the reference electrode through the cell. This voltage is recorded on the horizontal axis of the plotter. The resulting Faradaic current at the working electrode is recorded on the vertical axis of the plotter.  Chapter 2. General Experimental Methods ^  44  3— Epo = 314 mV  2—  1)  0-  L  -1-  —2—  Epp = 260 mV —3  0  100^200^300^400^500^600  Eapp  (mV versus SHE)  Figure 2.8: A sample cyclic voltammogram of iso-2-cytochrome c at 25 °C at a sweep rate of 50 mV/s is plotted. The broken lines were drawn tangentially to the baselines of the voltammogram. The applied voltage when current peaked at both the positive anode (E pa ) and positive cathode (E pc ) are indicated by dotted lines. (287 mV) of the applied voltage at the two peak currents (25 °C, pH 6.0 and p=0.1 M). The peak voltage difference (54 mV) is similar to the theoretical voltage (57 mV) expected for a fully reversible system (Greef et al., 1985). The method used to calculate the enthalpy and entropy from the temperature dependence of the midpoint reduction potentials was obtained from Taniguchi et al. (1980) and Rafferty, (1992). Since only the temperature of the cytochrome c reaction couple was changed while the temperature of the reference was held constant, the en0 tropy (AS eyt ) of the cytochrome c half reaction was computed directly from the slope of a plot of the midpoint potential (E7 ) versus temperature (T) using the equation:  45  Chapter 2. General Experimental Methods ^  0 0^DE„ ,ASeyt nF aT  (2.12)  where n is the number of electrons transferred in the reaction and F is the Faraday constant. The standard conditions were /1 = 0.1 M and pH 6. Since n = 1 for cytochrome c, the value 0^0 of nF is 23.06 cal mot -1 mV -1 . By definition the AG H and AH H of the standard hydrogen electrode (SHE) are zero, however, the practical entropy (ASH ) of the SHE is 15.6 e.u. (Latimer  et al., 1938; Latimer, 1952) and the entropy of the complete reaction was computed by: 0^0 ^ AS = .AScyt — AS H  0 ^ = AScyt 15.6 e.u.  (2.13) (2.14)  The free energy and enthalpy of the complete reaction was then derived from the equations: 0^0 AG = —n.FE n,^  (2.15)  0^0^0 OH = AG + T.AS^  (2.16)  Chapter 3  Structure - Function Analyses of Yeast Iso-2-Cytochrome c and the Composite Mutant Protein B-2036  3.1 Experimental Procedures Crystals of yeast iso-2-cytochrome c were initially grown by hair seeding hanging drops with crushed yeast iso-l-cytochrome c crystals as previously described (Leung et al., 1989). Crystals used in diffraction analyses were transferred into a freshly prepared solution of 97% saturated (NH4)2SO4, 0.3 M NaC1, 0.1 M sodium phosphate buffer (pH 6.0), and 0.03 M dithiothreitol, to maintain the reduced state of the heme group. A complete diffraction data set to 1.9  A  was collected using a single crystal on an Enraf Nonius diffractometer using continuous Q scans of 0.4° at 0.55°/min, at an ambient temperature of 15 °C. Three standard reflections were measured every 2.2 hours of X-ray exposure time to monitor crystal decay and slippage. Crystal specific data collection statistics are presented in Table 3.11. An initial model for the structure of iso-2-cytochrome c was constructed by using the coordinates of the refined iso-1 structure as a template (Louie & Brayer, 1990). An alignment of the iso-1 and iso-2 primary sequences is presented in Table 3.12. Amino acid sequence differences were modeled with the computer program MUTATE (R,.J. Read, unpublished results). In this procedure, at positions where amino acid substitutions have occurred, the template side chain was replaced by a new side chain with a conformation that follows the path of the template side chain as far as possible. Where this is not possible, the conformation is determined from a library of preferred side chain conformations (Bhat et al., 1978: Janin et al., 1978; James Sielecki, 1983; Moult & James, 1986). All of the new amino acid side chains were accommodated into the iso-2-cytochrome c fold without the formation of unacceptably close van der  46  Chapter 3. Iso-2-Cytochronle c and a Iso-1/Iso-2 Composite Mutant Protein^47  Table 3.11: Data collection statistics of iso-2 and B-2036 cytochromes c  Iso-2  B-2036  0.2 x 0.4 x 0.4 mm  0.2 x 0.25 x 0.25 mm  36.44(1) 137.86(4) 1.9 8159 7946 1.3 3.0  36.39(1) 137.28(4) 1.95 10866 7352 1.5 1.4  30.4%  20.9%  Parameter Crystal size Unit cell parameters (A): a, b c  Resolution (A) No. of reflections measured No. of unique reflections Max. decay correction Max. absorption correction R-scale with iso-1t t This R-scale is defined as:^  2 E I Foi —^I E (Fit, Fj)  where Foi and F'of are the observed structure factor amplitudes of wild-type iso-l-cytochrome c and the data set being compared, respectively. Waals contacts. As a result, no manual adjustments were applied to the iso-2 starting model. The four extra amino terminal residues of iso-2-cytochrome c were not included in the starting model. The resulting iso-2 starting model was placed in the unit cell in the same position and orientation as the refined iso-1 structure. The model was refined by a restrained parameter least-squares procedure with the program PROLSQ (Hendrickson & Konnert, 1981) using data with F > 2o- (F). Fragment deleted, Fo — Fc , 2Fo — Fe and 3F0 — 2F, maps were examined at intervals during the course of refinement as a guide for manual adjustment of side chains in the structure. The four additional amino terminal residues were added to the model in groups of two. Water molecules, modeled as oxygen atoms, were added to the model periodically by searching for peaks in Fo — F, maps. A water molecule was included in the refinement if at least one hydrogen bond was formed to the existing structure, and if 2F, — F, or 3F, — 2F, maps had significant electron density at the water position. In total, 58 bound water molecules with  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein ^48  Table 3.12: Sequence alignment of iso 1, B 2036 and iso 2 cytochromes c -  -  -  -  1^10 20 -9 Iso-1^ T E FK A GSAKKGATLFKTRC L QCHT V E K G G P H K V G QCHT I E E GGP N KVG E FK A GSAKKGATLFKTRC B2036 E E GGP N KVG Iso-2^AK ES T G FK P GSAKKGATLFKTRC Q QCHT -^-^-^-  -^-^-  -  50 60 30^ 40 Iso-1 PNLHGIFGRHSGQ A E GYSYTDANI K K N V L W D E NN M B2036 PNLHGIFGRHSGQ VK GYSYTDANI N KNV K WDE DS M Iso-2 PNLHGIFGRHSGQ V K GYSYTDANI N K N V K W DE DS M 70^80 90 100 SEYLTNPKKYIPGTKMAF G GLKKEKDRNDLITY L K KA C EB2036 SEYLTNPKKYIPGTKMAF G GLKKEKDRNDLITY L K KA C E'so-2 SEYLTNPKKYIPGTKMAF A GLKKEKDRNDLITY MT KA AK-  Iso-1  The primary sequences of iso-1 (Smith et al., 1979), B-2036 (Ernst et al., 1982) and iso-2 (Montgomery et al., 1980) cytochromes c have been aligned. Boxes enclose regions of sequence identity. For the composite B-2036 mutant protein, the bold sequence represents the region derived from the yeast iso-2-cytochrome c gene. an average thermal factor of 25.5  A 2 were identified. An exceptionally large peak of density  appeared at the amino terminal end of an a-helical segment, involving residues 2 to 4, and was modeled by a SO  —  ion as in the iso-1 structure (Louie Si Brayer. 1990). Over the course of  refinement, the conventional crystallographic 11-factor was reduced from 34.8 to 19.0% (22.4% for Fo > a(F0))• Crystals of B-2036 were obtained by techniques similar to those used in the growth of iso-2 crystals. These crystals were grown in a solution of 94% saturated (NH4)2SO4, 0.1 M sodium phosphate buffer (pH 6.2), and 0.04 M dithiothreitol. Free interface diffusion capillaries were used to obtain the largest crystals (Saleinme, 1972). Shortly before data collection, the crystals were transferred into a freshly prepared solution of mother liquor. Data collection and subsequent corrections were performed as described in Chapter 2 and the resulting data statistics are also presented in Table 3.11. A starting model for B-2036 was prepared from the coordinate sets of the iso-1 and iso-2 structures. The sulfate ion and 15 water molecules  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^49  conserved in the iso-1 and iso-2 structures were also included in the starting model. During the course of refinement, 7 of the original 15 water molecules were later removed from the structure and 47 additional water molecules were identified for a total of 55 bound water molecules (average thermal factor of 28.5 A 2 ) in the final structure. The conventional R-factor was reduced from 35.3 to 17.5% (18.8% for Fo > F0 )) using the same techniques as described for the iso-2-cytochrome c protein. The agreement of the iso-2 and B-2036 structures with ideal stereochemical geometry and the corresponding refinement weights used in the final refinement cycle are outlined in Table 3.13. For both structures, the deviations from ideal stereochemistry are well within the estimated coordinate error. Figure 3.9 shows a plot of the conventional R-factor as a function of resolution, Table 3.13: Final stereochemistry of iso-2 and B-2036 cytochromes c Restraint  r.m.s. deviation from ideal values (restraint weighting values) Iso-2^B-2036  Distances (A) Bond Angle Planar 1-4 Plane (A) Chiral volume (A 3 ) Torsional angle (deg.) Planar Staggered Orthonormal Non-bonded contact (A)t Single torsion Multiple torsion Possible hydrogen bonds  0.020 0.041 0.050 0.016 0.205  (0.020) (0.030) (0.050) (0.020) (0.160)  0.019 0.037 0.047 0.016 0.196  (0.020) (0.030) (0.045) (0.020) (0.150)  2.4 (2.5) 25.7 (20.0) 14.5 (15.0)  2.6 (3.0) 22.8 (20.0) 12.0 (15.0)  0.213 (0.250) 0.202 (0.250) 0.249 (0.250)  0.218 (0.250) 0.186 (0.250) 0.227 (0.250)  The r.m.s. deviations from ideality for this class of restraint incorporates a reduction of 0.2 from the radius of each atom involved in a contact.  A  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^50 10.0 A  5.0  A  3.3  Resolution A^2.5 A  1.7  A o  -  0.05  0.10  Figure 3.9: A plot of the conventional R-factor as a function of resolution at the end of refinement. The fit to the data of iso-2 (0) is represented by the thick line and the fit to the B-2036 data (^) is represented by a thin line. The theoretical dependence of R-factor on resolution, assuming various levels of r.m.s. error in the atomic positions of the model (Luzzati, 1952), are drawn in broken lines. The fraction of data used in refinement is presented above, using the same data point and curve scheme. along with theoretical curves calculated by assuming various r.m.s. coordinate errors (Luzzati, 1952). Inspection of this plot suggests a coordinate error for both structures of approximately 0.2  A. This value is a statistical estimate of the overall coordinate error, whereas the actual  positional error of an individual atomic coordinate will depend on the location of the atom in the structure. The errors of atoms in the core of the protein are likely to be less than 0.2  A,  whereas highly mobile atoms on the protein surface are less accurately determined. The r.m.s. coordinate errors of residues —5 to 103 and the heme group by an individual atom method (Cruickshank, 1949; Chambers & Stroud, 1979) are 0.19  A and 0.16 A for iso-2 and B-2036,  respectively. Also included in Figure 3.9 is a plot of the fraction of structure factors included in the refinement as a function of resolution.  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein ^51  3.2 Polypeptide Chain Conformations From the coordinates of the iso-2 and B-2036 cytochrome c structures, hydrogen bonds were defined by geometrical criteria (Table 3.14). The majority of the hydrogen bonds are conserved between the iso-2 and B-2036 structures. Many of the observed differences in main chain hydrogen bonds result from the formation of bifurcated interactions within a-helical segments, where the amide of a residue hydrogen bonds with both of the carbonyls of the i + 3 and i + 4 residues. Changes in hydrogen bonding involving side chain atoms result from amino acid substitutions and alternative placement of amino acid side chains. The latter is particularly evident for poorly ordered residues such as G1u21, Lys58, Glu61 and Lys86. Plots of the main chain 0 and 0 angles for both iso-2 and B-2036 cytochrome c are presented in Figure 3.10. In total, ten non-glycyl residues have values that fall outside the permissible regions of these two plots. The residue with angles the furthest from a permissible region is Lys27 (0, = —158°, —138° in iso-2; —137°. —127° in B-2036) which is part of a 7-turn as in iso-1-cytochrome c (Louie & Brayer, 1990). The other eight residues are not far from permissible regions of the plot. Asn56 is the only non-glycyl residue found in the aL conformation. This residue is part of a 310 turn that terminates an a-helix and has a comparable conformation in iso-l-cytochrome c (Louie & Brayer, 1990). Main chain hydrogen bonds and 0, 0 angles have been used to classify sections of the iso-2 polypeptide chain into secondary structure elements (Table 3.15). In total, five a-helices, three type I, three type II and one 7 turn were identified. The i + 2 position of each type II /3turn is a conserved glycine residue (Table 3.12). The secondary structure assignments for the B-2036 structure are identical with the exception of Helix III, where the last turn is in a 310 conformation and Helix V which ends at residue Cys102. An a-carbon tracing of yeast iso-2-cytochrome c with the heme iron ligands, His18 and Met80, as well as the heme thioether linkages to residues Cys14 and Cysl7, is illustrated in Figure 3.11. In Figure 3.12, all atoms for the iso-2 protein are presented in the same orientation as in Figure 3.11. The amino acid sequence numbering scheme is as described in Table 3.12.  Chapter 3. Iso-2-Cytochrome c and a Iso-1/1 so-2 Composite Mutant Protein^52 -  Table 3.14: Hydrogen bond analysis of iso-2, B-2036 and iso-1-cytochromes c I. Common hydrogen bondst (iso-2/13-2036/iso-1) A. Main main 6N-20 7N-30 8N-40 9N-50 10 N - 6 0 11 N - 7 0 12 N - 8 0 14 N - 10 0 15 N - 10 0 17N-140 18 N - 14 0  24 N - 21 0 27 N - 29 0 29 N - 17 0 32 N - 19 0 34 N - 102 0 35 N - 32 0 38 N - 35 0 40 N - 57 0 53 N - 49 0 54 N - 50 0 59 N - 38 0  B. Main side 1N Thr96 OG1 2 N - Asp93 OD1 5 N - Ser2 OG Thr8 0G1 - 5 0 Thrl2 OG1 - 8 0 Thrl2 OG1 - 9 0 Hisl8 ND1 - 30 0 Thr19 OG1 - 25 0  Asn31 ND2 - 21 0^Tyr46 OH - 28 0^73 N - Asn70 OD1 33 N - Asn31 OD1^49 N - Hem 02D^Lys79 NZ - 47 0 His33 ND1 - 20 0^52 N - Thr49 OG1^79 N - Hem OlD Arg38 NH1 - 33 0^Lys55 NZ - 74 0^80 N - Thr78 OG1 Arg38 NH2 - 33 0^57 N - Ser40 OG^Lys86 NZ - 69 0 Ser40 OG - 52 0^63 N - Asp60 OD1^Arg91 NH2 - 85 0 41 N - Hem 02A^Thr69 OG1 - 65 0^Thr96 OG1 - 92 0 43 N - Tyr48 OH  -  64 N - 60 0 65 N - 61 0 67 N - 63 0 68 N - 64 0 69 N - 66 0 70 N - 67 0 74 N - 70 0 75 N - 71 0 78N-750 85 N - 68 0 91 N - 87 0  92 N - 88 0 93 N - 89 0 94 N - 90 0 95 N - 91 0 96 N - 92 0 97 N - 93 0 98 N - 94 0 99 N - 95 0 100 N - 96 0 101 N - 97 0 102 N - 98 0  -  -  C. Side side Thr19 OG1 - Asn31 ND2 Thr49 OG1 - Hem OlD Tyr67 OH - Met80 SD Arg91 NE - Ser65 OG Tyr48 OH - Hem 01A Asn52 ND2 - Hem 02A Thr78 OG1 - Hem OlD -  II. Unique hydrogen bonds to iso-2 and B-2036 A. Main main 13 N - 10 0^55 N - 51 0 37 N - 34 0 -  B. Main side 31 N - Asn26 OD1^Ser63 OG - 58 0  56 N - 53 0  66 N - 62 0  -  Lys86 NZ - 83 0  III. Unique hydrogen bonds to iso-2 and iso-1 A. Main side Lys27 NZ 15 0^62 N Asp60 OD1 -  -  -  Arg91 NH2 - 86 0  B. Side side Trp59 NE1 - Hem 02.,A, -  Continued  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^53  IV. Unique hydrogen bonds to B 2036 and iso 1 -  -  A. Main main -  13 N 9 0^82 N 80 0 -  -  B. Main side -  Cys102 SG - 98 0 V. Hydrogen bonds present only in iso-2 A. Main main (-5) N - (-9) 0 (-4) N - (-8) 0 10 N - 7 0 -  34 N - 103 0 70 N - 66 0  92 N - 89 0 96 N - 93 0  100 N - 97 0 103 N - 100 0  Thr(-5) 0G1 - (-9) 0 Lys27 NZ - 16 0  Ser47 OG - 47 0  Thr69 OG1 - 66 0  Tyr74 OH - Ser63 OG  Asn92 ND2 - G1u88 0E2  B. Main side -  (-9) N - G1u66 0E2 Thr(-5) OG1 - (-8) 0 C. Side side -  Ser63 OG - Tyr74 OH  VI. Hydrogen bonds present only in B-2036 A. Main side -  46 N - 43 0 B. Main side -  (-5) N - Thr(-5) 0G1^Lys58 NZ - 36 0^60 N - Ser63 OG^Ser63 OG - 60 0 Asn26 ND2 - 44 0^Lys58 NZ - 59 0^62 N - Asp60 OD2 C. Side Side Thr(-5)0G1 - Asp62 OD1 Lys5 NZ - Asp93 OD2 Ser63 OG - Asp60 OD1 Lys99 NZ - Glu61 0E2 -  VII. Hydrogen bonds present only in iso-1 A. Main main -  37 N - 59 0^55 N - 52 0^69 N - 65 0^73 N - 70 0 48 N - 46 0 B. Main-side (-3) N - Thr(-5) OG1^His26 NE2 - 44 0^31 N - His26 ND1^Lys99 NZ - 96 0 20 N G1u21 OE1 C. Side side Thr(-5)OG1-G1u61 OE1 Thr(-5)0G1-Asp62 OD1 Asn63 ND2-Asp60 OD2 Tyr74 OH-Asn63 OD1 -  Where main chain atoms are listed, only the residue number is given. The abbreviations iso-1, iso-2 and B-2036 refer to yeast iso-1, yeast iso-2, and B-2036 cytochromes c, respectively. tHydrogen bonds were defined by the following criteria: a H...A distance < 2.60 A, 2.70 A or 3.05 A where A is an oxygen, nitrogen or sulfur atom respectively; a D-H...A angle > 120°; and, a C-A...H angle > 90°. The hydrogen bonds listed are defined by geometry only and do not account for disorder in side chain positions in the structures.  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^54  a  180  0  D OD °°^ ..•. • 0 .•  0^0 S  120  o  0  0(000 0  0  0^  60  1p  .....  '',, I  r  /  J  r : I CI^*  ,...*  *Sx '^ •• • a^r,^\ 0o 0 ,0^-.0 0 • C8 )^ 0^  IC  o  -60  -120 0 *  -180 -180  b  ^  cc  60^120  ^  180  180  )o ° 6 °*°621carii) o CS 0^o ®^ ^0 Q  120  0v  0  60  ^\ix  •  -120^-60^0  ..  .  ,  ..'  0^,D^....."'...-"j /  4^I  o4 ,..^I  gfr 0  -60  0 00  **  &^ 0 0 \s 0^6?^o° 0 ", . •^,, 0 ,^ I^ t^  -120  -180 -180  0  **  •  *  -120^-60^0  60^120  180  SO  Figure 3.10: Ramachandran plots of the and angles of (a) iso-2 and (b) B-2036 cytochromes c (Ramakrishnan Ramachandran, 1965). Glycine residues are denoted by an asterix (*) and all other residues by (0). Main chain conformations for an alanine residue in poly(L-alanine) which are fully allowed, and allowed assuming shortened permissible minimum interatomic distances are enclosed by solid and dashed lines, respectively.  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^55  Table 3.15: Secondary structural elements in iso-2-cytochrome c  Secondary structural  Residues  Main chain torsional  element  involved  angles (deg.)t  A. Average angles a-Helix I ,Q-Turn (type I) /3-Turn (type II) -y Turn P-Turn (type II) /3-Turn (type I) /3-Turn (type I)  2-14 14-17 21-24 27-29 32-35 34-37 35-38  (-70, -38) (-62, -38), (-46, -44) (-59, 131), (71, 17) (-158. -138), (-59, -46), (-106, 170) (-54, 136), (81, 5) (-50, -56), (-51, -33) (-51, -33), (-93, 33)  a-Helix IIt a-Helix III a-Helix IV P-Turn (type II)  49-56 60-70 70-75 75-78  (-63, -39) (-67, -39) (-65, -40) (-59, 130), (105, -25)  a-Helix Vt  87-103  (-61, -43)  -  B. Overall averages a-Helix P-Turn (type I) /-Turn (type II)  45 residues 3 turns 3 turns  (-63, -40) (-54, -42), (-63, -15) (-57, 132), (86, -1)  t The main chain torsional angles refer to the average (0,0) angles for residues in each a-helical segment, excluding the 2 terminal residues. For /3-turns, the angles listed correspond to (0,0)2 and (0,0)3, respectively. All three of (00/0 1 , (0,0)2 and (0,0)3 are given for the ry-turn. Overall averages for each class of structures are also listed. The a-helices have been numbered from the N-terminal end of the polypeptide chain. t The last turn of these a-helices is in a 310 conformation. The heme component is almost completely buried by the surrounding polypeptide chain, with only part of one edge exposed to bulk solvent.  3.3 Comparison of Iso-2, B-2036 and Iso-1-Cytochromes c A comparison of the superimposed structures of iso-2. B-2036 and iso-l-cytochromes c reveals the strong conservation of the main chain fold in these closely related proteins. A detailed  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein  it  1# T -5  K5  ^  56  K5  tD90  4twa  F 10 Q 15 K 100 1 20  A -9  *A  D 50  Figure 3.11: Stereo-drawing of the a-carbon backbone of yeast iso-2-cytochrome c. Also drawn in thick lines are the heme group, the two covalent thioether linkages from the heme to Cysl4 and Cys17, as well as the side chains of the axial iron ligands, Hisl8 and Met80. The heme moiety is viewed edge on in this orientation. Every 5th residue and the two termini are labeled according to the sequence alignment in Table 3.12.  Figure 3.12: Stereographic representation of iso-2-cytochrome c with side chains drawn in thin lines and the polypeptide backbone and heme group in thick lines. The orientation and labeling are similar to that in Figure 3.11.  Chapter 3. Iso-2-Cytochrome c and a Iso-.I/Iso-2 Composite Mutant Protein ^57  2.5  2.0-  1  1.0-  0.5-  kt  -rwit vwf 0.0  k er^ViSfry  -6^5^15^25^35^45^55^65  ^  75^85^95^105  Residue Number  Figure 3.13: The average positional deviations of main chain atoms between iso-2 and B-2036 (thick line), iso-2 and iso-1 (thin line), and B-2036 and iso-1 (dotted line) cytochromes c along the course of the polypeptide chain. The horizontal lines represent the overall average deviations for the main chain atoms of residues —3 to 103 for each comparison. Vertical arrows denote positions of amino acid substitutions and the horizontal bar indicates the region of B-2036 derived from the iso-2 gene. analysis of main chain positional differences between the three structures is presented in Figure 3.13. The average displacement of main chain atoms between iso-1 and iso-2-cytochromes c for residues —3 to 103 is 0.30  A. The region of greatest structural difference is at the amino ter-  minus, where the iso-2 protein contains an extension of four extra amino acid residues. These additional residues are placed on the surface away from the heme crevice (Figures 3.11 and 3.12) and as Figure 3.13 shows, cause the first two residues in common with the iso-1 protein (-5 and —4, Table 3.12) to be in an altered conformation. The next largest main chain displacement is centered around Gly37. The conformations of iso-1, iso-2 and B-2036 in this region are depicted in Figure 3.14. Two distinct main chain conformations are observed in electron density maps of the iso-2 structure. One of these is similar  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein ^58  Figure 3.14: Stereographic plot of iso-2 (thick lines), B-2036 (medium lines), and iso-1 (thin lines) cytochromes c in the region near G1y37. The a-carbon of G1y37 is displaced 2.4 A in iso-2 relative to iso-l-cytochrome c. In iso-l-cytochrome c, G1y37 N forms a hydrogen bond (broken lines) with Trp59 0; however, in the iso-2 protein, G1y37 N hydrogen bonds with Gly34 0. The hydrogen bond between Arg38 N and 11e35 0 is present in both structures. The B-2036 structure is analogous to the iso-2 protein in this region. to that observed in iso-l-cytochrome c where a hydrogen bond is formed between G1y37 N and Trp59 0 (Table 3.14). The second conformation is generated by rotating of Phe36 by 180°, and the formation of a new hydrogen bond between G1y37 N and Gly34 0, generating a type I 0-turn (Table 3.15). As part of this movement, the geometry of the nearby G1y38 N to G1y35 hydrogen bond is improved and the 0-turn from residues 35 to 38 is converted from type II in iso-l-cytochrome c to type I in iso-2-cytochrome c. This second conformation was chosen for structure refinement, since in this conformation the thermal factors of the atoms involved refined to lower values. The conformation of Gly37 in the B-2036 structure is similar to that in the preferred iso-2 conformation. An amino acid substitution in this region of the structure may provide an explanation for the observed structural differences. This occurs at position 58, where there is a leucine in iso-1 and a lysine residue in iso-2 and B-2036 cytochromes c. The Leu58 side chain in the iso-1 structure stabilizes the conformation of Gly37 by hydrophobic packing interactions. The side chain of Lys58 does not form similar interactions in the iso-2 and B-2036 structures. In B-2036, the alternative conformation is further stabilized by Lys58 NZ  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^59  interacting with Phe36 0 and Trp59 0 (Table 3.14). Another region that displays significant structural heterogeneity is near positions 26 and 43, which are neighbors in the folded structure (Figure 3.15). In the iso-1 structure, His26 forms two hydrogen bonds through its NE2 and ND1 atoms with G1u44 0 and Asn31 N respectively (Table 3.14). The iso-2 and B-2036 structures have an asparagine at position 26 that also interacts with Asn31 N through its OD1 atom. The Asn26 ND1 atom forms a further weak interaction to Lys44 0 in these two latter structures. The decreased volume of an asparagine side chain relative to that of histidine at position 26 is compensated for by the substitution of A1a43 for a valine in the iso-2 and B-2036 structures. Small shifts in the overall atomic positions of residues 23 to 26 and 42 to 45 allow the optimal packing of the different amino acid side chains in this region. The influence of these changes on the average main chain thermal factors is illustrated in Figure 3.16. At position 63, there is an asparagine in iso-1, but a serine in B-2036 and iso-2-cytochromes c. The hydrogen bond between Asn63 OD1 and Tyr74 OH in iso-1 is maintained in the iso-2 structure with a hydrogen bond between Ser63 OG and Tyr74 OH. Alternatively, in the B-2036  Figure 3.15: Stereo-diagram of the region about residue 26 in iso-2 (thick lines) and iso-1 (thin lines) cytochromes c. Hydrogen bonds are represented by broken lines. His26 in the iso-1 structure forms hydrogen bonds that bridge the main chain at G1u44 and Asn31. Only one of these hydrogen bonds is present in iso-2-cytochrome c as a result of the His26Asn substitution. The nearby substitution of an alanine for a valine at position 43 in iso-2-cytochrome c is also illustrated.  -  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^60  e-N  aQ  40  L  0 0  30 6  EL -  20  a>  0) 0  a)  >  10  -6^5^15^25^35^45^55^65^75^85^95^105  Residue Number  Figure 3.16: The average thermal factor of the four main chain atoms of each amino acid in iso-2 (thick line), B-2036 (thin line) and iso-1 (dotted line) cytochromes c are plotted as a function of residue number. The vertical arrows indicate positions of amino acid substitutions between iso-1 and iso-2-cytochromes c and the horizontal bar represents that region of B-2036 that is derived from the iso-2 gene. Differences in the thermal factor profiles between iso-2 and iso-1 (thick line), iso-2 and B-2036 (thin line) and B-2036 and iso-1 (dotted line) cytochromes c are plotted above (scale to the upper right). Data for residues —9 to —6, which occur only in iso-2-cytochrome c, are not shown. structure a hydrogen bond is made from Ser63 OG to Asp60 OD1 replacing the interaction between Asn63 ND2 and Asp60 OD1 in iso-l-cytochrome c. Since a serine residue is smaller than an asparagine, small changes in main chain conformation are required to maintain the observed hydrogen bonds (Figure 3.13). There is also a small peak in the region of Phe82 in the positional deviation plot of Figure 3.13. The nearest sequence substitution is the replacement of Gly83 for an alanine residue in iso-2-cytochrome c. The phenyl ring of Phe82 is observed to shift 0.5  A outwards toward the  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein ^61  protein surface in iso-2 and B-2036. As a consequence, this phenyl ring also packs less tightly with the adjacent side chain of Leu68. The polypeptide main chain shifts with the phenyl ring in the case of iso-2-cytochrome c, and as a result, the hydrogen bond between Phe82 N and Met80 0 is not present in the iso-2 structure (Table 3.14). The conformation of the main chain is more conserved between B-2036 and iso-1-cytochrome c. An examination of the B-2036 starting model revealed a steric clash between 11e20 CD1 and Cys102 SG, two residues differing between iso-1 and iso-2-cytochromes c (Table 3.12). Cys102 is near the end of Helix V (Table 3.15) and 11e20 is located between the His18 ligand and a type II 0-turn (residues 21-24). Iso-1 has valine and cysteine at positions 20 and 102, whereas in iso-2, these residues are isoleucine and alanine, respectively. This steric clash in the B-2036 protein is relieved by displacements of up to 0.8  A in atoms of the side chains of residues 20 and  102. There is little displacement in the a-carbon atoms of the residues involved (Figure 3.17), although the shift in the side chain atoms of Cys102 in B-2036 forces a rotation of 15° in the main chain  b angle of this residue leading to some distortion at the end of Helix V. Presumably,  the His18 ligand and the /3-turn composed of residues 21 to 24 constrain the position of the main chain atoms of 11e20. The side chain atoms of 11e35 and Leu98, two nearby residues that pack against Cys102 and 11e20, shift up to 0.7  A to accommodate the extra volume of the two  substituted side chains.  3.4 The Buried Cavity in B-2036 and Iso-l-Cytochromes c Leu98 is one of the residues that line a small buried cavity located behind the heme group in the iso-1 structure (Figure 3.18). The substitution of this residue by a methionine in iso-2 eliminates this cavity. As in iso-1, B-2036 also has a leucine at position 98 and an internal cavity. The displacements described previously in the region of Cys102 and 11e20 increase the volume of this cavity from about 30 A 3 in iso-1, to 50 A 3 in B-2036. This enlarged cavity is illustrated in Figure 3.18(b). Both cavities are large enough to accommodate a water molecule; however, no significant electron density appears in this region and the lack of hydrogen bond  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^62  Ile 35  he 36  Phe 10^Leu 98  Phe 10^Leu 98  Figure 3.17: The structures of iso-2 (thick lines), B-2036 (medium lines) and iso-1 (thin lines) cytochromes c are plotted in stereo in the region about residues 20 and 102. The substitution of a cysteine for an alanine at position 102 in B-2036 relative to iso-2-cytochrome c results in small shifts in neighboring residues to accommodate the larger cysteine side chain. partners prevents favorable enthalpic interactions to offset the large entropic energy required to isolate a water molecule from the bulk solvent. A larger cavity could be expected to decrease the stability of the B-2036 protein (Eriksson  et al., 1992); however, it appears to be preferable to the steric clash between 11e20 and Cys102. Conversely the substitution Leu98Met in the iso-2 structure completely fills this cavity. The methionine side chain adopts the same conformation as the leucine side chain it replaces and the X3 torsional angle of 73° is close to a staggered conformation (Figure 3.18). Two heme atoms, CMA and CMB, form part of the internal cavity of both iso-1 and B-2036 cytochromes c. In iso-2, however, Met98 packs directly against the rear of the heme. The cavity in iso-2-cytochrome c seems to be filled without the addition of significant structural strain and, therefore, would be expected to be a stabilizing mutation (Karpusas et al., 1989). Substituting methionine for leucine in iso-l-cytochrome c by site-directed mutagenesis could be used to test the destabilizing effect of the internal cavity in the iso-1 structure. It is of interest that only leucine and methionine are observed at position 98 in mitochondrial cytochromes c (Hampsey et al., 1986).  Chapter 3. Iso-2-Cytochrome c and a Iso-1/1"so-2 Composite Mutant Protein ^63  a  b Leu98 CD1 Hem  Leu98 CD1 Hem  Figure 3.18: Both the B-2036 and iso-l-cytochromes c contain a buried cavity behind the heme as viewed in the standard orientation (Figure 3.11). In (a) the structures of iso-2 (thick lines), B-2036 (medium lines), and iso-1 (thin lines) cytochromes c are superimposed, with the cavity found in iso-1 depicted as a dotted surface. The atoms of the iso-1 structure that border the cavity are labeled. In (b) the surface representation and labels are those of B-2036. In both plots, the Met98 CE atom of the iso-2 structure fills this cavity.  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^64  3.5 Heme Geometry and Environment The overall environment of the heme in iso-2, B-2036 and iso-1-cytochromes c is hydrophobic and highly conserved, with only the heme edge facing the viewer in Figures 3.11 and 3.12 being exposed to the bulk solvent. The solvent exposure of the heme is similar in all known mitochondrial cytochrome c structures, as documented in Table 3.16. In addition, all heme propionate hydrogen bonds are conserved with the exception of the Trp59 NE1 hydrogen bond to the 02A atom of the heme, which is absent in B-2036 according to the definitions outlined in Table 3.14. In this protein, the indole ring of Trp59 has moved away from the heme propionate possibly in response to the changes centred at G1y37 described above. The conjugated heme porphyrin ring of yeast iso-2-cytochrome c is distorted from planarity. However, each of the five-membered pyrrole rings, as well as the pyrrole N ligands (pyrrole N plane) are nearly planar. In Table 3.17, the angular deviations of the pyrrole ring planes from a plane fit to the entire porphyrin ring and the pyrrole N plane are presented along with iron ligand distances. The angular deviations and ligand distances are similar to those observed  Table 3.16: Heme solvent accessibility of mitochondrial cytochromes c  Cytochrome c structure  Iso-2  B-2036  Iso-1  Tuna  Horse  Rice  0.0 8.5 0.0 20.9 11.7  2.2 8.3 3.0 18.6 10.7  2.9 9.4 3.7 18.0 10.4  4.6 8.2 1.9 23.5 10.1  0.0 8.3 5.2 17.3 3.9  0.0 10.4 0.0 12.8 8.1  41.4  42.9  44.4  48.3  34.7  31.3  8.9  9.2  9.5  10.5  7.4  6.8  Solvent accessible heme atoms and surface area exposed (A 2 ) CHD CMC CAC CBC CMD Total heme exposure (A 2 ) % Heme surface area exposed  Computations were done using the method of Connolly (1983) with a probe radius of 1.4  A.  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^65  Table 3.17: Heme conformation and ligand geometry in yeast iso-2 and B-2036 cytochromes c A. Angular deviations between both plane normals of individual pyrrole rings and the heme co-ordinate bonds, and both the pyrrole nitrogen plane and the porphyrin ring planet  Pyrrole ring A B C D  Pyrrole N plane  Porphyrin ring plane  10.3° 11.5° 12.3° 12.3°  (9.1°) (11.2°) (8.8°) (10.9°)  3.8° 10.5° 10.6° 5.8°  (4.8°) (10.7°) (10.7°) (6.6°)  3.2° 2.0°  (3.8°) (4.3°)  9.8° 6.7°  (2.8°) (7.1°)  Heme coordinate bonds Fe-His18 NE2 Fe-Met80 SD  B. Henze iron coordinate bond distances (A) Hisl8 NE2 Met80 SD Hem NA Hem NB Hem NC Hem ND  1.86 2.42 2.02 2.06 1.97 2.02  (2.06) (2.33) (1.99) (2.03) (2.02) (1.99)  The values for B-2036 cytochrome c are in parentheses. t Each pyrrole ring plane is defined by 9 atoms, which include the 5 ring atoms plus the first carbon atom bonded to each ring carbon. The porphyrin ring plane is defined by all the atoms in the 4 pyrrole ring planes and the iron atom. The pyrrole N plane is defined by only the 4 pyrrole nitrogens. These latter two planes differ in orientation by an angle of 6.8°.  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein ^66  in other cytochrome c structures (Bushnell et al., 1990; Louie & Brayer, 1990). The greatest planar distortion in yeast iso-2, iso-1, B-2036 and horse cytochromes c occurs at pyrrole rings B and C which are attached to Cys14 and Cys17 through thioether linkages.  3.6 Conserved Water Molecules An analysis of bound water molecules was performed for the iso-2, B-2036 and iso-1 structures. In this procedure a water molecule was considered structurally conserved if its atomic displacement from a counterpart in iso-2-cytochrome c was less than 2  A and conserved water-protein  hydrogen bonds were present. In total, 15 such conserved water molecules were observed (Table 3.18). Many of these interact with main chain amide and carbonyl groups. Further analysis shows only three waters are conserved between all known cytochrome c structures completed to date. In the yeast proteins, only four waters are completely buried, and as such, form an integral part of the protein structure. Three of these (Wat106, 118 and 131) may play functional roles in stabilizing the alternative oxidation states of these proteins (Berghuis  & Brayer, 1992).  3.7 Comparison with Other Cytochromes c The structure of yeast iso-2-cytochrome c can also be compared to the high resolution structures of horse (Bushnell et al., 1990), tuna (Takano & Dickerson, 1981a) and rice cytochromes c (Ochi et al., 1983). A plot of the overall average deviations of these cytochromes c from that of the iso-2 protein is presented in Figure 3.19. At each individual residue in the polypeptide chain a further indication of the range of pairwise deviations between these cytochrome c is given by vertical bars. This comparison shows the structurally most conserved region of cytochrome c stretches from residues 61 to 101. As reference to Figure 3.11 shows, this segment of polypeptide chain forms most of the Met80 ligand side of the molecule. Despite the overall high degree of structural homology in this and other regions of the polypeptide chain, there are points of substantial structural difference between these cytochromes c. These include residues 21 to 28, 36 to 38 and 55 to 57. All three of these regions occupy positions on the protein surface  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^67  Table 3.18: Conserved water molecules in yeast iso-2, B-2036 and iso-l-cytochromes c Water Number Iso-2^B-2036^Iso-1 106tt 107 1091: 112 115 1 117 Mitt 123 130 131t 144 145 153 156 158  106 107 108 139 110 118 122 149 158 109 145 126 119 123 156  166 154 110 197 122 207 121 133 138 168 215 142 158 208 140  Hydrogen bonds  Asn52 ND2, Tyr67 OH, Thr78 0G1 100 0, 103 OXT 19 N, 29 0 His39 ND1 79 0, 81 N 66 0, 70 0 39 0, 42 N, Hem O1A 23 N 50 N Arg38 NH1, Asn31 0 Hem O1A 77 N G1u88 0E1 86 N, 87 N Asn92 OD1, Ser65 OG 46 0  Indicates water and hydrogen bonds observed in all of the structures of yeast iso-1, yeast iso-2, rice, tuna and horse cytochromes c. tIndicates a water molecule buried in the protein matrix. (Figure 3.11) and their positioning is strongly influenced by nearby amino acid substitutions which occur between the compared cytochromes c (Louie & Brayer, 1990). 3.8 Electrochemical Properties  Direct electrochemistry of iso-2 and B-2036 cytochromes c was reversible from 10 to 35 °C (Figure 3.20). The midpoint reduction potential and the derived thermodynamic parameters at 25 °C are presented in Table 3.19 along with data for iso-1-cytochrome c. Although the midpoint potentials at 25 °C are similar, the enthalpic and entropic contributions to the free energy change of reduction of iso-2 and B-2036 cytochromes c have increased. The enthalpic term has been associated with the polarity of the heme environment through electrostatic effects  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^68  3.0  Ir  ^ferrtrrerrirrilrr■r tIrslIttlItItellterstIrrreir esti,  2.5  2.0C 0 •-  - 'a  `>  0 1.5 (Zt  0^-  (3)  0.5-  0.0 10^20^30  40^50^60  70  80  90^100  Residue Number  Figure 3.19: A plot of the overall average deviations of main chain atoms of reduced yeast iso-1 (Louie & Brayer, 1990: PDB file lYCC), reduced tuna (Takano & Dickerson, 1981a: PDB file 5CYT), oxidized horse (Bushnell et al., 1990) and oxidized rice (Ochi et al., 1983: PDB file 1CCR) cytochromes c from those of yeast iso-2-cytochrome c along the course of the polypeptide chain. Only residues 1 to 103, which are common to all five cytochromes c, are represented. The vertical bars represent the range of individual pairwise average deviations of main chain atoms between yeast iso-2 and the other cytochromes c. Structures with pairwise deviations both larger than 0.7 A and 30% greater than the overall average deviation of all structures are labeled at the particular residue involved (T, tuna; H, horse; R, rice cytochromes c). The peak at position 37 is not labeled because all of the structures deviate significantly from iso-2-cytochrome c. (Kassner, 1973). The increase in reduction enthalpy in the iso-2 and B-2036 proteins may be due to a decrease in the polarity of the heme environment as a result of the structural changes resulting from the substitutions Va120Ile and Leu98Met. The structural features that influence the entropy of reduction are less well defined. Previous studies have shown that the reduced cytochrome c structure is more compact and thermally less mobile (Eden et al., 1982; Trewhella et al., 1988; Berghuis & Brayer, 1992). Mutations that alter the packing of the hydrophobic  core may also increase the entropy of reduction.  Chapter 3. Iso-2-Cytochrome c and a Iso-1/1 so-2 Composite Mutant Protein^69 -  300.0  295.0  0  290.0  C 0 O  a_ C  0 285.0  280.0  275.0  50  10.0^15.0^20.0^25.0^30.0  ^  35.0^40.0  Temperature (°C)  Figure 3.20: The midpoint reduction potential of the iso-2 (0), B-2036 (0) and wild-type iso-1 (A) cytochromes c as a function of temperature. Data for iso-1-cytochrome c is from Rafferty, (1992).  Table 3.19: Electrochemical properties of the yeast iso-2, B-2036 and iso-l-cytochromes c  Protein  Iso-2 B-2036 Iso-1  0  0  0  (mV)  OH (kcal/mol)  AScyt (e.u.)  AG (kcal/mol)  286 ± 2 288 ± 2 290 ± 2  -15.9 ± 0.4 -16.5 ± 0.4 -14.0 ± 0.2  -16 ± 1 -17 ± 1 -9.1 ± 0.4  -6.6 ± 0.1 -6.7 ± 0.1 -6.7 ± 0.1  En,  Experimental conditions were 25 °C, pH 6.0, it = 0.1 M and SHE reference. Data for iso-lcytochrome c is from Rafferty et al., (1990).  Chapter 3. Iso-2-Cytochrorne c and a Iso-1/Iso-2 Composite Mutant Protein^70  3.9 Stability of Yeast Cytochromes c The thermal transition temperature (T in ) of iso-l-cytochrome c has been measured to be 52.3 ± 0.3 °C (Dumont et al., 1990). Iso-2-cytochrome c is slightly more stable with a Tni of 54.2 ± 0.3 °C, while the Tin of the B-2036 protein is 48.9 ± 0.3 °C. Recently, a study of cavity creating mutations in lysozyme found that the free energy of folding increases with the size of a resident cavity and this can be expressed as 24 to 33 cal mot -1  A -3 (Eriksson et al., 1992). Thus, the  filling of the buried cavity by Met98 without additional strain in the iso-2 structure would be expected to increase this protein's stability relative to iso-1 by about 0.8 kcal mot -1 and may partially account for the increase in Tin observed. The steric clash between residues 11e20 and Cys102 in B-2036 cytochrome c and the resulting enlargement of the buried cavity may account for the decrease in thermostability of this protein. The structural studies described here also identify further individual amino acid substitutions resulting in alterations in the folding and packing of the three-dimensional structure of cytochrome c. Of the 16 amino acid substitutions between iso-1 and iso-2-cytochromes c, five of these cause changes in interactions between well ordered components of the structure: Va120Ile, His26Asn, Ala43Val, Leu98Met and Cys102Ala, and may significantly alter protein stability. Two other substitutions, Leu58Lys and Gly83Ala, affect less well ordered sections of polypeptide chain. The remaining 11 substitutions do not appear to alter how the polypeptide chain is folded or those interactions that maintain that fold. All 11 of these latter substitutions occur at the protein surface and are not expected to alter protein stability. The B-2036 composite protein may be an example of an intermediate of protein evolution since it is the product of a non-allelic recombination. Although the overall fold is strongly preserved, the composite is less stable than either of the wild-type proteins. This loss of thermodynamic stability appears to be a result of incongruous amino acid substitutions. One example described here is the substitution Va120Ile which appears to destabilize the protein if the structurally complementary substitution Cys102Ala is not also present, as in the iso-2  Chapter 3. Iso-2-Cytochrome c and a Iso-1/Iso-2 Composite Mutant Protein^71  structure. Systematic substitution of amino acid residues that differ between iso-1 and iso2-cytochromes c will better define structural determinants of both thermal stability and the midpoint reduction potential. Those amino acid substitutions that cause structural perturbations of the well ordered sections of the polypeptide chain, as described here, are particularly good candidates for study by site-directed mutagenesis. Another direction of study is to examine the structural effects of replacing secondary structural elements of cytochrome c (eg. Q-loops) with those of related proteins to further understand how large components of polypeptide chain are assembled and packed on the core of the protein.  Chapter 4  Structure - Function Analyses of Omega Loop A Replacements in Yeast Iso-1-Cytochrome c  4.1 Experimental Procedures Q-loop swap mutagenesis and subsequent protein purifications were performed by Dr. J. Fetrow (State University of New York). An alignment defining the primary sequence of the Q-loop swap proteins RepA2 and RepA2(Va120) with respect to those of yeast iso-l-cytochrome c and Rhodospirillum rubrum cytochrome c2 is given in Table 4.20. RepA2 crystals were produced by hair seeding liquid-liquid diffusion capillaries with crushed iso-1-cytochrome c crystals. Crystals of yeast RepA2(Va120) cytochrome c were initially grown by hair seeding hanging drops with crushed yeast iso-1-cytochrome c crystals as previously described (Leung et al., 1989). Crystals used for diffraction analyses were subsequently produced by macro-seeding hanging drops with small RepA2(Va120) crystals. For both proteins, the final crystallization conditions were 95% (NH4)2504, 0.07 M Na2S204 and 0.1 M sodium phosphate buffer adjusted to pH 5.5. Crystals used in diffraction analyses were first transferred into a freshly prepared solution of 97% (NH4)2SO4, 0.1 M sodium phosphate buffer at pH 5.5, and 0.04 M dithiothreitol, to maintain the reduced state of the heme group. These were then mounted in thin wall glass capillaries. A single RepA2 crystal was utilized to collect diffraction data to 2.25  A resolution (91%  complete) on a Rigaku R-AXIS II imaging plate detector system. Each frame had a 0 oscillation of 1.0° and was exposed for 15 minutes to CuK a X-rays from a rotating anode generator operating at 80 mA and 50 kV. A total of 99 frames were collected after 25 hours of X-ray exposure time. The resulting images were processed to structure factor amplitudes and the merging R-factor for both full and partial intensities was 7.3%. Other data collection parameters  72  Chapter 4. Replacements of Q-Loop A in Iso-l-Cytochrome c^  73  Table 4.20: Sequence alignment of the yeast iso-1, RepA2 and RepA2(Va120) cytochromes c with cytochrome c2 from Rhodospirillum rubrum ^ 30 20 -5^1^10 Iso-i^TEFKAGSAKKGATLFKTRCLQC H TVEKGGPHKVGPNL HG I FDQ^AN RepA2 RepA2(Va120) DQ AN R. rubrum - - - - EGDAAAGEKVSK - KCLAC H TFDQGGANKVGPNL HGV 40^50^ 60^70 Iso-1^FGRHSGQAEGYSYTDAN I K K NV - - - LWDENNMSEYLTNP R. rabrumFENTAAHKDNYAYS ESYTEMKAKGLTWTEAN L AAYVKNP 80^90^100 Iso-1^KKYIP ^ GTKMAFGGLKKEKDRNDLITYLKKACE R. rubrum KAFVLEKSGDPKAKSKMTF - KLTKDDE I ENVIAYLKTLK -  The primary sequences of yeast iso-1 (Smith et al., 1979) cytochrome c and R. rubrum (Dus et al., 1968) cytochrome c2 have been aligned based on a comparison of their superimposed threedimensional structures. The amino acid residues of the RepA2 and RepA2(Va120) mutants are given only where they differ from the yeast iso-1 sequence. The box encloses the sequence of 1k-Loop A. are listed in Table 4.21. The absolute scale of the resultant structure factors was derived by a linear rescale against a wild-type iso-l-cytochrome c data set having the same resolution range. The overall thermal factor of 20 A 2 was derived by inspection of a Wilson (1942) plot. From a single RepA2(Va120) crystal, a complete diffraction data set to 1.9  A was collected  on an Enraf Nonius CAD4-F11 diffractometer using continuous Q scans of 0.6° at 0.55°/min. The incident CuK c radiation was Ni filtered and generated from a X-ray tube operated at 26 mA and 40 kV. Intensity backgrounds were measured by extending the scans by 25% on either side. Three standard reflections were measured every 8 hours of X-ray exposure time to monitor crystal decay and slippage. Diffraction intensities were corrected for background radiation, absorption, decay, Lorentz and polarization effects as described in Section 2.1.3. Crystal specific data collection statistics are presented in Table 4.21. The initial scale and overall thermal factor (16 A 2 ) were determined as described for RepA2 cytochrome c.  Chapter 4. Replacements of Q-Loop A in Iso-l-Cytochrome c ^  74  Table 4.21: Data collection statistics of RepA2 and RepA2(Va120) cytochromes c  Parameter Crystal size (mm) Space group Unit cell parameters (A): a, b c Resolution (A) No. of reflections measured No. of unique reflections  RepA2  RepA2(Va120)  Iso-lt  0.1 x 0.1 x 0.05 P43212  0.48 x 0.38 x 0.2 P43212  P43212  36.38 137.63 2.25 25731 4498  36.37 137.64 1.9 9290 7918  36.46 136.86  t The cell dimensions and space group of wild-type yeast iso-l-cytochrome c are provided only for comparison purposes (Louie & Brayer, 1990). A starting model of RepA2(Va120) cytochrome c was constructed from the coordinates of yeast iso-l-cytochrome c (Louie & Brayer, 1990). At the position of each differing amino acid, the residue present was initially modeled as an alanine (Table 4.20). A sulfate ion and a selection of 52 water molecules of the iso-l-cytochrome c structure were included in the initial model. The model was then refined by a restrained parameter least-squares method (Hendrickson & Konnert, 1981) using data with F > 2o (F). Fragment deleted, F0 — Fe , 2F,— F, and 3F, — 2Fe -  maps were examined at intervals during the course of refinement as a guide for the addition and manual adjustment of side chains in the structure. Water molecules were added to the refinement model if at least one hydrogen bond was formed to the existing structure and if 2F, — I', or 3F, — 2F, maps had significant electron density at the water position. At the end of refinement, 74 water molecules and one sulfate ion (average solvent thermal factor of 34 A 2 ) were included into the model and the conventional R-factor was 19.1%. The final refined overall thermal factor was 22 A 2 . The final RepA2(Va120) structure was used as an initial model for the refinement of the RepA2 structure, with residue Va120 converted to an alanine by deletion of the coordinates of the two 7-carbons. The same selection of 52 water molecules and a sulfate ion from the  Chapter 4. Replacements of a-Loop A in Iso-l-Cytochrome c^  75  iso-1 structure were used as the initial solvent model. This model was refined as described for RepA2(Va120) with the eventual inclusion of the Phe20 side chain to a final R-factor of 18.3%. The final RepA2 structure contained 51 water molecules and a comparable sulfate ion to that found in the iso-1 and RepA2(Va120) cytochromes c (overall solvent thermal factor of 27 A 2 ). The final refined overall thermal factor was 17 A 2 . The agreement of the RepA2 and RepA2(Va120) structures with ideal stereochemical geometry and the corresponding refinement weights used in the final refinement cycles of each are outlined in Table 4.22. To estimate coordinate errors, a plot of the conventional R-factor as a function of resolution along with theoretical curves calculated by assuming various r.m.s. coordinate errors (Luzzati, 1952) was constructed (Figure 4.21). Inspection of this plot suggests  Table 4.22: Final stereochemistry of RepA2 and RepA2(Va120) cytochromes c  Stereochemical parameter  r.m.s. deviation from ideal values  Restraint weight  RepA2^RepA2(Va120) Distances (A) Bond Angle Planar 1-4 Plane (A) Torsional (deg.) Planar Staggered Orthonormal Chiral volume (A 3 )  0.018 0.042 0.053 0.013  0.018 0.039 0.048 0.015  0.020 0.030 0.050 0.020  2.3 24.5 16.5 0.210  2.5 23.9 13.8 0.213  2.5 20.0 15.0 0.160  Non-bonded contact (A)t Single torsion Multiple torsion Possible hydrogen bonds  0.217 0.195 0.224  0.209 0.195 0.192  0.250 0.250 0.250  t The r.m.s. deviations from ideality for this class of restraint incorporates a reduction of 0.2 from the radius of each atom involved in a contact.  A  Chapter 4. Replacements of C1-Loop A in Iso-l-Cytochrome c^  10.0 A  ^  5.o A  ^  Resolution 3.3 A^2.5  A  ^  2.0  A  ^  76  1.7 A 1.0 0.9  O  ^  ^ IL  s... 0 0.8 j o 0 0.7 C 0  ^  47-  0.6 0 a  ^^  u_  0.24 A  0.30-^  0.20  0.25-  0 "  A  0.16 A  0 0.20I  0.15-  0.10 0.05^0.10  0.15^0.20 SIN(0) /  0.25  ^  0.30  A  Figure 4.21: A plot of the conventional R-factor as a function of resolution at the end of refinement. The fit to the data of RepA2 (Q) is represented by the thick line and the fit to the RepA2(Va120) data (^) is represented by a thin line. The theoretical dependence of R-factor on resolution, assuming various levels of r.m.s. error in the atomic positions of the model (Luzzati, 1952), are drawn in dashed lines. The fraction of data used in refinement is presented above, using the same data point and curve scheme. a coordinate error for both structures of approximately 0.20 to 0.24  A. This value is a statistical  estimate of the overall coordinate error, whereas the actual positional error of an individual atomic coordinate will depend on the location of the atom in the structure. The error of atoms in the core of the protein is likely to be less, whereas highly mobile atoms on the protein surface are less accurately determined. A further analysis by an individual atom method (Cruickshank, 1949; Chambers & Stroud, 1979) suggests r.m.s. coordinate errors of 0.16  A for both RepA2  Chapter 4. Replacements of Q-Loop A in Iso-l-Cytochrome c ^  77  and RepA2(Va120) cytochrome c. 4.2 Results 4.2.1 Comparison of the Structures of RepA2, RepA2(Va120) and Iso-1-Cytochromes c  a-Loop A is located adjacent to the heme prosthetic group and is an integral component of the heme pocket (Figures 4.22 and 4.23). Part of this loop is adjacent to the solvent exposed edge of the heme and may be involved in the binding of redox partners. The structural differences present were examined by the superposition of the main chain and heme atom coordinates of RepA2 and RepA2(Va120) cytochromes c onto those of iso-1-cytochrome c by a least-squares fit. The two amino terminal residues (Thr-5 and Glu-4) were not included in this process since they are substantially disordered. The average main chain positional deviation between the three superimposed structures was computed on a per residue basis and plotted as shown in Figure 4.24. The average deviations of the 11-Loop A main chain atoms from the iso-1 structure are 0.21 A and 0.16 A for the RepA2 and RepA2(Va120) cytochromes c, respectively. Both of these average deviations are similar to the overall averages for all main chain atoms which are 0.22 A and 0.17 A for the two mutant proteins, respectively (Figure 4.24). Other than the disordered amino terminal residues —5 and —4, the region of Figure 4.24 with the most significant deviation is centered at position 44. The structures of RepA2 and iso-l-cytochromes c in this region are plotted in Figure 4.25. Note that as a result of S2-Loop A substitution, residue 26 is changed from a histidine to an asparagine in the RepA2 protein. The histidine side chain in iso-l-cytochrome c forms two hydrogen bonds to Asn31 N and Glu44 0 bridging these two sections of polypeptide chain. The OD1 atom of asparagine 26 in RepA2 is able to occupy the same location as His26 ND1 in iso-1 and forms the first hydrogen bond. However, due to a shorter side chain length, Asn26 ND2 cannot occupy the same location as the original His26 NE2 atom of the iso-1 protein. As a result, the polypeptide chain from Gln42 to G1y45 shifts so that G1u44 0 is positioned within hydrogen bonding distance.  Chapter 4. Replacements of 52-Loop A in Iso-l-Cytochrome c^  78  Figure 4.22: A space-filled representation of yeast iso-l-cytochrome c. The atoms of the central heme group are shown as black spheres. All other atoms are colored white except those of a-Loop A (residues 18 to 32) which are shown as grey, with Va120 and His26 highlighted in black.  Chapter 4. Replacements of CI-Loop A in Iso-l-Cytochrome c ^  79  Figure 4.23: A stereo plot of iso-l-cytochrome c with C2-Loop A drawn with thick lines. Every tenth residue is labeled with the one letter amino acid code. The orientation of this plot is similar to that of Figure 4.22. A further analysis of the positional deviation of well ordered side chains with average thermal factors less than 25  A 2 reveals three side chains with different conformations between RepA2  and iso-1-cytochromes c. These residues are Tyr97, Leu98 and A1a101 all of which pack against the side chain at position 20. As can be seen in Figure 4.26, the side chain of Phe20 in the RepA2 structure is positioned in a direction defined by the vector between atoms Va120 CB and Va120 CG2 of the wild-type iso-l-cytochrome c. This conformation has the phenyl ring directed towards the protein surface. While the presence of a phenylalanine at position 20 does not markedly perturb the main chain atoms of C/-Loop A (Figure 4.24), the side chain atoms of Tyr97, Leu98 and Ala101 are displaced on average by 0.8, 0.7 and 0.7  A, respectively  (Figure 4.26). In the RepA2(Va120) structure, Leu98 is one of the residues that line an internal hydrophobic buried cavity large enough to contain a solvent molecule. A similar cavity is observed in the iso-l-cytochrome c structure (Louie & Brayer, 1990). The structural and functional role of this cavity is unknown, but amino acid substitutions in the hydrophobic core of the protein have been shown to affect the size of this cavity (see Section 3.4). The RepA2 structure does not  •  Chapter 4. Replacements of a-Loop A in Iso-l-Cytochrome c^  80  1.0  0.8  O 0.6 0  a) 0 (1) -) O  N  0.4  0.2  ,^AA  IlliMPLOINSISEVAINMEMMI IMERMANIMINIIMEM  T '^fit^Vim  0.0 15^25^35^45^55  ^  65  ^  75  ^  85  ^  95  Residue Number  Figure 4.24: The average positional deviations of main chain atoms between RepA2 and wild-type yeast iso-1 (thick line), RepA2(Va120) and wild-type yeast iso-1 (thin line), and RepA2 and RepA2(Va120) (dotted line) cytochromes c along the course of the polypeptide chain. The horizontal lines represent the overall average deviations for the main chain atoms of residues (-3) to 103 for each comparison: RepA2 and isol, 0.22 A; RepA2(Va120) and iso-1, 0.17 A; RepA2(Va120) and RepA2, 0.18 A. contain an internal cavity observable with a 1.4  A radius probe. The loss of this well defined  cavity appears to be a result of the repositioning of the Leu98 side chain. None of the atoms of the Phe20 side chain occupy the space of the original Va120 CG1 methyl group which is buried in the iso-1 structure. Movement of the side chain of Leu98 in the RepA2 structure, however, partially fills the space vacated by Va120 CG1. The substitution of valine for phenylalanine at position 20 in the RepA2(Va120) protein eliminates most of the structural changes described above. Residues Va120, Tyr97, Leu98 and Ala101 are all positioned in a manner similar to that observed in the iso-1 structure (Figure 4.26). Furthermore, in the RepA2(Va120) structure, an internal cavity comparable to the wild-type iso-l-cytochrome c structure is present. However, as observed in the RepA2  Chapter 4. Replacements of 11-Loop A in Iso-l-Cytochrome c ^  81  Gln 42  Ala 43  '•••—iGlu 44  -  Asn. 31^  Pro 30  Gly 45  His 26  Asn 31  Pro 30  Figure 4.25: The structures of wild-type yeast iso-1 (thin lines), RepA2 (thick lines) and RepA2(Va120) (medium lines) cytochromes c are plotted in the region of residue 26. In the iso-1 structure, His26 bridges these two segments of polypeptide chain by forming two hydrogen bonds (dashed lines). In the RepA2 and RepA2(Va120) structures, the main chain surrounding G1u44 shifts to form the same two hydrogen bonds to the shorter replacement Asn26 side chain.  Phe 20  Phe 20  Val 20  Val 20 Leu 98  Figure 4.26: Superimposed structures showing the packing of residue 20 against the nearby carboxy terminal a-helix in wild-type yeast iso-1 (thin lines), RepA2 (thick lines) and RepA2(Va120) (medium lines) cytochromes c. In the RepA2 structure, three side chains (Tyr97, Leu98, A1a101) are repositioned to accommodate the larger phenylalanine side chain at position 20.  Chapter 4. Replacements of Q-Loop A in Iso-l-Cytochrome c^  82  structure, the substitution of asparagine for histidine at position 26 results in similar shifts of the main chain atoms of residues G1n42 through G1y45 (Figure 4.24).  4.2.2 Electrochemistry of RepA2 and RepA2(Va120) Cytochromes c Direct electrochemistry of RepA2 and RepA2(Va120) cytochromes c appears to be reversible with a peak to peak separation of 62 ± 7 mV for all measurements, over the temperature range from 10 to 25°C. However, measurements above 25 °C could not be made since protein solutions became turbid resulting in greatly increased peak to peak separation in the voltammograms and a loss of reversibility. A plot of the temperature dependence of the midpoint potential of these two Q-loop replacement proteins and iso-1-cytochrome c is presented in Figure 4.27. Thermodynamic parameters derived from this data are listed in Table 4.23 along with midpoint  300.0  5 290.0E  a C  (1)  0 0_ 0  a 280.0-  270.0 50  10.0  ^  15.0^20.0  ^  25.0  30.0  Temperature (°C)  Figure 4.27: The midpoint reduction potential of the RepA2 (0), RepA2(Va120) (0) and wild-type iso-1 (,A) cytochromes c are plotted as a function of temperature. Data for iso-l-cytochrome c is from Rafferty (1992).  ^  Chapter 4. Replacements of 12-Loop A in Iso-1-Cytochronle c^  83  Table 4.23: Electrochemical properties of the yeast iso-1, RepA2, and RepA2(Va120) cytochromes c  Protein  0^0^0 Em^OH^AScyt^AG (mV)^(kcal/mol)^(e.u.)^(kcal/mol)  ^  Iso-1 RepA2 RepA2(Va120)  290 2 271 ± 2 290 ± 2  -14.0 ± 0.2 -18.2 ± 0.7 -15.2 ± 0.1  -9.1 ± 0.4 -24 ± 2 -13.0 ± 0.2  -6.7 ± 0.1 -6.3 ± 0.1 -6.7 ± 0.1  Experimental conditions were 25 °C, pH 6.0, it = 0.1 M and SHE reference. Data for iso-1cytochrome c is from Rafferty et al., 1990.  potentials under standard conditions. The midpoint potential for the RepA2(Va120) protein at 25 °C is the same as that observed for wild-type iso-l-cytochrome c within experimental error. In contrast, the midpoint potential of RepA2 differs markedly and is 19 mV lower than that of the wild-type cytochrome c. The enthalpy and entropy of reduction of the RepA2 and RepA2(Va120) proteins are less than that of wild-type iso-1-cytochrome c and partially compensate for each other.  4.3 Discussion 4.3.1 Comparison of the Wild Type Yeast Iso-1 and It. rubrum Structures Overall, the amino acid sequence identity between yeast iso-l-cytochrome c and R. rubrum cytochrome c2 is 38% as shown by the sequence alignment in Table 4.20. A comparison of the amino acid sequences of their respective 12-Loop A segments (residues 18 to 32) reveals that 10 of the 15 residues present are identical (67% of the total). There are 18 additional residues from other polypeptide chain segments that contact S2-Loop A in iso-l-cytochrome c. Nine of these (50%) are identical with those of R. rubrum cytochrome c2. The main chain and heme coordinates of R. rubrum cytochrome c2 can be superimposed onto the iso-1 structure with an average deviation of 1.1  A, excluding residues 57 and 76 which differ greatly in conformation between  84  Chapter 4. Replacements of Q-Loop A in Iso-l-Cytochrome c^  Val 20 His 18  Leu 32  Lys 22  Lys 22^ Val 28^  His 26  Val 28  His 26  Figure 4.28: A stereographic plot of a-Loop A from yeast iso-l-cytochrome c (thick lines) superimposed on that of cytochrome c9 from R. rubrum (thin lines). The overall path of the polypeptide chain is similar in both loops, but the precise conformation differs. The termini of this loop, His18 and Leu32, and every fifth residue of yeast iso-1-cytochrome c are labeled. these two proteins. The Q-Loop A portion of these superimposed structures is illustrated in Figure 4.28. If only the main chain atoms for Q-Loop A are considered, the average deviation observed is 0.53  A.  Many of the residues of a-Loop A conserved between the yeast iso-1 and R. rubrum cytochromes form intra-loop hydrogen bonds which stabilize this folded structure (Table 4.24). Note that two of the four intra-loop hydrogen bond differences listed in Table 4.24 are equivalent since the hydrogen bond between the side chain of residue 26 and the main chain amide of residue 31 is conserved despite a change in amino acid sequence. The other two hydrogen bond differences are a result of an amino acid difference at position 21 and the absence of a ry-turn in the R. rubrum structure. A further understanding of the structural heterogeneity in C2-Loop A can be found by examining the interaction of this loop with the remainder of the protein molecule. Hydrogen bond interactions involving only one loop atom are more varied due to amino acid substitutions occurring at positions outside of a-Loop A (Table 4.24). Substitutions at positions outside of S2-Loop A particularly affect the packing of buried loop side chains. One prominent example of packing differences in a side chain group between the yeast iso-1 and R. rubrum cytochromes involves residue 20 which forms part of the hydrophobic core of  Chapter 4. Replacements of a-Loop A in Iso-l-Cytochrome c  ^  85  Table 4.24: Hydrogen bond interactions of a-Loop A in yeast iso-1, RepA2, RepA2(Va120) cytochromes c and R. rubrum cytochrome c2 Cytochrome  ^  Intra-a-Loop A interactions  ^  Involving other protein residues  Yeast iso-1 18 N - 14 0 His26 NE2 - 44 0 Lys27 NZ - 15 0 29 N - 17 0 33 N - Asn31 OD1 His33 ND1 - 20 0 35 N - 32 0 Tyr46 OH - 28 0  His18 ND1 - 30 0 Thr19 0G1 - 25 0 Thr19 OG1 - Asn31 ND2 20 N - G1u21 OE1 24 N - 21 0 27 N - 29 0 31 N - His26 ND1 Asn31 ND2 - Thr19 OG1 Asn31 ND2 - 21 0 32 N - 19 0 Yeast RepA2  e e  Yeast RepA2(Va120)  e e  20 N - Glu21 OE1 31 N - Asn26 OD1 31 N - His26 ND1 20 N - Glu21 OE1 31 N - Asn26 OD1 31 N - His26 ND1  e  e  e  Asn26 ND2 - 44 0 His26 NE2 - 44 0  Asn26 ND2 - 44 0 His26 NE2 - 44 0 33 N - Asn31 OD1  R. rubrum  e e e  20 N - Glu21 OE1 27 N - 29 0 31 N - Asn26 OD1 31 N - His26 ND1  ED  e e ED  Asn26 ND2 - Asn45 ND2t His26 NE2 - 44 0 28 N - 17 0 His33 ND1 - Asn31 OD1 Lys43 NZ - 31 0 Lys43 NZ - Asn31 OD1  C2-Loop A is comprised of the amino acids from His18 to Leu32. Where main chain atoms are involved only the sequence number and atom name are given. All hydrogen bonds are listed for wild-type iso-l-cytochrome c. For the other three structures, the symbol 6 indicates an additional hydrogen bond in a particular structure whereas e indicates the absence of a hydrogen bond relative to the iso-1 structure. Hydrogen bonds are defined by the following criteria: a H...A distance < 2.60 A. 2.70 A or 3.05 A where A is an oxygen, nitrogen or sulfur atom respectively; a D-H...A angle > 120°; and, a C-A...H angle > 90°. tIn the structure determined for R. rubrum cytochrome c2 (Salemme et al., 1973), if the side chain amide group of Asn45 is rotated 180°, the OD1 atom would be positioned within hydrogen bonding distance.  Chapter 4. Replacements of a-Loop A in Iso-l-Cytochrome c^  86  both molecules. This residue is a valine in the iso-1 structure and a much larger phenylalanine in R. rubrum cytochrome c2. The larger side chain of the R. rubrum protein is accommodated in the hydrophobic core by the complementary substitution of PhelOSer (Table 4.20). In addition, the presence of Phe33 eliminates a hydrogen bond to the carbonyl oxygen of residue 20 formed in the iso-1 structure by His33 ND1. These factors serve to optimize the fit of the phenyl group of residue 20 into the hydrophobic core of the R. rubrum protein. Another major packing difference involves residue 26. In one instance, the hydrogen bond between the side chain of residue 26 and the main chain of residue 31 is in fact conserved between the two proteins. However, the interaction of this residue with the nearby polypeptide chain segment (involving primarily residues 44 and 45) is substantially different. In the iso-1 structure, a hydrogen bond is formed between His26 NE2 and Glu44 0. In the R. rubrum structure, Asn26 ND2 interacts with the side chain of Asn45 leading to a different folding of the polypeptide chain in this area.  4.3.2 Structural Effects of Q-Loop A Replacements In RepA2 cytochrome c, Q-Loop A from R. rubrum has been spliced into the yeast iso-lcytochrome structure in place of the normally resident residues. An examination of this structure reveals that the new Q-Loop A has an overall main chain conformation very similar to that observed for the original loop in the iso-1 structure (Figure 4.24). In some cases, such as the Q-Loop A amino acid differences at positions 21 and 22 on the surface of the protein, these substitutions are easily accommodated into the yeast iso-1 structure without any obvious structural perturbations. However, in other cases, such as the side chain substitutions at positions 20 and 26, which occur in the molecular interior where these residues form close molecular interactions, the a-Loop A substitution has had a significant structural effect. For example, if the R. rubrum loop is inserted without adjustment of the new phenylalanine side chain at position 20, a steric clash with the side chain of Phe10 results. This conflict is relieved in the RepA2 structure by redirecting the Phe20 side chain towards the molecular surface by  Chapter 4. Replacements of a-Loop A in Iso-1-Cytochrome c^  87  a 120° rotation about the xi torsional angle of this residue. Nonetheless, this reorientation is not entirely sufficient to avoid disruption of the packing in the hydrophobic core of the yeast iso-l-cytochrome c structure and as a consequence the side chain of Leu98 shifts to fill some of the internal empty space generated by the Va120Phe substitution. At the molecular surface, the large phenyl group at position 20 in the RepA2 mutant also displaces the side chains of Tyr97 and Ala101 which pack against the original Va120 side chain (Figure 4.26). The end result is that residues not part of a-Loop A, along with the Phe20 side chain, undertake positional adjustments which allow the swapped loop to have a main chain conformation nearly identical to the loop it replaces. In the RepA2(Va120) mutant, residue 20 has been substituted back to a valine as in the wild-type iso-l-cytochrome c. The side chain of Va120 in the RepA2(Va120) structure is in a conformation comparable to that of the iso-1 protein (Figure 4.26). As a result, this substitution restores the packing of the hydrophobic core and does not lead to the displacement of Tyr92, Leu98 or Ala101. Although the substitution of asparagine for histidine at position 26 in both loop replacement proteins is conservative, as Figures 4.24 and 4.25 show, it does result in alterations in the main chain fold centered at the adjacent residue G1u44. An analysis shows that the main chain atomic positions of residue 26 appear to be more tightly fixed by hydrogen bond interactions with other groups than residues 42 through 45 which occupy a more solvent exposed position. As a result, this latter segment of polypeptide chain has greater flexibility to shift position in order to maintain a hydrogen bond between the side chain of residue 26 and the carbonyl group of residue 44. It is of interest that in yeast iso-2-cytochrome c there is also an asparagine at position 26 (see Section 3.3). Although the polypeptide chain in the region of residue 44 is slightly displaced relative to that of the iso-1 structure, the hydrogen bond between Asn26 ND2 and G1u44 0 is not formed (Table 3.14). This appears to be a consequence of the replacement of alanine at the adjacent residue 43 by a valine which fills the space vacated as a result of the substitution  Chapter 4. Replacements of a-Loop A in Iso-l-Cytochrome c^  88  of the smaller asparagine side chain for the histidine side chain at position 26. This second substitution therefore appears to prevent the type of displacement observed in the polypeptide chain of RepA2 and RepA2(Va120) cytochromes c which is required to form a hydrogen bond to the carbonyl group of G1u44. 4.3.3 In Vivo Functional Consequences of a-Loop A Replacements Data concerning the in vivo function and further measurements of cytochrome c levels in intact cells were completed and kindly provided by Dr. J. Fetrow (State University of New York). In vivo function was assessed by measuring the rate of growth of yeast strains containing wildtype, RepA2 and RepA2(Va120) as well as a strain containing no cytochrome c (Figure 4.29). The substantive difference in functional activity of RepA2 at various temperatures could be caused by a decreased level of intact cytochrome c present within the cell. This possibility was examined by determining the levels of holo-cytochrome c present within intact cells by low temperature spectroscopy (Figure 4.30). Note that this spectroscopic technique can only determine levels of holoprotein, that is, protein with a heme group covalently attached. Levels of apoprotein are not determined using this technique.  In vivo, low temperature spectroscopy shows that the amount of RepA2(Va120) holoprotein present in yeast cells is slightly less than wild-type protein at 25, 30 and 37 °C; however, the function of these proteins is very similar at all three temperatures (Figures 4.29 and 4.30). In total. RepA2(Va120) cytochrome c differs from the wild-type yeast iso-1 protein by four amino acids (Table 4.20). The RepA2 mutant, on the other hand, which differs from RepA2(Va120) cytochrome c by a single amino acid, displays very different functional behavior and appears to be a temperature sensitive mutant. This protein is functional at levels comparable to the wild-type at 25 °C, somewhat less at 30 °C (Fetrow et al., 1989), and only at very low levels at 37 °C. Low temperature spectroscopy indicates that there is less of this protein present in intact yeast cells at all three temperatures, but that the relative ratios are similar. However the RepA2 mutant does contain holoprotein in quantities sufficient to allow growth of cells in  ^  Chapter 4. Replacements of C2-Loop A in Iso-l-Cytochronie c ^  1000  ^  89  A. 25°C  100  10  0_00^7:30^15.00^22:30  0:00  ----  ^co  1000  ^  7:30  ^  15:00  —^  ^  22:30  ^  30:00  C. 37°C  nf ■  100  ,--e— e-  •  10  -  112 0 ^0:00  ^  7:30^15:00^22:30^30:00  Culture Time (Hours)  Figure 4.29: Growth curves of yeast strains in liquid lactate media containing RepA2 (•) RepA2(Va120) ), wild-type iso-1 (0) and no (A) cytochromes c are plotted. Growth curves were determined by a variation of the method of Schweingruber et al. (1979). Data and figure were kindly provided by of Dr. J. Fetrow, State University of New York.  Chapter 4. Replacements of 12-Loop A in Iso-l-Cytochrome c ^  90  0.13  0.1187  0.1075  0.09625  0.085 500  527.5  555  582.5  610  O (0 2 0.285 O  -  610  0.305  0.295 CO 0.285 03 cri .0  0.275  0.265  0.255 ^ 500^527.5^555^582.5 610  Wavelength (nm)  Figure 4.30: Spectra of intact yeast cells, both experimental and control strains, at —196 °C. Spectra of RepA2 (thick line), RepA2(Va120) (dotted line), wild-type iso-1 (thin line) and no (dashed line) cytochromes c are plotted. The peak at 550 nm (indicated with an arrow) is one of the herne absorbance peaks in reduced cytochrome c. Spectra were recorded as described in Fetrow et al. (1989). Data and figure were kindly provided by of Dr. J. Fetrow. State University of New York.  Chapter 4. Replacements of a-Loop A in Iso-l-Cytochrome c^  91  liquid lactate media. The presence of significant amounts of RepA2 protein in intact cells suggests that the mutation has not interfered with protein production. Cytochrome c in yeast is transcribed in the nucleus, translated on a ribosome in the cytosol, and transported across the mitochondrial membrane where the heme is covalently attached by heme lyase (Pettigrew & Moore, 1987). The messenger RNA, the apoprotein and the holoprotein of RepA2 cytochrome c appear to be nearly as stable as those of iso-1-cytochrome c to degradation at higher temperature. The more likely explanation for the reduced function of RepA2 cytochrome c at higher temperatures is that it is a functionally less efficient protein. The average main chain thermal factors of a-Loop A in the RepA2 structure are significantly higher than in the RepA2(Va120) and iso-1 structures (Figure 4.31). The replacement of amino acids at the protein surface combined with the increase in mobility of Q-Loop A in the RepA2  30  oQ  L 0  U  0 20a) 0  L  a)  0  E  H. 10 a) 0") 0 L  a)  0 20^22^24^26^28^30^32  Residue Number  Figure 4.31: The average thermal factor of the four main chain atoms of each amino acid in RepA2 (thin line). RepA2(Va120) (dotted line) and wild-type iso-1 (thick line) cytochromes c are plotted as a function of residue number for Q-Loop A. The thermal factors have been normalized over all protein atoms by an additive factor. The greatest main chain thermal factor difference between the RepA2 structure and the other two structures is within the 1-Loop A region.  Chapter 4. Replacements of C2-Loop A in Iso-l-Cytochrome c^  92  structure may interfere with the binding of yeast cytochrome c with physiologically important electron transfer partners thereby diminishing cytochrome c function. This loss of function is ultimately expressed in terms of slower cell growth. 4.3.4 The Relationship Between Heme Reduction Potential and Temperature Sensitivity The RepA2(Va120) cytochrome c has a midpoint potential comparable to that of wild-type yeast iso-1-cytochrome c (Table 4.23). It therefore appears that the amino acid substitutions present in comparison to the original iso-1 Il-Loop A have had no effect on this functional parameter. This includes the Lys22Gln substitution which has caused a change in the surface charge of the molecule. However, the single substitution of valine for a phenylalanine at position 20 in the RepA2(Va120) cytochrome c did restore the midpoint potential of the RepA2 cytochrome c to comparable values found for the wild-type iso-1 protein at 25 °C (Figure 4.27). Clearly, Va120 is important for the maintenance of the heme reduction potential in wild-type iso-1-cytochrome c. Nonetheless, the mechanism by which residue 20 modulates midpoint potential is not readily apparent. Previous studies have linked a change in the solvent accessibility of the heme (Stellwagen, 1978; Louie et al., 1988b) or polarity of the heme environment (Kassner, 1973; Louie Sc Brayer, 1989) to a change in reduction potential. However, the solvent accessibilities of the porphyrin ring atoms of the heme are the same for RepA2, RepA2(Va120) and iso-l-cytochromes c. Furthermore, no other changes in the position of polar groups near the heme are observed in either the RepA2 or RepA2(Va120) structures relative to the wild-type protein. The thermodynamic parameters derived from the temperature dependence of the midpoint reduction potential may provide insight into the mechanism of reduction potential modulation by residue 20 (Table 4.23). The larger decrease in the entropy of reduction is partially offset by a decrease in the enthalpic contribution. A change in the polypeptide contribution to the thermodynamic properties of cytochrome c reduction has been suggested to be primarily due to electrostatic effects (Schejter et al., 1982), however, the substitution at position 20 does not  Chapter 4. Replacements of a-Loop A in Iso-l-Cytochrome c ^  93  involve a change in net molecular charge. The change in enthalpy could be accounted for by the increase in hydrophobic bulk of the protein (Kassner, 1973). Taniguchi et al. (1980) have suggested that polypeptide chain ordering and protein solvent interactions are also likely important in determining the entropy of reduction. The Phe20 side chain is largely solvent accessible in the RepA2 structure which may alter solvent ordering upon reduction. The increased mobility of a-Loop A as seen in the temperature factor values (Figure 4.31) may also contribute to this effect. In addition, the increase in thermal factors may result in an increase in dynamic heme solvent exposure.  Chapter 5  Structural Analysis of Mutants of an Invariant Proline in Yeast Iso-1-Cytochrome c  5.1 Experimental Procedures Samples of the Pro7lAla, Pro7lIle, Pro7lSer and Pro71Val cytochromes c were provided by Dr. F. Sherman (University of Rochester). Crystals of these mutant proteins were produced by hair seeding liquid-liquid diffusion capillaries with crushed iso-l-cytochrome c crystals (Salemme, 1972; Leung et al., 1989). The final crystallization conditions for the Pro7lAla, Pro7lIle and Pro7lSer mutants were: 92% saturated (NH4)2SO4, 0.07 M dithionite and 0.1 M sodium phosphate buffer adjusted to pH 6.8. The Pro7lVal crystals were grown under similar conditions: 90% saturated (NH4)2SO4, 0.01 M dithiothreitol and 0.1 M sodium phosphate buffer adjusted to pH 6.0. Crystals used in diffraction analyses were transferred into a freshly prepared solution of 95% saturated (NH4)2SO4, 0.1 M sodium phosphate buffer of the same pH as the crystallization buffer, and 0.04 M (0.01 M for Pro7lVal) dithiothreitol, to maintain the reduced state of the heme group. Each of the data sets listed in Table 5.25 was collected from a single crystal. Diffractometer data sets were collected of the Pro7lAla, Pro7lSer and Pro71Val cytochromes c with an Enraf Nonius CAD4 instrument using continuous S2 scans of 0.5 to 0.6° at 0.55°/min at an ambient temperature of 15 °C. Standard reflections were measured every 8 hours of X-ray exposure time to monitor crystal decay and slippage. Two different area detector systems were used to collect two independent data sets of the Pro7lIle mutant. The first data set was collected with a Rigaku R-AXIS II imaging plate detector system. Each of the 60 frames was exposed for 15 minutes to CuIC, X-rays from a rotating anode generator operating at 80 mA and 50 kV. The  94  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c^95  Table 5.25: Data collection statistics for Pro71 mutant cytochromes c  Parameter Crystal volume (mm 3 ) Space group Unit cell parameters (A): a, b  c Instrument Resolution (A) No. of reflections measured No. of unique reflections  Pro7lAla  Pro7lIle(1)  Pro7lIle(2)  Pro7lSer  Pro71Val  0.04 P43212  0.01 P43212  0.005 P43212  0.03 P43212  0.06 P43212  36.45 137.3 CAD4 1.8 12376 9266  36.45 137.1 FAST 1.7 25290 8899  36.54 137.3 R-AXIS 1.7 40082 8546  36.44 137.3 CAD4 1.8 15374 9295  36.45 137.0 CAD4 1.9 10215 7864  Max. decay correctiont  0.81  0.68  0.68  Max. absorption correctiont  0.54  0.62  0.61  R-scale with iso-1t (%)  11.7  14.5  15.3  tThis R-scale is defined as:  8.6  2 E I Fo E (F61 ±  9.2  -  where Foi and F0J are the observed structure factor amplitudes of wild-type iso-l-cytochrome c and the data set being compared, respectively. t These corrections are handled by inter-frame scaling for area detector data sets. crystal was oscillated through a cb angle of 1.0° for each frame. Due to the high symmetry of space group P43212, a nearly complete data set could be collected with an overall 60° rotation of the angle 0. The second data set was collected with an Enraf Nonius FAST detector using the same X-ray radiation. Data collection statistics for both data sets are presented in Table 5.25. The two Pro7lIle mutant data sets were then merged together giving an R-factor of 7.7% based on 7246 duplicate intensities, to produce a 92% complete data set to 1.7  A having 10198 unique  reflections. The absolute scale of the structure factors of each mutant diffraction data set was derived by a linear rescale against an iso-l-cytochrome c data set having the same resolution range.  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c^96  The starting model of each mutant protein was constructed from the coordinates of yeast iso1-cytochrome c (Louie & Brayer, 1990) by deleting all the side chain atoms except the 3-carbon at position 71 to create an alanine residue. A sulfate ion and a selection of 52 water molecules of the iso-l-cytochrome c structure were also included in the initial model. The model was then refined by a restrained parameter least-squares method (Hendrickson & Konnert, 1981) using all data with F > 2a(F) and a resolution greater than 6  A. Fragment deleted, F0 — Fe,  2F0 — I', and 3Fo — 2F, maps were examined at intervals during the course of refinement to fit the complete side chain at position 71 and to adjust the conformations of other side chains in the structure. Peaks along the protein surface in F, — I', maps were considered water molecules if at least one hydrogen bond was formed to the existing structure and the refined thermal factors were less than 55 A 2 . The R-factor, number of solvent molecules, thermal factor statistics and stereochemistry of the final refined mutant structures are presented in Tables 5.26 and 5.27. Plots of the conventional R-factor as a function of resolution, along with theoretical curves calculated by assuming various r.m.s. coordinate errors (Luzzati, 1952), indicate a r.m.s. error of approximately 0.20  A for all four structures (Figure 5.32). If a complete model is assumed,  the r.m.s. coordinate errors range from  0.11 A for Pro7lIle to 0.16 A  for Pro7lVal using the  Cruickshank approach (Cruickshank, 1949; Chambers & Stroud, 1979).  5.2 Results 5.2.1 The Environment of Pro71 in Yeast Iso-1-Cytochrome c The effects of amino acid replacements at position 71 in yeast iso-1-cytochrome c were analyzed through comparison with the wild-type protein (Louie & Brayer, 1990). In yeast cytochrome c, Pro71 is adjacent to the carboxy terminus of one a-helix and initiates another a-helix (Figure 5.33). One of these is Helix III formed by residues 60 through 70 and the second helix is Helix IV consisting of residues 70 to 74 (Table 1.4). Pro71 prevents the elongation of the first helix because the pyrrole ring removes the amide functional group and prevents the formation of a hydrogen bond to the carbonyl group of Tyr67. Furthermore, a steric clash between the  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c^97  Table 5.26: Final refinement statistics for Pro71 mutant cytochromes c Refinement Statistic  Pro7lAla  Pro7lIle  Pro7lSer  Pro7lVal  6868 18.9 17.2 891 73 28.2  9805 18.6 19.4 894 80 36.0  6197 18.3 17.0 892 67 29.6  5381 18.9 17.7 893 61 26.6  No. of reflections R-factort(%) Overall thermal factor (A 2 ) No. of protein atoms No. of solvent molecules Avg. solvent thermal factor (A 2 ) t This R-factor is defined as: R  =  ^  hkl  liFohkri ohial  Table 5.27: Final stereochemistry for Pro71 mutant cytochromes c Stereochemical restraint Distances (A) Bond Angle Planar 1-4 Plane (A) Chiral volume (A 3 ) Non-bonded contact (A)t Single torsion Multiple torsion Possible hydrogen bonds Torsional angles (deg.) Planar Staggered Orthonormal  r.m.s. deviation from ideal values Pro7lAla Pro7lIle Pro7lSer Pro7lVal  Restraint weight  0.019 0.039 0.051 0.015 0.209  0.019 0.038 0.056 0.016 0.199  0.019 0.040 0.051 0.016 0.200  0.019 0.040 0.052 0.015 0.230  0.020 0.030 0.050 0.020 0.180  0.219 0.185 0.217  0.211 0.182 0.190  0.225 0.172 0.220  0.218 0.214 0.260  0.250 0.250 0.250  2.4 21.4 19.0  2.7 20.3 19.3  2.4 23.5 20.2  2.4 22.8 20.6  2.5 20.0 20.0  t The r.m.s. deviations from ideality for this class of restraint incorporates a reduction of 0.2 A from the radius of each atom involved in a contact.  ^  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c^98  Resolution 3.3 A^2.5 A^2.0 A^1.7 A  10.0 A^5.0 A  I^I  ,^  ,^f^i^r^  I  1.4 A '^ 1.0 -  er a)  -0.9 L. -0.8 0 - 0 - 0.7 ti  0  0.35-  0.24 A 0.300.20 A 0  0.25 -  13  0.16 A 0.200 0.15-  'o• ^I  /  ^ / /^/^.... I /^I^ /  0.10^ t^1^t^t ^ I^' 0.05^0.10^0.15^0.20^0.25 0.30^0.35 SIN(0)  /x  Figure 5.32: A plot of the conventional R-factor as a function of resolution at the end of refinement. The fit to the data of the ■ Pro71Ala, ^ Pro71Ile, • Pro7lSer, 0 Pro71Val cytochrome c structure determinations are represented by solid lines. The theoretical dependence of the R-factor on resolution, assuming various levels of r.m.s. error in the atomic positions of the model (Luzzati, 1952), are drawn with dashed lines. Above, the fraction of data used in refinement for each structure is presented, using the same data point and curve scheme.  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c ^99  Figure 5.33: The region near Pro71 in yeast iso-l-cytochrome c. Main chain atoms in the region of Pro71 of yeast iso-l-cytochrome c are depicted as ribbons. The ribbon corresponding to Helices III and IV (Table 1.4) is shaded darker. Residues within 4 A of the Pro71 side chain are displayed and labeled. The heme moiety, the heme iron ligands (His18 and Met80) and the heme thioether linkages to Cys14 and Cys17 are also displayed for reference.  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c^100  pyrrole ring of Pro71 and the side chain of Asn70 would exist if these residues were in an ahelical conformation. Conversely, the same pyrrole side chain locks the main chain torsional angle q5 near —60° initiating helical segment IV. In this way, the carbonyl group of residue 71 interacts with 11e75 N forming the first hydrogen bond in Helix IV. It has been proposed that one function of Pro71 is to direct the proper folding of the polypeptide chain in this region (Takano & Dickerson, 1981a; Louie et al., 1988a). The overall polypeptide chain fold of cytochrome c completely occludes the main chain and side chain atoms of Pro71 from bulk solvent. Thus, the side chain of Pro71 is in the hydrophobic core of this protein and packs against Tyr67, the Met80 side chain, Phe82 and Gly83 (Figure 5.33). Of these residues, the heme iron ligand Met80 would be expected to be the most functionally sensitive to amino acid replacements at position 71. Furthermore, the polypeptide chain in the region of both Pro71 and Gly83 is observed to be more flexible when cytochrome c is in the oxidized state suggesting that these portions of the polypeptide chain may be of functional importance in oxidation state dependent conformational changes (Berghuis & Brayer, 1992).  5.2.2 Structural Differences of Pro71 Mutant Cytochromes c Matrices showing main chain distance differences and thermal factor differences of each position 71 mutant structure from the values of wild-type iso-l-cytochrome c are presented in Figure 5.34. These analyses indicate that observed main chain structural differences are localized to two regions. These are in the immediate vicinity of the mutation site and about residue 83. In addition, the four Pro71 mutant structures have been superimposed onto the wild-type structure by a least-squares fit of the main chain and heme atoms. The average deviations for all the main chain atoms of Pro7lAla, Pro71Ile. Pro7lSer and Pro71Val cytochromes c are presented in Table 5.28. Like the difference matrices, these results indicate that the main chain fold is perturbed in the vicinity of Pro71 and Gly83 but is otherwise comparable to the wild-type protein. Analysis of mutant protein heme solvent accessibility and heme ligand geometry shows  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c  60^70^80^90^100  100  ^  101  40^50^60^70^80^90^100  C  Pro71 Ser  a  90 80  0  _aE  0.>  60  0^I  C4) 0  Z 50  a)  40  a)  CC 30 20  0  10 0^  0  10^20^30^40^50^60^70^80  Residue Number  100  10^20^30^40^50  60^70  80  90  100  Residue Number  Figure 5.34: Positional and thermal factor difference matrices for the comparison of Pro7lAla, Pro7lIle, Pro7lSer and Pro71Val cytochromes c with the wild-type protein. The upper left triangular matrix contains the a-carbon distance differences and the lower right triangle contains the average main chain thermal factor differences. Each triangular matrix is contoured at one standard deviation intervals above the mean difference. The first five residues (-5 to —1) are not included in the matrices due to positional disorder in this region. See Section 2.2.2 for a complete description of difference matrix construction.  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c^102  Table 5.28: Positional deviations of groups in the vicinity of residue 71  Group  Positional deviation (A) Pro7lAla Pro7lSer Pro7lIle Pro71Val  A. Main chain Asn70 71 Phe82 G1y83 G1y84  0.27 0.10 0.31 0.75 0.40  0.11 0.24 0.32 0.84 0.45  0.38 0.34 0.28 0.44 0.19  0.57 0.45 0.17 0.68 0.47  B. Side chain Tyr67 Met80 Phe82  0.19 0.12 0.27  0.10 0.15 0.24  0.16 0.19 0.66  0.22 0.20 0.33  C. All main chain atoms  0.15  0.16  0.15  0.19  these to be within experimental error the same as those of wild-type iso-l-cytochrome c. The average main chain deviations of residues near the mutation site in all four mutant structures are presented in Table 5.28. Only average deviations greater than two times the overall average deviation are considered significant in the present discussion. The four mutant proteins can be divided into two groups based on the size and shape of the replacement side chain. In the first group, Pro7lAla and Pro7lSer cytochromes c, the replacement side chains are smaller in volume than the original proline side chain. In Figure 5.35, these two mutant structures have been superimposed onto the structure of iso-l-cytochrome c and the region about Pro71 drawn. The main chain atom positions of residue 71 are unaffected by the alanine and serine replacements (Table 5.28), however, the space vacated by these smaller side chains is partially filled by G1y84 0 as a result of the reorganization of the main chain atoms about G1y83. Despite these main chain shifts the packing of the hydrophobic core in the region of residue 71 is particularly open in the Pro7lAla structure. The CB atom in this structure is positioned similar to that of the wild-type protein and does not shift to compensate for the loss  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c^103  Figure 5.35: Stereo-diagram of the structures of Pro7lAla, Pro7lSer (thin lines) and wild-type iso-1 (thick lines) cytochromes c. Substitution of smaller side chains at position 71 results in shifts about the main chain atoms of residues 82 to 84. of the other two side chain atoms present in the wild-type structure. The side chain of Ser71 adopts an unusual conformation to fit into the hydrophobic core. As shown in Figure 5.35, the Ser71 OG atom in Pro7lSer cytochrome c is in a position similar to that of the Pro71 CG atom in the iso-1 structure. The xi angle of residue 71 in Pro7lSer cytochrome c is —17° as compared to —33° for the same residue in the wild-type protein. This xi angle in the Pro7lSer structure is distant from frequently observed angles for serine residues (Ponder & Richards, 1987; James & Sielecki, 1983). A potential explanation for this conformation is the formation of a new bifurcated hydrogen bond between the hydroxyl group of Ser71 and the main chain carbonyl group of Tyr67. The second group of mutants, Pro71Ile and Pro71Val cytochromes c, possess side chains that are either larger or differently shaped than the normally resident Pro71. The valine side chain, although constructed from the same number of atoms as a proline residue, has a ,Qbranch that results in a differently shaped side chain than the ring structure of a proline. The isoleucine side chain is both /3-branched and larger, containing an additional methyl group. The structures of Pro7lIle and Pro71Val cytochromes c in the vicinity of the mutation site are plotted in Figure 5.36 along with the corresponding wild-type iso-l-cytochrome c structure.  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c ^104  Figure 5.36: Stereo-diagram of the structures of Pro71Ile, Pro71Val (thin lines) and wild-type iso-1 (thick lines) cytochromes c in the same orientation as Figure 5.35. Larger side chains at position 71 displace the main chain atoms of residues 69 to 71 and 81 to 83. In the Pro71Val mutant, one of the 7-carbons is positioned near the corresponding atom of the proline residue in the wild-type structure. The second 7-carbon is directed into the hydrophobic core of the molecule. The xi torsional angle of Va171 is —40° and is within 20° of a frequently observed torsional angle for that residue (Ponder & Richards, 1987; James Si Sielecki, 1983). The 11e71 side chain is in a similar conformation to that of Va171, however, the additional methyl group, 11e71 CD1, is directed into the hydrophobic core. (Figure 5.36). The xi and X2 torsional angles are 177°and —52°, respectively. These angles are also commonly observed for this residue. Unlike the smaller alanine and serine replacements at Pro71, both the Pro71Val and Pro7lIle mutant structures display positional perturbations in the region of the mutation site, in addition to the region about G1y83 (Figure 5.34). As detailed in Table 5.28, the average main chain deviations of residues 70 and 71 range from 0.34 to 0.57  A to accommodate the bulkier side  chains in these mutants. In particular, these displacements appear to be necessary to allow for a second 7-carbon atom to fit within the hydrophobic core without the development of unacceptable steric clashes with residues Tyr67 and Met80 (Figure 5.33). Note that the side chains of Met80 and Tyr67 are not displaced by any of the mutations studied at position 71  Chapter 5. Replacements of an Invariant Proline in Isa-l-Cytochrome c ^105  (Table 5.28). The displacement of G1y83 is less pronounced than in the Pro7lAla and Pro7lSer proteins, however the phenyl ring of Phe82 is displaced towards the molecular surface by 0.7 and 0.3  A in the Pro7lIle and Pro7lVal structures, respectively. The displacement of the Phe82  side chain is proportional to the size of the side chain at position 71. Movements occurring in the Pro71Val mutant protein lead to the formation of a hydrogen bond between Va171 N and G1y83 0 (3.4 A). A similar but longer range interaction (4.0 A) is seen in the Pro7lIle structure. Inspection of the difference matrices in Figure 5.34 shows that the positional perturbations centered at G1y83 are accompanied by a change in the main chain thermal factors in this region. These values are presented in Table 5.29. The average increase in main chain thermal factor for G1y83 in all four Pro71 mutant structures is about 8.4  A2 .  The largest increase is  observed in the Pro7lSer structure (A = +11 A 2 ) when compared to wild-type protein which has an average thermal factor of 19.0  A 2 . In contrast, the main chain thermal factors at the  mutation site, residue 71, do not differ greatly from the wild-type protein values (Table 5.29). The largest deviation is observed in the Pro7lVal structure in which the average increase in main chain thermal factor is found to be 5.5  A 2 . The thermal factors of the Phe82 side chain in  the Pro7lSer structure have decreased in contrast to the other mutants which display a modest increase in these thermal factors.  5.3 Discussion The three-dimensional structures of Pro7lAla, Pro7lIle, Pro7lSer. Pro71Val and wild-type iso-l-cytochromes c provide insight into the role of Pro71 in the folding and function of cytochrome c. Mutations at Pro71 are found to lead to perturbations in the atomic positions and thermal factors of residues 82 through 84 (Figure 5.34). If the replacement amino acid contains a larger side chain, the main chain atoms at the mutation site are also displaced. However, the nearby buried side chains of Tyr67 and Met80, which pack against the Pro71 side chain, are not affected by such substitutions and appear to be more rigidly held in place than residues  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrorne c ^106  Table 5.29: Average thermal factors of groups in the region of residue 71  Group  Wild-type  Average thermal factor (A 2 ) Pro7lAla Pro7lSer Pro7lIle  Pro71Val  A. Main chain Asn70 71 Phe82 G1y83 G1y84  15.1 12.7 15.2 19.0 17.4  16.7 12.8 24.2 29.3 23.2  17.4 17.0 21.8 30.0 22.6  18.6 16.9 21.6 24.6 19.5  20.7 18.2 21.5 25.7 26.7  B. Side chain Tyr67 71 Met80 Phe82  13.2 14.2 5.2 16.9  14.7 8.0 9.4 19.2  13.9 22.9 7.7 14.7  11.2 24.0 9.6 20.3  17.4 18.5 9.4 20.4  The thermal factors of the protein atoms of the Pro71 mutants were normalized to those of iso-1-cytochrome c for these comparisons. 82 through 84 which are located at the protein surface. There is a further correlation between the size of the amino acid replacement and the displacement of the invariant Phe82 side chain (Table 5.28). Larger replacements of Pro71 result in an increased displacement of the phenyl ring of Phe82 towards the protein surface. The four Pro71 mutant structures studied all displayed positional perturbations at residues 82 and 83. Phe82 and Gly83 are both located at the protein surface near to the solvent exposed heme edge (Figure 5.33). These residues are part of the proposed interface in modeled electron transfer complexes between cytochrome c and electron transfer partners such as cytochrome c peroxidase (Poulos & Kraut, 1980; Lum et al., 1987) and cytochrome b5 (Salemme, 1976; Mauk et al., 1986). The invariance and location of Phe82 suggests that this residue may be important in the mechanism of electron transfer (Dickerson & Timkovich, 1975). Site-directed mutagenesis has shown that an aromatic group at position 82 is required for efficient electron transfer (Pielak et al., 1985: Liang et al., 1988). Structural studies of the Phe82Gly mutant  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c ^107  of yeast iso-l-cytochrome c revealed that the polypeptide chain in this region had refolded (Louie & Brayer, 1989). This refolding, which altered the surface contour of cytochrome c, was suggested to account for the reduced steady state electron transfer kinetics of this mutant with cytochrome c peroxidase (Louie & Brayer, 1989). Similarly, structural investigation of Phe82Ser cytochrome c suggested that the phenyl ring of Phe82 must be present for optimal electron transfer function (Louie et al., 1988b). Mutations at Phe82 have also been shown to decrease the alkaline transition pK, indicating that this residue is important for maintaining the heme crevice structure (Pearce et al., 1989). Therefore these mutations at position 71 that substantially alter the conformational positioning of Phe82 could be expected to affect the overall structure of cytochrome c as well as potentially disrupting the putative redox partner complex interface. The functional properties of Pro7lIle, Pro7lLeu, Pro7lSer, Pro7lThr and Pro71Val cytochromes c have been determined in vivo by measuring growth rates of yeast cells on lactate media (Ernst et al., 1985). The thermodynamic stability of Pro7lIle, Pro7lThr and Pro7lVal cytochromes c have been also measured by guanidine hydrochloride induced equilibrium unfolding studies (Ramdas et al., 1986). The functional and stability measurements are summarized in Table 5.30. The variance in function and stability cannot be readily correlated to one structural parameter. The functional capacity of an amino acid replacement at Pro71 appears to depend on the size, shape and chemical character of the replacement side chain. The integration of residue 71 into the cytochrome c fold is presented in Figure 5.37 for wild-type and four Pro71 replacement structures. The Pro71 side chain in the wild-type iso-1 cytochrome c structure is surrounded by other sections of polypeptide chain. Inspection of the Pro7lAla mutant reveals the creation of additional space surrounding the A1a71 side chain resulting in a looser packing of the hydrophobic core in this region. The increased thermal mobility and reorganization of the main chain atoms about Gly83 do not entirely fill the space vacated by the Pro7lAla substitution. This inability to optimize packing of the hydrophobic core could be expected to reduce the stability of the protein.  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c ^108  Table 5.30: Functional and stability studies of Pro71 mutant cytochromes c  Cytochrome c Wild-type Pro7lIle Pro7lLeu Pro7lSer Pro7lThr Pro71Val  Function (%) 100 20 0 30 60 90  Stability (kcal/mol) 3.6 1.9 ND ND 1.9 2.6  Function was estimated by cell growth on lactate at 22 °C (Ernst et al., 1985). Thermodynamic stability of these cytochromes c was determined by denaturation with guanidine hydrochloride at pH 6.0 and 20 °C (Ramdas et al., 1986). In these latter experiments, Cys102 was modified with methyl methanethiosulfonate. Examination of the Pro7lSer packing interactions reveals that the additional Ser71 OG atom fills more of the vacated space than CB atom of the Pro7lAla mutant protein (Figure 5.37). However, the function of this replacement is 30% of that of wild-type cytochrome c (Table 5.30). The poor functional capacity of this mutant may be a consequence of the increased polarity and the hydrogen bonding potential of the Ser71 side chain. In the reduced structure, the highly mobile Ser71 OG atom (thermal factor of 27 A 2 ) is hydrogen bonded to Tyr67 0. By rotating Xi, the Ser71 side chain could hydrogen bond to Tyr67 OH which is a component of a hydrogen bonding network implicated in mediating electron transfer (Berghuis & Brayer, 1992). It is possible that this alternate hydrogen bond involving Ser71 OG could be formed in the more flexible oxidized state and thereby diminish electron transfer capacity. It is of interest to note that Pro7lThr cytochrome c functional capacity is twice that of the Pro7lSer protein (Table 5.30). If the Pro7lThr structure is modelled by comparison to the Pro71Val structure, the 0G1 atom is positioned similar to the Ser71 OG atom. However, the additional methyl group in the Pro7lThr protein relative to the Pro7lSer structure could be expected to lock the orientation of the hydroxyl group of the Thr71 side chain such that only the hydrogen bond to the Tyr67 main chain is formed in both the reduced and oxidized states.  Chapter 5. Replacements of an Invariant Proline in Iso-I-Cytochrome c  ^  109  Figure 5.37: Space-filled representations of wild-type and Pro71 mutant cytochromes c. The residue 71 side chain is shaded black, the heme group is shaded gray and all other protein groups are drawn in white. Atoms in front of the plane of view were removed to expose residue 71. The modelled placement of the Leu71 side chain results in a steric clash with the side chains of Met80 and Phe82.  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c^110  The stability of the Pro7lThr mutant has also been measured and found to be 2.7 kcal/mol less than that of the wild-type protein (Ramdas et al., 1986). In addition, the apparent pl( c, of the alkaline transition of the iso-2 Pro7lThr mutant has also been measured by examination of the 695 nm absorption band and found to decrease from 8.45 to 6.63 (White et al., 1987). These results indicate that despite its higher functional capacity the exchange of proline for threonine at position 71 leads to a considerable loss of protein stability. Of the four replacements of known structure, the Pro7lVal mutant protein has the highest functional capacity (Table 5.30). In addition, Pro7lVal cytochrome c is the most stable replacement of the mutants studied, undoubtedly because the valine side chain is best able to mimic the size and chemical character of the Pro71 side chain in iso-l-cytochrome c (Figure 5.37). Also, the formation of a hydrogen bond between Va171 N and Gly83 0 may help stabilize the Pro7lVal mutant protein. The least functional and stable replacement for which a three-dimensional structure is available is Pro7lIle cytochrome c (Table 5.30). As shown in Figures 5.36 and 5.37, the 11e71 side chain does not fit into the cytochrome c fold without substantial displacements in adjacent groups. Furthermore, the 695 nm absorbance band has been found to be significantly reduced in the Pro7lIle mutant protein (Ramdas et al., 1986). This absorbance band is an indicator of the presence of the Met80 heme iron ligand (Schechter & Saludjian, 1967) and the integrity of the heme pocket. These results suggest that the 11e71 side chain disrupts the heme environment leading to substantial disruption of the electron transfer mechanism of cytochrome c. The folding and functional requirements of residue 71 of yeast iso-l-cytochrome c have also been characterized by the examination of functional revertants of yeast strains containing a leucine at this position (Ernst et al., 1985). Of the 29 revertants examined, only four different partially functional proteins were observed, Pro7lIle, Pro7lSer, Pro7lThr and Pro71Val cytochromes c. Six other amino acid replacements, Pro7lGlu, Pro7lGln, Pro7lLys, Pro7lArg, Pro7lTyr and Pro7lPhe, could have been generated by single base pair substitutions but were not observed and are presumed to be non-functional. All of these residues are larger than  Chapter 5. Replacements of an Invariant Proline in Iso-1-Cytochrome c^111  the wild-type proline and most contain polar functional groups which would be expected to significantly alter the character of the hydrophobic heme pocket. This suggests that most mutations in this region cannot be tolerated. A model constructed by replacing the side chain of Pro71 with that of a leucine is shown in Figure 5.37. The Leu71 side chain could not be positioned such that a steric clash with protein groups surrounding residue 71 does not occur. Although leucine and isoleucine side chains contain the same number of atoms, the difference in the arrangement of these atoms greatly effects the ability of these side chains to fit into the hydrophobic core at position 71. As shown in Figure 5.37, a minimalist modelling of the Leu71 side chain results in steric clashes with both the Met80 and Phe82 side chains. The difference in packing of the Leu71 and Ile71 side chains appear to define the functional limitation of the cytochrome c fold to accommodate replacement side chains at position 71. A further factor in the instability of Pro71 mutant proteins may be related to amino acid preferences for specific helix positions. The occurrence of an amino acid residue at a given position in an a-helix was examined in 215 a-helices in 45 globular proteins (Richardson & Richardson, 1988). The amino acid preference ratio is derived by normalizing the number of occurrences by the expected number based on the overall percentage of each amino acid found in a sample of 135 proteins of known structure. In particular, there is a strong preference for the first and second residues of an a-helix to be asparagine (3.5:1) and proline (2.6:1), respectively. Helix IV of yeast cytochrome c is initiated by the residues Asn70 and Pro71. Of the Pro71 replacements discussed, two have preference ratios greater than one: alanine (1.2:1) and valine (1.1:1). Leucine and isoleucine side chains are equally undesirable (0.9:1), whereas a serine is the least preferred residue (0.7:1) at the second helix position. All of the partially functional amino acid replacements have near neutral preference for the second helix position as compared to the strong preference for proline. The structural results obtained for the four Pro71 mutants clearly show the limitations imposed on substitutions at position 71. The most acceptable by functional and thermal stability  Chapter 5. Replacements of an Invariant Proline in Iso-l-Cytochrome c^112  criteria appears to be the Pro71Val substitution (Ernst et al., 1985: Ramdas et al., 1986). The valine side chain is best able to mimic the size and chemical character of the Pro71 side chain in iso-l-cytochrome c (Figure 5.36). It seems likely that the structural perturbations observed at residues 82 through 84 as a result of this substitution are responsible for the partial loss of function observed and may be related to changes in the surface contour of the protein at the redox partner interface. The other mutants investigated are less functional due to additional structural perturbations. For example, the serine substitution introduces a polar group adjacent to the Met80 ligand which was formerly in a highly hydrophobic environment. Smaller replacements, such as Pro7lAla, result in poor packing in the hydrophobic core which could be expected to lead to reduced thermal stability. The larger replacement, Pro7lIle, is only tolerated by rearrangements of the main chain at the mutation site and a displacement of the Phe82 side chain. The combination of both these displacements seems to lead to a low level of functional activity. As evident from our studies (Figure 5.37), even larger side chains at this position such as Leu71 would require large structural reorganizations which would abolish cytochrome c function probably as a result of disrupting the Met80 ligand bond. It is also entirely possible that the substitution of even larger side chains would prevent polypeptide chain folding altogether.  Chapter 6  Summary  In this work, a total of eight cytochrome c structures were elucidated to atomic resolution. One of these, iso-2-cytochrome c, is a wild-type yeast isozyme differing from iso-l-cytochrome c at 17 amino acid positions. The composite mutant protein, B-2036 cytochrome c, possesses 10 of these 17 amino acid replacements relative to the iso-1 protein. The a-Loop A replacement protein, RepA2 cytochrome c, contained five amino acid substitutions, one of which was back substituted to that of the wild-type iso-1 protein to produce RepA2(Va120) cytochrome c. Finally, four single site mutants were structurally characterized at position 71. In all, these proteins have 25 different substitutions at 20 sites (Table 6.31). Table 6.31. Amino acid additions and replacements in the eight cytochrome c structures studied in this work Cytochrome c No. Residue additions and replacements Iso-2  B-2036 RepA2 RepA2(Va120) Pro71Ala Pro7lIle Pro71Ser Pro71Val  21^Ala(-9), Lys(-8), Glu(-7), Ser(-6), Glu(-3)Gly, Ala(-1)Pro, Leul5Gln, Va120Ile, Lys22Glu, His26Asn, Ala43Val, Glu44Lys, Lys54Asn, Leu58Lys, Asn62Asp, Asn63Ser, Gly83Ala, Leu98Met, Lys99Thr, Cys102Ala, Glu103Lys 10 Leul5G1n, Va120Ile, Lys22Glu, His26Asn, Ala43Val, Glu44Lys, Lys54Asn, Leu58Lys, Asn62Asp, Asn63Ser 5^Va120Phe, Glu2lAsp, Lys22G1n, Pro25Ala, His26Asn 4^Glu21Asp, Lys22Gln, Pro25Ala, His26Asn 1^Pro71Ala 1^Pro7lIle 1^Pro71Ser 1^Pro7lVal  113  Chapter 6. Summary^  114  An overview of the positional deviations in the various cytochromes c examined in this work from wild-type iso-l-cytochrome c is presented in Figure 6.38. The overall average main chain deviation of the eight structures is 0.25  A. Despite the large number of amino acid substitutions  present in these structures, the main chain fold is highly conserved. The largest deviation occurs at G1y37 and is due to an alternative conformation in main chain atom positions in the iso-2 and B-2036 structures (Figure 3.14). The overall average positional deviations of side chain atoms (0.45  A) is almost twice that observed for main chain atoms. The majority of large deviations  involve residues with disordered solvent accessible side chains (Figure 6.38). Additionally, the two buried side chains with the largest deviations, Leu9 and G1u61, are disordered in the iso1-cytochrome c structure (Louie & Brayer, 1990). The iso-2, B-2036 and RepA2 structures share some common features with respect to residue 20 and the subsequent alterations in hydrophobic core packing when compared to iso-l-cytochrome c. In iso-2-cytochrome c, the replacement of isoleucine for valine at position 20 is compensated for by the substitution Cys102Ala. In the B-2036 protein where the Cys102 is retained, disruptions of the hydrophobic core (residues Ile20, 11e35, Leu98 and Cys102) occur to accommodate the addition of the buried Ile20 CD1 group (Figures 3.17 and 6.38). These alterations in hydrophobic core packing also lead to an increase in the volume of a buried cavity located adjacent to the heme group (Figure 3.18). In RepA2 cytochrome c, there is a phenylalanine at position 20. The larger side chain of this amino acid is redirected towards the surface of the protein (Figure 4.26). This Va120Phe substitution creates a void in the hydrophobic core and Leu98 moves to fill the vacated space. In addition, Tyr97 and Ala101 are displaced to accommodate the phenylalanine side chain at the protein surface. These results indicate that there is a limit as to the size of the amino acid replacement that may be accommodated into the hydrophobic core by adjustment of neighboring side chain positions. At position 20 in yeast iso-l-cytochrome c, whereas an extra methyl group was accommodated, a phenyl ring cannot be accommodated and is redirected away from the hydrophobic core. Even with the disruptions observed to the hydrophobic core in the case of Va120Phe it is remarkable  Chapter 6. Summary  2.5  ^  II^1^1  ^  115  ^ 11111I^II^11^1111^11111^1^1^1 1^1^1 1^1 1^1^i^1111  ^  1^1^1I^1^1^1^1^1^1^ 11  2.0L' 1.5 1.0 0.50.0 0  0  a)  D  45^55^65^75 0.0  85 1195 11  1^1^11^111i1^1^1 1^1^1  0.5 7 -  a)  0) 0 L  a)  1.5 2.02.5 3.0  -  3.5 4.0  1^1^1^1^1^I^1^1^1^1^I^1^1^1^1^1^1^1^1^1  ^11111111  Residue Number Figure 6.38: A plot of the overall average deviation of main (above) and side (below) chain atoms of the structures examined in this work (Table 6.31). The vertical bars in the upper graph represent the range of individual pairwise average deviations. The dashed and solid vertical bars in the lower graph represent solvent accessible and inaccessible side chains, respectively. A side chain was considered solvent inaccessible if less then 20% of its surface area was solvent exposed in any one of the structures examined. The overall average deviations for the main and side chain atoms are 0.25 and 0.45 A, respectively.  Chapter 6. Summary^  116  how little the overall conformation of the main chain was perturbed. The reduction potential of the RepA2 protein is observed to be 19 mV less than that of either RepA2(Va120) or wild-type iso-l-cytochrome c (Table 4.23). The largely solvent exposed Phe20 side chain in the RepA2 structure may modify the reduction potential by altering solvent ordering upon reduction. The presence of a phenylalanine and the associated structural perturbations also diminish the in vivo function of cytochrome c. This loss in function may be due to the replacement of amino acids at the protein surface combined with the increase in mobility of Q-Loop A in the RepA2 structure thereby interfering with the binding of cytochrome c with physiologically important electron transfer partners. All of iso-2, B-2036, RepA2 and RepA2(Va120) cytochromes c have an asparagine at position 26 as compared to a histidine in the iso-1 protein. His26 forms two hydrogen bonds, one to each of the main chain atoms of residues 31 and 44 (Table 3.14). This forms a crosslink between the main chain backbones of the polypeptide segments involved. The substituted asparagine side chain of the iso-2 and mutant cytochromes c possesses equivalent functional groups to form these hydrogen bonds, but because of its smaller size the hydrogen bonded groups cannot be placed in the same position as observed in wild-type iso-l-cytochrome c. As a consequence, the main chain about Glu44 shifts (Figure 6.38) in the a-Loop A mutants to allow the formation of a hydrogen bond between Asn26 ND2 and G1u44 0 (Figure 4.25). In the iso-2 and B-2036 proteins this hydrogen bond is not formed. This appears to result from a second substitution, Ala43Val, which fills the space resulting from the His26Asn substitution (Figure 3.15). In the end this double substitution provides stronger hydrophobic interactions in this region. The positions of the main chain atoms surrounding G1u44 are adjusted in the iso-2 and B-2036 proteins to optimize the fit of Va143 (Figures 3.13 and 6.38). The invariant residue Pro71 is located near the Met80 ligand (Figure 5.33). It is a completely solvent inaccessible residue that bridges Helices III and IV (Table 1.4). The unique properties of proline residues are thought to be important for the stabilization of this structurally conserved region of cytochrome c. The structures of four substitutions at this position  Chapter 6. Summary^  117  revealed that changes in side chain size, shape and chemical character resulted in displacements in the positions of residues 70, 71 and 82 through 84 (Figure 5.34). Replacement of Pro7l with the smaller side chains, Pro7lAla and Pro7lSer, required only adjustment of residues 82 through 84 (Figure 5.35), however, larger side chain replacements, for example Pro7lIle and Pro71Val (Figure 5.36), were accommodated only at the expense of altering the structure of the main chain at the mutation site. It appears that unlike residues 20 and 26 as discussed above, the internal positioning and rigid polypeptide chain structure at the mutation site prevents the protein from adapting to the presence of a wider range of residues at position 71. The structural perturbations observed at residues 82 through 84 as a result of substitutions at position 71 are suggested to be responsible for the observed partial loss of function in these mutant proteins. Of the 25 amino acid substitutions listed in Table 6.31, only a fraction give rise to the substantial structural perturbations. The majority do not appear to result in observable structural differences. Those amino acid substitutions that cannot be incorporated into the cytochrome c fold without structural perturbations occur in conformationally restricted regions. In addition, the displacement of groups by these replacements indicates the limits present in specific regions in terms of conformational flexibility. For example, the substitution Va120Ile results in small alterations in the packing of the hydrophobic core, however, the Va120Phe substitution results in the redirection of the side chain of this residue apparently because the hydrophobic core is incapable of adjusting to accommodate the larger phenyl ring. The interaction of the two segments of polypeptide chain bridged by hydrogen bonds to His26 represents a conformational restriction in the polypeptide chain at this point. The observed shifts of residues 42 to 44 in the iso-2, B-2036. RepA2 and RepA2(Va120) mutant proteins as a result of the His26Asn replacement suggests that this segment of polypeptide chain is more flexible than the polypeptide chain segment that contains residue 26. The small range of acceptable substitutions at position 71 reflects the low tolerance for side chain replacements in this conformationally restricted and rigid region of the protein.  Bibliography  Armstrong, F.A., Hill, A.O. & Walton, N.J. (1988). Direct electrochemistry of redox proteins. Acc. Chem. Res., 21, 407-413. Arndt, U.W. & Wonacott, A.J., eds. (1977). The Rotation Method in Crystallography. NorthHolland Publishing Company, Amsterdam. Barker, P.D., Hill, A.O. & Walton, N.J. (1989). Fast second order electron transfer reactions coupled to redox protein electrochemistry. J. Electroanal. Chem., 260, 303-326. Berghuis, A.M. (1993). The Nature and Role of Oxidation State Dependent Conformational Differences in Cytochrome c. 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