Tryptophan stabilizes His-heme loops in the denatured state only when it is near a loop end

We use a host-guest approach to evaluate the effect of Trp guest residues relative to Ala on the kinetics and thermodynamics of formation of His-heme loops in the denatured state of iso-1-cytochrome c at 1.5, 3.0, and 6.0 M guanidine hydrochloride (GdnHCl). Trp guest residues are inserted into an alanine-rich segment placed after a unique His near the N-terminus of iso-1-cytochrome c. Trp guest residues are either 4 or 10 residues from the His end of the 28-residue loop. We find the guest Trp stabilizes the His-heme loop at all GdnHCl concentrations when it is the 4th, but not the 10th, residue from the His end of the loop. Thus, residues near loop ends are most important in developing topological constraints in the denatured state that affect protein folding. In 1.5 M GdnHCl, the loop stabilization is ~0.7 kcal/mol, providing a thermodynamic rationale for the observation that Trp often mediates residual structure in the denatured state. Measurement of loop breakage rate constants, k(b,His), indicates that loop stabilization by the Trp guest residues occurs completely after the transition state for loop formation in 6.0 M GdnHCl. Under poorer solvent conditions, approximately half of the stabilization of the loop develops in the transition state, consistent with contacts in the denatured state being energetically downhill and providing evidence for funneling even near the rim of the folding funnel.     

Conformational properties of the iso-1-cytochrome C denatured state: dependence on guanidine hydrochloride concentration

Production of seven single surface histidine variants of yeast iso-1-cytochrome c allowed measurement of the apparent pK(a), pK(a)(obs), for histidine-heme loop formation for loops of nine to 83 amino acid residues under varying denaturing conditions (2 M to 6 M guanidine hydrochloride, gdnHCl). A linear correlation between pK(a)(obs) and the log of the loop size is expected for a random coil, pK(a)(obs) proportional to k log(n), where k is a scaling factor and n is the number of monomers in the loop. For small loops of nine, 16, and 22 monomers, no dependence of pK(a)(obs) on loop size was observed at any denaturant concentration indicating effects from chain stiffness. For larger loops of 37, 56, 72, and 83 monomers, the dependence of pK(a)(obs) on log(n) was linear and the slope of that dependence decreased with increasing concentration of denaturant. The scaling factor obtained at 5 M and 6 M gdnHCl for the larger loop sizes was approximately -2.0, close to the value of -2.2 expected for a random coil with excluded volume. However, scaling factors obtained under less harsh denaturing conditions (2 M to 4.5 M gdnHCl) deviated strongly from that expected for a random coil, being in the range -3 to -4. The gdnHCl dependence of pK(a)(obs) at each loop size was also evaluated to obtain denaturant m-values. Short loops where chain stiffness dominates had similar m-values of approximately 0.25 kcal/mol M. For larger loops m-values decrease with increasing loop size indicating that less hydrophobic area is sequestered when larger loops form. It is known that the earliest events in protein folding involve the formation of simple loops. The data from these studies provide direct insight into the relative probability with which loops of different sizes will form, as well as the factors which affect loop formation.     

Importance of Contact Persistence in Denatured State Loop Formation: Kinetic Insights into Sequence Effects on Nucleation Early in Folding

Protein folding is dependent on the formation and persistence of simple loops early in folding. Ease of loop formation and persistence is believed to be dependent on the steric interactions of the residues involved in loop formation. We have previously investigated this factor in the denatured state of iso-1-cytochrome c using a five-amino-acid insert in front of a unique histidine in the N-terminal region of the protein. Previously, we reported that the apparent pK_a values of loop formation for the most flexible (all Gly) and least flexible (all Ala) insert were, within error, the same. We evaluate whether this observation is due to differences in the persistence of loop contacts or due to effects of local sequence sterics and main-chain hydration on the persistence length of the chain. We also test whether sequence order affects loop formation. Here, we report kinetic results coupled to further mutagenesis of the insert to discern between these possibilities.We find that the amino acid—glycine versus alanine—next to the loop forming histidine has a dominant effect on loop kinetics and equilibria. A glycine in this position speeds loop breakage relative to alanine, resulting in less stable loops. At high percentage of Gly in the insert, rates of loop formation and breakage exactly compensate, leading to a leveling out in loop stability. Loop formation rates also increase with glycine content, inconsistent with poly-Gly segments being more extended than previously suspected due to main-chain hydration or local sterics. Unlike loop breakage rates, loop formation rates are insensitive to local sequence. Together, these observations suggest that contact persistence plays a more important role in defining the “folding code” than rates of loop formation.     

Propensities of Aromatic Amino Acids versus Leucine and Proline to Induce Residual Structure in the Denatured-State Ensemble of Iso-1-cytochrome c

Histidine-heme loop formation in the denatured state of a protein is a sensitive means for probing residual structure under unfolding conditions. In this study, we use a host-guest approach to investigate the relative tendencies of different amino acids to promote residual structure under denaturing conditions. The host for this work is a 6-amino-acid insert of five alanines, followed by a lysine engineered immediately following a unique histidine near the N-terminus of yeast iso-1-cytochrome c. We substitute the fourth alanine in this sequence HAAAXAK (with X = Trp, Phe, Tyr, and Leu). The effects of proline are tested with substitutions at positions 1 and 5 in the insert (HPAAAAK and HAAAAPK, respectively). Thermodynamic studies on His-heme loop formation in 3 M guanidine hydrochloride reveal significant stabilization of residual structure by aromatic amino acids, particularly Trp and Phe, and minimal stabilization of residual structure by Leu. Prolines slightly disfavor His-heme loop formation, presumably due to enhanced chain stiffness. Kinetic studies reveal that much of the change in His-heme loop stability for the aromatic amino acids is caused by a slowdown in the rate of His-heme loop breakage, indicating that residual structure is preferentially stabilized in the closed-loop form of the denatured state.     

Effects of topology and excluded volume on protein denatured state conformational properties

The conformational constraints on protein denatured states are of prime importance in modulating early events in protein folding. Although structural studies have demonstrated residual structure in protein denatured states, much remains poorly understood with regard to the conformational properties of this state. Here, we investigate topological effects on loop formation probabilities in denatured iso-1-cytochrome c by comparing histidine-heme binding affinities for histidines on the N- versus the C-terminal side of the heme. For histidines N-terminal to the heme (preceding cysteine 14), the polypeptide emerges from the edge of the heme and must simply fold over to bind to the heme. For histidines C-terminal to the heme (following histidine 18), the polypeptide emerges from the back side of the heme and must wrap around the heme for the histidine to bind to the heme. Thus, the steric constraints on this wrap-around topology are expected to be much more demanding than for the heme-edge topology of the N-terminal histidines. Evaluation of loop formation probabilities in 3 M guanidine hydrochloride, conditions that fully denature the variants studied, demonstrates that N-terminal histidine-heme loop formation is 10-25-fold more favorable than C-terminal histidine-heme loop formation, for similar loop sizes. A two-dimensional square lattice model indicates that excluded volume is important in this topological preference. These data provide direct evidence that denatured state topology affects contact probability, and thus probable folding pathways, in a disordered protein.     

Sequence composition effects on denatured state loop formation in iso-1-cytochrome c variants: polyalanine versus polyglycine inserts

Protein folding is dependent on the formation and persistence of simple loops during the earliest events of the folding process. Ease of loop formation and persistence is believed to be dependent on the steric properties of the residues involved in loop formation. We have investigated this conformational factor in the denatured state of iso-1-cytchrome c using a five alanine insert in front of a unique histidine in the N-terminal region of the protein. The alanine residues have then been progressively substituted with sterically less-constrained glycine residues. Guanidine-HCl unfolding shows that all variants have a free energy of unfolding of approximately 2 kcal/mol. The low stability of these variants is well accounted for by stabilization of the denatured state by histidine-heme loop formation. The stability of the 22 residue histidine-heme loop has been measured in 3 M guanidine hydrochloride for all variants. Surprisingly, relative to alanine, glycine has only a very modest effect on equilibrium loop stability. Thus, the greater flexibility that glycine confers on the main-chain provides no advantage in terms of the persistence of simple loops early in folding. The underlying basis for the similar behavior of loops with polyalanine versus polyglycine inserts is discussed in terms of the current knowledge of the structure and loop formation kinetics of glycine versus alanine-rich peptides.     

Measuring denatured state energetics: deviations from random coil behavior and implications for the folding of iso-1-cytochrome c

The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stability relative to the wild-type protein. The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions. Significant deviations from random coil behavior are observed. Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied. The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding. The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c.     

Effect of pH on the iso-1-cytochrome c denatured state: changing constraints due to heme ligation

The effect of pH on the denatured state (3 M guanidine hydrochloride) was evaluated with fluorescence spectroscopy for four variants of iso-1-cytochrome c, AcTM (no surface histidines), AcH26 (surface histidine at position 26), AcH54 (surface histidine at position 54), and AcH54I52 (stabilizing I52 mutation added to AcH54). Changes in the compactness and the heme ligation of the denatured state, as a function of pH, were monitored through changes in Trp 59-heme fluorescence quenching. With the AcTM and AcH26 variants, no change in the fluorescence intensity occurs from pH 4 to 10. However, for the AcH54 and AcH54I52 variants the fluorescence intensity drops significantly between pH 4 and 6, consistent with His 54 binding to the heme of cytochrome c. Between pH 8 and 10 fluorescence intensity increases again, indicating that the His 54 is displaced from the heme. The data are consistent with lysines 4 and 5 being the primary heme ligands at alkaline pH, under denaturing conditions. This conclusion was confirmed by site-directed mutagenesis. Thermodynamic analysis indicates that heme-ligand affinity in the denatured state is controlled primarily by sequence position (loop size) and that when histidines are present they inhibit lysine ligation until approximately pH 8.5-9.0 as compared to pH 7.5 with the AcTM variant. Thus, at physiological pH, histidine ligands provide the primary constraint on the denatured state of cytochrome c. The heme-Trp 59 distance in the denatured state of iso-1-cytochrome c, derived from analysis by F?rster energy transfer theory, is approximately 26 A at pH 4 and 10, much shorter than the random coil prediction of 56 A. Surprisingly, the heme-Trp 59 distance in the His 54 bound conformation only drops to approximately 21 A, consistent with an extended conformation for the short polypeptide segment separating heme and Trp 59.     

Probing the dynamics of a His73-heme alkaline transition in a destabilized variant of yeast iso-1-cytochrome c with conformationally gated electron transfer methods

The alkaline transition of cytochrome c involves substitution of the Met80 heme ligand of the native state with a lysine ligand from a surface Ω-loop (residues 70 to 85). The standard mechanism for the alkaline transition involves a rapid deprotonation equilibrium followed by the conformational change. However, recent work implicates multiple ionization equilibria and stable intermediates. In previous work, we showed that the kinetics of formation of a His73-heme alkaline conformer of yeast iso-1-cytochrome c requires ionization of the histidine ligand (pK(HL) ~ 6.5). Furthermore, the forward and backward rate constants, k(f) and k(b), respectively, for the conformational change are modulated by two auxiliary ionizations (pK(H1) ~ 5.5, and pK(H2) ~ 9). A possible candidate for pK(H1) is His26, which has a strongly shifted pK(a) in native cytochrome c. Here, we use the AcH73 iso-1-cytochrome c variant, which contains an H26N mutation, to test this hypothesis. pH jump experiments on the AcH73 variant show no change in k(obs) for the His73-heme alkaline transition from pH 5 to 8, suggesting that pK(H1) has disappeared. However, direct measurement of k(f) and k(b) using conformationally gated electron transfer methods shows that the pH independence of k(obs) results from coincidental compensation between the decrease in k(b) due to pK(H1) and the increase in k(f) due to pK(HL). Thus, His26 is not the source of pK(H1). The data also show that the H26N mutation enhances the dynamics of this conformational transition from pH 5 to 10, likely as a result of destabilization of the protein.     

Denatured state thermodynamics: residual structure, chain stiffness and scaling factors

A set of nine variants of yeast iso-1-cytochrome c with zero or one surface histidine have been engineered such that the N-terminal amino group is acetylated in vivo. N-terminal acetylation has been confirmed by mass spectral analysis of intact and proteolytically digested protein. The histidine-heme loop-forming equilibrium, under denaturing conditions (3 M guanidine hydrochloride), has been measured by pH titration providing an observed pK(a), pK(a)(obs), for each variant. N-terminal acetylation prevents the N-terminal amino group-heme binding equilibrium from interfering with measurements of histidine-heme affinity. Significant deviation is observed from the linear dependence of pK(a)(obs) on the log of the number of monomers in the loop formed, expected for a random coil denatured state. The maximum histidine-heme affinity occurs for a loop size of 37 monomers. For loop sizes of 37-83 monomers, histidine-heme pK(a)(obs) values are consistent with a scaling factor of -4.2+/-0.3. This value is much larger than the scaling factor of -1.5 for a freely jointed random coil, which is commonly used to represent the conformational properties of protein denatured states. For loop sizes of nine to 22 monomers, chain stiffness is likely responsible for the decreases in histidine-heme affinity relative to a loop size of 37. The results are discussed in terms of residual structure and sequence composition effects on the conformational properties of the denatured states of proteins.     

Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases

The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody-detected (ad) kinetic phase accompanying epitope formation has k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)). N5I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.     

Helical Propensity Affects the Conformational Properties of the Denatured State of Cytochrome c′

Changing the helical propensity of a polypeptide sequence might be expected to affect the conformational properties of the denatured state of a protein. To test this hypothesis, alanines at positions 83 and 87 near the center of helix 3 of cytochrome c′ from Rhodopseudomonas palustris were mutated to serine to decrease the stability of this helix. A set of 13 single histidine variants in the A83S/A87S background were prepared to permit assessment of the conformational properties of the denatured state using histidine-loop formation in 3 M guanidine hydrochloride. The data are compared with previous histidine-heme loop formation data for wild-type cytochrome c′. As expected, destabilization of helix 3 decreases the global stabilities of the histidine variants in the A83S/A87S background relative to the wild-type background. Loop stability versus loop size data yields a scaling exponent of 2.1 ± 0.2, similar to the value of 2.3 ± 0.2 obtained for wild-type cytochrome c′. However, the stabilities of all histidine-heme loops, which contain the helix 3 sequence segment, are increased in the A83S/A87S background compared to the wild-type background. Rate constants for histidine-heme loop breakage are similar for the wild-type and A83S/A87S variants. However, for histidine-heme loops that contain the helix 3 sequence segment, the rate constants for loop formation increase in the A83S/A87S background compared to the wild-type background. Thus, residual helical structure appears to stiffen the polypeptide chain slowing loop formation in the denatured state. The implications of these results for protein folding mechanisms are discussed.     

Hydrogen exchange behavior of [U-15N]-labeled oxidized and reduced iso-1-cytochrome c

Heteronuclear NMR spectroscopy was used to measure the hydrogen-deuterium exchange rates of backbone amide hydrogens in both oxidized and reduced [U-15N]iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. The exchange data confirm previously reported data [Marmorino et al. (1993) Protein Sci. 2, 1966-1974], resolve several inconsistencies, and provide more thorough coverage of exchange rates throughout the cytochrome c protein in both oxidation states. Combining the data previously collected on unlabeled C102T with the current data collected on [U-15N]C102T, exchange rates for 53 protons in the oxidized state and 52 protons in the reduced state can now be reported. Most significantly, hydrogen exchange measurements on [U-15N]iso-1-cytochrome c allowed the observation of exchange behavior of the secondary structures, such as large loops, that are not extensively hydrogen-bonded. For the helices, the most slowly exchanging protons are found in the middle of the helix, with more rapidly exchanging protons at the helix ends. The observation for the Omega-loops in cytochrome c is just the opposite. In the loops, the ends contain the most slowly exchanging protons and the loop middles allow more rapid exchange. This is found to be true in cytochrome c loops, even though the loop ends are not attached to any regular secondary structures. Some of the exchange data are strikingly inconsistent with data collected on the C102S variant at a different pH, which suggests pH-dependent dynamic differences in the protein structure. This new hydrogen exchange data for loop residues could have implications for the substructure model of eukaryotic cytochrome c folding. Isotopic labeling of variant forms of cytochrome c can now be used to answer many questions about the structure and folding of this model protein.     

Scaling properties of glycine-rich sequences in guanidine hydrochloride solutions

The intrinsic polymer properties of glycine-rich sequences are evaluated with a set of iso-1-cytochrome c variants with N-terminal inserts of the sequence (GGGGGK)(n) for n = 1-5. The thermodynamics and kinetics of His-heme loop formation are measured as a function of guanidine hydrochloride (GdnHCl) concentration for loop sizes ranging from 22 to 46 residues. The scaling exponent for loop formation, ν(3), evaluated using the Jacobson-Stockmayer equation is near 1.8, at 1.5 and 3.0 M GdnHCl, but it increases to 2.2 in 6.0 M GdnHCl. Previous work on a set of iso-1-cytochrome c variants with (AAAAAK)(n) inserts gave ν(3) = 2.2 for alanine-rich sequences in both 3.0 and 6.0 M GdnHCl. Chain stiffness was evaluated from the relative magnitude of Flory's characteristic ratio, C(n), for alanine-rich versus glycine-rich sequences. In 3.0 M GdnHCl, C(n)(Ala)/C(n)(Gly) is 1.6, decreasing to 1.3 in 6.0 M GdnHCl. The data suggest that solvent-backbone interactions dominate polypeptide conformational properties under good solvent conditions whereas side-chain-dependent properties are more important under poor solvent conditions. The results provide a direct experimental assessment in terms of polymer properties of the distinct roles of Gly versus Ala in the folding code.     

Evaluation of cooperative interactions between substructures of iso-1-cytochrome c using double mutant cycles

A double mutant cycle has been used to evaluate interaction energies between the global stabilizer mutation asparagine 52 --> isoleucine (N52I) in iso-1-cytochrome c and mutations producing single surface histidines at positions 26, 33, 39, 54, 73, 89, and 100. These histidine mutation sites are distributed through the four cooperative folding units of cytochrome c. The double mutant cycle starts with the iso-1-cytochrome c variant AcTM, a variant with no surface histidines and with asparagine at position 52. Isoleucine is added singly at position 52, AcTMI52 variant, as are the surface histidines, AcHX variants, where X indicates the histidine sequence position. The double mutant variants, AcHXI52, provide the remaining corner of the double mutant cycle. The stabilities of all variants were determined by guanidine hydrochloride denaturation and interaction energies were calculated between position 52 and each histidine site. Six of the seven double mutants show additive (AcH33I52, AcH39I52, AcH54I52, AcH89I52, and AcH100I52) stability effects or weak interaction energies (AcH73I52) of the histidine mutations and the N52I mutation, consistent with cooperative effects on protein folding and stability being sparsely distributed through the protein structure. The AcH26I52 variant shows a strong favorable interaction energy, 2.0 +/- 0.5 kcal/mol, between the N52I mutation in one substructure and the addition of His 26 to an adjacent substructure. The data are consistent with an entropic stabilization of the intersubstructure hydrogen bond between His 26 and Glu 44 by the Ile 52 mutation.     

Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.     

Changes in conformation and slow refolding kinetics in mutant iso-2-cytochrome c with replacement of a conserved proline residue

Using oligonucleotide-directed mutagenesis, we have produced a mutant form of iso-2-cytochrome c of yeast in which threonine (Thr-71) replaces a conserved proline residue (Pro-71) located between two short alpha-helical segments in the native protein. Optical spectroscopy indicates that, at pH 7.2, Thr-71 iso-2-cytochrome c folds to a nonnative conformation possibly related to the alkaline form of the native protein. On titration to pH 5.2, Thr-71 iso-2-cytochrome c regains many of the optical properties of the normal protein. We have shown that the proline residue at position 71 has no effect on the kinetics of fluorescence-detected slow refolding. However, between pH 5 and pH 7.2 the amplitude for absorbance-detected slow folding is strongly pH dependent in the mutant protein but is largely independent of pH in the normal protein. We believe this to be due to the folding of Thr-71 iso-2-cytochrome c to a nonnative conformation at pH 7.2 that does not require the slow, absorbance-detected conformational changes observed in folding to the more native-like state at pH 5-6.     

The structure and function of omega loop A replacements in cytochrome c.

The structural and functional consequences of replacing omega-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three-dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-1-cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of omega-loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, omega-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core. Also, as a result of omega-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 degrees C. This is 19 mV less than the wild-type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of omega-loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.     

Yeast iso-1-cytochrome c: genetic analysis of structural requirements

We describe the use of classical and molecular genetic techniques to investigate the folding, stability, and enzymatic requirements of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. Interpretation of the defects associated with an extensive series of altered forms of iso-1-cytochrome c was facilitated by the recently resolved three dimensional structure of iso-1-cytochrome c [(1987) J. Mol. Biol. 199, 295-314], and by comparison with the phylogenetic series of eukaryotic cytochromes c. Residue replacements that abolish iso-1-cytochrome c function appear to do so by affecting either heme attachment or protein stability; no replacements that abolish electron transfer function without affecting protein structure were uncovered. Most nonfunctional forms retained at least partial covalent attachment to the heme moiety; heme attachment was abolished only by replacements of Cys19 and Cys22, which are required for thioether linkage, and His23, a heme ligand. Replacements were uncovered that retain function at varying levels, including replacements at evolutionarily conserved positions, some of which were structurally and functionally indistinguishable from wild type iso-1-cytochrome c.     

Network of interactions between unlinked genes: synergistic and antagonistic regulation of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 synthesis]

Five chromosomal genes, CYPI to CYP5 involved in the regulation of the synthesis of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 are described. The function of these genes was studied either by varying the proportion of the mutated and wild type alleles in the cell vy varing the growth conditions, or else by transforming the mutants into sigma-cytoplasmic petites. We have shown a network of genetic interactions which regulate the synthesis of three structurally different proteins : iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2, by two unlinked genes : CYC1 and CYP1, one of which (CYC1) is the structural gene by iso-1-cytochrome c. Within this network the interactions are proportional to the gene dosage and are either antagonistic or synergistic depending on the allele combination and the protein studied. The mutated alleles cyp1 stimulate the synthesis of iso-2-cytochrome c, inhibit the synthesis of iso-1-cytochrome c, while the cytochrome b2 synthesis is also inhibited but by a combination of cyp1 mutated alleles CYC1 wild type allele. Other loci, CYP2, CYP3, CYP4 and CYP5 were also studied in various allelic combinations. They show some interactions between them or with CYC1 locus but these interactions are different and less pronounced than those involving loci CYP1 and CYC1.