Regulation of interprotein electron transfer by residue 82 of yeast cytochrome c.

Yeast iso-1-cytochrome c (Cc) mutants have been constructed with Phe, Tyr, Gly, Ser, Leu, and Ile at position 82, each with Thr substituted for Cys at position 102. Their long-range electron transfer with zinc-substituted cytochrome c peroxidase (ZnCcP) has been studied by two kinetic techniques. The charge-separated complex, [(ZnCcP)+,FeIICc] converts to [ZnCcP,FeIIICc] by a single, intracomplex electron transfer step that is not governed by "gating" through possible rapid dissociation of the complex or isomerization (for example, heme-ligand) by FeIICc subsequent to its formation from FeIIICc. In every variant with an aliphatic residue at position 82 of Cc, the rate of this electron transfer process is approximately 10(4) slower at approximately 0 degrees C than for the two variants with aromatic residues.

Temperature-sensitive variants of Saccharomyces cerevisiae iso-1-cytochrome c produced by random mutagenesis of codons 43 to 54.

In vitro random mutagenesis within the CYC1 gene from the yeast Saccharomyces cerevisiae was used to produce a library of mutants encompassing codons 43 to 54 of iso-1-cytochrome c. This region consists of an evolutionarily conserved structure within an evolutionarily diverse sequence. The library, on a low-copy-number yeast shuttle phagemid, was introduced into a yeast strain lacking cytochrome c. The ability of transformants harboring a functional cytochrome c to grow on the non-fermentable carbon source glycerol at 30 degrees C and 37 degrees C was used to determine the phenotype of nearly 1000 transformants. Approximately 90% of the missense mutants present in the library give rise to the wild-type phenotype, 7% result in the temperature-sensitive (Cycts) phenotype, and 3% give rise to the non-functional (Cyc-) phenotype. Phagemids from 20 Cycts and 30 Cyc- transformants were subjected to DNA sequence analysis. All the mutations occur within the targeted region. One-third of the mutants from Cyc- transformants and all the mutants from Cycts transformants are missense mutants. The remaining mutants from Cyc- transformants are nonsense or frame-shift mutants. Missense mutations within the codons for Gly45, Tyr46, Thr49, Asn52 or Ile53 alone are sufficient to produce temperature-sensitive behavior both in vivo and in the variant proteins. The deduced amino acid substitutions correlate remarkably well with side-chain dynamics, secondary structure and tertiary structure of the wild-type protein.

Native tertiary structure in an A-state.

The A-state is an equilibrium species that is thought to represent the molten globule, an on-pathway protein folding intermediate with native secondary structure and non-native, fluctuating tertiary structure. We used yeast iso-1-ferricytochrome c to test for an evolutionary-invariant tertiary interaction in its A-state. Thermal denaturation monitored by circular dichroism (CD)spectropolarimetry was used to determine A-state and native-state stabilities, delta GA reversible D and delta GN reversible D. We examined the wild-type protein, seven variants with substitutions at the interface between the N and C-terminal helices, and four control variants. The controls have the same amino acid changes as the interface variants, but the changes are close to, not at, the interface. We also examined the pH and sulfate concentration dependencies and found that while these factors affect the far-UV CD spectra of the least stable variants, they do not alter the difference in stability between the wild-type protein and the variants. A delta GA reversible D versus-delta GN reversible D plot for the interface variants has a slope near unity and the control variants have near-wild-type stability. These results show that the helix-helix interaction stabilizes the A-state and the native state to the same degree, confirming our preliminary report. We determined that the heat capacity change for A-state denaturation is approximately 60% of the value for native-state denaturation, indicating that the A-state interior is native-like. We discuss our results in relation to ferricytochrome c folding kinetics.

Partially formed native tertiary interactions in the A-state of cytochrome c.

Considerable insight into protein structure, stability, and folding has been obtained from studies of non-native states. We have studied the extent of native tertiary contacts in one such molecule, the A-state of yeast iso-1-ferricytochrome c. Previously, we showed that the interface between the N and C-terminal helices is completely formed in the A-state. Here, we focus on interactions essential for forming the heme pocket of eukaryotic cytochromes c. To determine the extent of these interactions, we used saturation mutagenesis at the evolutionarily invariant residue leucine 68, and measured the free energy of denaturation for the native states and the A-states of functional variants. We show that, unlike the interaction between the terminal helices, the native interactions between the 60s helix and the rest of the protein are not completely formed in the A-state.

Stability of yeast iso-1-ferricytochrome c as a function of pH and temperature.

Absorbance-detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2-state model. Values for free energy (delta GD), enthalpy (delta HD), and entropy (delta SD) of denaturation were determined as functions of pH and temperature. The value of delta GD at 300 K, pH 4.6, is 5.1 +/- 0.3 kcal mol-1. The change in molar heat capacity upon denaturation (delta Cp), determined by the temperature dependence of delta HD as a function of pH (1.37 +/- 0.06 kcal mol-1 K-1), agrees with the value determined by differential scanning calorimetry. pH-dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a delta HD of 1.30 kcal mol-1 residue-1 at 373.6 K and a delta SD of 4.24 cal mol-1 K-1 residue-1 at 385.2 K. These values agree with those for other single-domain, globular proteins.

Second virial coefficients as a measure of protein--osmolyte interactions.

The cytoplasm contains high concentrations of cosolutes. These cosolutes include macromolecules and small organic molecules called osmolytes. However, most biophysical studies of proteins are conducted in dilute solutions. Two broad classes of models have been used to describe the interaction between osmolytes and proteins. One class focuses on excluded volume effects, while the other focuses on binding between the protein and the osmolyte. To better understand protein--smolyte interactions, we have conducted sedimentation equilibrium analytical ultracentrifugation experiments using ferricytochrome c as a model protein. From these experiments, we determined the second virial coefficients for a series of osmolytes. We have interpreted the second virial coefficient as a measure of both excluded volume and protein--osmolyte binding. We conclude that simple models are not sufficient to understand the interactions between osmolytes and proteins.

Baseline length and automated fitting of denaturation data.

To understand relationships between protein sequence and stability, we often compare data from proteins that differ by the substitution of one amino acid. Frequently, an amino acid change causes the cooperative denaturation transitions to shift to lower temperatures, diminishing the signal from the native state. Here we show that apparent stability changes, i.e., the free energy of denaturation, deltaGD, can also be caused by a deficiency of points in the low temperature end of the transition. In addition, we suggest a method for overcoming this problem.

Design, synthesis, expression, and characterization of the genes for mouse Fc gamma RIIb1 and Fc gamma RIIb2 cytoplasmic regions.

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, mFc gamma RIIb1, and mFc gamma RIIb2, play a key role in signal transduction by mediating different cellular functions. mFc gamma RIIb1 has a 94-residue cytoplasmic region, whereas mFc gamma RIIb2 has a 47-residue cytoplasmic region. Genes encoding the cytoplasmic regions of mFc gamma RIIb1 (b1-94) and mFc gamma RIIb2 (b2-47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli. A sequence-specific protease, thrombin, was used to release the b1-94 peptide, which was purified by using HPLC. The b2-47 peptide was synthesized chemically. CD spectropolarimetry was employed to examine the secondary structures of b1-94 and b2-47. These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature. The results indicate that the b1-94 and b2-47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant alpha-helical structure.

Solvent-induced collapse of alpha-synuclein and acid-denatured cytochrome c.

The effects of solution conditions on protein collapse were studied by measuring the hydrodynamic radii of two unfolded proteins, alpha-synuclein and acid-denatured ferricytochrome c, in dilute solution and in 1 M glucose. The radius of alpha-synuclein in dilute solution is less than that predicted for a highly denatured state, and adding 1 M glucose causes further collapse. Circular dichroic data show that alpha-synuclein lacks organized structure in both dilute solution and 1 M glucose. On the other hand, the radius of acid-denatured cytochrome c in dilute solution is consistent with that of a highly denatured state, and 1 M glucose induces collapse to the size and structure of native cytochrome c. Taken together, these data show that alpha-synuclein, a natively unfolded protein, is collapsed even in dilute solution, but lacks structure.

Probing weakly polar interactions in cytochrome c.

Theoretical, statistical, and model studies suggest that proteins are stabilized by weakly polar attractions between sulfur atoms and properly oriented aromatic rings. The two sulfur-containing amino acids, methionine and cysteine, occur frequently among functional alleles in random mutant libraries of Saccharomyces cerevisiae iso-1-cytochrome c genes at positions that form a weakly polar aromatic-aromatic interaction, the wild-type protein. To determine if a weakly polar sulfur-aromatic interaction replaced the aromatic-aromatic interaction, the structure and stability of two variants were examined. Phenylalanine 10, which interacts with tyrosine 97, was replaced by methionine and cysteine. The cysteine was modified to form the methionine and cysteine analog, S-methyl cysteine (CysSMe). Proton NMR studies indicate that changing Phe 10 to Met or CysSMe affects only local structure and that the structures of sulfur-containing variants are nearly identical. Analysis of chemical shifts and nuclear Overhauser effect data indicates that both sulfur-containing side chains are in position to form a weakly polar interaction with Tyr 97. The F10M and F10CSMe variants are 2-3 kcal mol-1 less stable than iso-1-cytochrome c at 300 K. Comparison of the stabilities of the F10M and F10CSMe variants allows evaluation of the potential weakly polar interaction between the additional sulfur atom of F10CSMe and the aromatic moiety of Tyr 97. The F10CSMe;C102T variant is 0.7 +/- 0.3 kcal mol-1 more stable than the F10M;C102T protein. The increased stability is explained by the difference in hydrophobicity of the sulfur-containing side chains. We conclude that any weakly polar interaction between the additional sulfur and the aromatic ring is too weak to detect or is masked by destabilizing contributions to the free energy of denaturation.

Amide proton exchange rates of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochrome c.

Proton NMR spectroscopy was used to determine the rate constant, kobs, for exchange of labile protons in both oxidized (Fe(III)) and reduced (Fe(II)) iso-1-cytochrome c. We find that slowly exchanging backbone amide protons tend to lack solvent-accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular secondary structure as well as in omega-loops. Furthermore, there is no correlation between kobs and the distance from a backbone amide nitrogen to the nearest solvent-accessible atom. These observations are consistent with the local unfolding model. Comparisons of the free energy change for denaturation, delta Gd, at 298 K to the free energy change for local unfolding, delta Gop, at 298 K for the oxidized protein suggest that certain conformations possessing higher free energy than the denatured state are detected at equilibrium. Reduction of the protein results in a general increase in delta Gop. Comparisons of delta Gd to delta Gop for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatured form. This persistent structure in high-energy conformations of the reduced form appears to involve the axially coordinated heme.

Change in charge of an unvaried heme contact residue does not cause a major change of conformation in cytochrome c.

The structure of the Ala38 variant of yeast iso-1-cytochrome c, in which the previously unchanged Arg38 has been replaced, has been characterised by NMR. The NMR data indicate that the structure of the Ala38 variant is very similar to that of the wild type protein. In particular, the heme environment and interactions of the heme macrocycle are shown to be preserved. Analysis of the chemical shift perturbations to the resonances of Ile35 is shown to be consistent with the change in charge at position 38. The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification. Therefore the redox potential change accompanying the modification [1988, Biochemistry 28, 3188-3197] appears to be a direct consequence of the altered side-chain of residue 38 and not a result of secondary conformational changes induced by the modification.

A rapid droplet method for Sanger dideoxy sequencing.

A method for performing the dideoxy sequence reaction on petri dishes is described. It allows rapid manipulation of clones and provides large amounts of sequence information quickly and without the need for elaborate laboratory equipment.

Dramatic stabilization of ferricytochrome c upon reduction.

By combining measurements of the free energy of denaturation of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c with determination of the formal potentials for the native and chemically-denatured states we have determined the free energy of denaturation of the ferro form of the protein. We report that the simplest of all chemical modifications, addition of an electron, increases the stability of ferricytochrome c by approximately 10 kcal mol-1 at 300 K, pH 4.6. This makes reduced cytochrome c one of the most stable proteins yet investigated.

Changes in global stability and local structure of cytochrome c upon substituting phenylalanine-82 with tyrosine.

We have examined the F82Y;C102T variant of Saccharomyces cerevisiae iso-1-cytochrome c using high-resolution proton nuclear magnetic resonance spectroscopy, chemical denaturation, and differential scanning calorimetry. Comparison of proton chemical shifts, paramagnetic shifts, and nuclear Overhauser effects indicates structural changes are localized to the vicinity of position 82. One alteration involves the rearrangement of the side chain of leucine-85. Using many more proton assignments than were available in the initial report [G. J. Pielak, R. A. Atkinson, J. Boyd, and R. J. P. Williams, Eur. J. Biochem. 177, 179-185 (1988)], a second alteration involving an interaction between arginine-13 and tyrosine-82 is observed. The interaction appears to involve a hydrogen bond with the eta-protons of arginine's guanido group acting as donor and tyrosine's phenolic eta-oxygen as acceptor. In spite of this potentially-stabilizing interaction, the free energy of denaturation decreases by approximately 2.4 kcal mol-1. Results are discussed with respect to alterations in the native and denatured states.

Exploring the interface between the N- and C-terminal helices of cytochrome c by random mutagenesis within the C-terminal helix.

Buried within cytochrome c lies a highly-conserved helix-helix interface formed by the perpendicular packing of the C-terminal helix against the N-terminal helix. This interface involves a peg-in-hole interaction between Gly-6 and Leu-94 and an aromatic-aromatic interaction between Phe-10 and Tyr-97. To gain insight into protein design, we investigated the relationship between the sequence of the interface and the physiological function of yeast iso-1-cytochrome c. A library of mutants at positions 94 and 97 of the C-terminal helix was created to examine the effect of novel amino acid combinations. We isolated 45 of the 400 possible amino acid combinations, 32 of which result in a functional cytochrome c. Contrary to evolutionary conservation of the peg-in-hole and aromatic-aromatic interactions, we find that side-chain volume and conservation of aromatic residues do not play an essential role in determining function. Additionally, we find negatively-charged residues within the interface that result in a functional cytochrome c. Examination of the 45 missense mutants indicates that approximately 120 unique combinations are compatible with function. These results show that the interface is flexible. However, truncation of the C-terminal helix at position 94 abolishes function, suggesting that the interface is essential. The correlation observed between our library of mutants and the mutation matrix compiled by Gonnet et al. [Gonnet, G. H., Cohen, M. A., & Benner, S. A. (1992) Science 256, 1443-1445] demonstrates the potential use of the matrix to predict the effect of sequence changes on natural proteins and to optimize the design of novel proteins.

Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis.

The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix. Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function. DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface. Basic residues are not tolerated at position 6 or 10. There is a broad volume constraint for amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone is sufficient to promote helical association. There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins.

The function of the Saccharomyces cerevisiae iso-1-cytochrome c gene is independent of the codon at invariant residue Phe82 when the gene is present on a low-copy-number vector.

Phe82 is the most studied invariant residue of cytochrome c. However, the physiological relevance of amino acid substitutions at this position is unclear because previous studies were either performed in vitro (i.e. using purified protein) or in yeast where the gene for the protein is present on a multi-copy vector. Multi-copy vectors yield a level of cytochrome c in yeast that is greater than the wild-type level. Oligodeoxyribonucleotide-directed mutagenesis was used to change the codon for Phe82 to that of the other 19 naturally occurring amino acids as well as the amber stop codon. The alleles are present on a yeast shuttle phagemid containing the CEN6 gene which ensures a vector copy number of one to two in yeast. All the missense alleles support growth under conditions requiring a functional iso-1-cytochrome c. However the F82C, F82P, and F82R variants grow at a significantly lower rate. After selection for function, phagemids were rescued from the transformants and the identity of the mutation verified. It is concluded that all 20 amino acids are capable of supporting function. Reasons for the evolutionary invariance of Phe82 are discussed.

A native tertiary interaction stabilizes the A state of cytochrome c.

Certain kinetic intermediates in protein folding are similar to the molten globule, or A state, an equilibrium state of many proteins that is populated under high salt and low pH conditions. Many A states are nearly as compact as native proteins and have native-like secondary structure, but the extent to which nonlocal interactions stabilize the A state is unclear. In this study, thermal denaturation, monitored by circular dichroism, was used to determine the free energy of denaturation of the A state (delta GA<-->D) for Saccharomyces cerevisiae iso-1-ferricytochrome c. Specifically, we examined the wild-type protein, seven variants with amino acid substitutions at the interface between the N- and C-terminal helices, and two variants with mutations at a position close to, but not involved in, the interface. A plot of delta GA<-->D versus delta GN<-->D (the free energy of denaturation of the native state) has a slope near unity, showing that the evolutionarily conserved helix-helix interaction stabilizes the A state to the same degree that it stabilizes the native state.