Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme–substrate complex is a matter of considerable debate. We present three structures at 1.7–2.0 Å resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6, and ω-undecylenyl-β-d-maltopyranoside, bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the axial heme ligand when cytochrome c functions as an electron carrier. The hydroxyl group of Tyr67 sits 3.6–4.0 Å from the nearest carbon of the detergents, positioned to act as a relay in radical abstraction during peroxidase activity. Docking studies with linoleic acid, the most common fatty acid component of cardiolipin, show that C11 of linoleic acid can sit adjacent to Tyr67 and the heme, consistent with the oxygenation pattern observed in lipidomics studies. The well-defined hydrocarbon binding pocket provides atomic resolution evidence for the extended lipid anchorage model for cytochrome c/cardiolipin binding. Dimer dissociation/association kinetics for yeast versus equine cytochrome c indicate that formation of mammalian cytochrome c dimers in vivo would require catalysis. However, the dimer structure shows that only a modest deformation of monomeric cytochrome c would suffice to form the hydrocarbon binding site occupied by these detergents.

•The rapid evolution of primate cytochromes c affects heme crevice stability.•The heme crevice stability of spider monkey and human cytochrome c is compared.•The Y46F substitution is the primary destabilizer of the heme crevice.•A 1.15 Å resolution structure of spider monkey cytochrome c is presented.•The Y46F substitution disrupts a hydrogen bond network connecting 3 surface Ω-loops.The hypothesis that the recent rapid evolution of primate cytochromes c, which primarily involves residues in the least stable Ω-loop (Ω-loop C, residues 40–57), stabilizes the heme crevice of cytochrome c relative to other mammals, is tested. To accomplish this goal, we have compared the properties of human and spider monkey cytochrome c and a set of four variants produced in the process of converting human cytochrome c into spider monkey cytochrome c. The global stability of all variants has been measured by guanidine hydrochloride denaturation. The stability of the heme crevice has been assessed with the alkaline conformational transition. Structural insight into the effects of the five amino acid substitutions needed to convert human cytochrome c into spider monkey cytochrome c is provided by a 1.15 Å resolution structure of spider monkey cytochrome c. The global stability for all variants is near 9.0 kcal/mol at 25 °C and pH 7, which is higher than that observed for other mammalian cytochromes c. The heme crevice stability is more sensitive to the substitutions required to produce spider monkey cytochrome c with decreases of up to 0.5 units in the apparent pKa of the alkaline conformational transition relative to human cytochrome c. The structure of spider monkey cytochrome c indicates that the Y46F substitution destabilizes the heme crevice by disrupting an extensive hydrogen bond network that connects three surface loops including Ω-loop D (residues 70–85), which contains the Met80 heme ligand.Destabilization of the heme crevice during the rapid evolution of primate cytochromes c is primarily due to a Y46F substitution, which disrupts a hydrogen bond network that knits together three surface Ω-loops.

Overall protein stability is thought to have an important impact on the millisecond time scale dynamics modulating enzyme function. In order to better understand the effects of overall stability on the substructure dynamics of mitochondrial cytochrome c, we test the effect of a destabilizing L85A mutation on the kinetics and equilibrium thermodynamics of the alkaline conformational transition. The alkaline conformational transition replaces the Met80 ligand of the heme with a lysine residue from Ω-loop D, the heme crevice loop, consisting of residues 70–85. Residues 67–87 are the most conserved portion of the sequence of mitochondrial cytochrome c, suggesting that this region is of prime importance for function. Mutations to Ω-loop D affect the stability of the heme crevice directly, modulating the pKapp of the alkaline transition. Two variants of yeast iso-1-cytochrome c, WT*/L85A and WT*/K73H/L85A, were prepared for these studies. Guanidine-HCl unfolding monitored by circular dichroism and pH titrations at 695 nm, respectively, were used to study the thermodynamics of global and local unfolding of these variants. The kinetics of the alkaline transition were measured by pH-jump stopped-flow methods. Gated electron transfer techniques using bis(2,2′,2″-terpyridine)cobalt(II) as a reducing reagent were implemented to measure the heme crevice dynamics for the WT*/K73H/L85A variant. Contrary to the expectation that dynamics around the heme crevice would be faster for the less stable WT*/K73H/L85A variant, based on the behavior of psychrophilic versus mesophilic enzymes, they were similar to those for a variant without the L85A mutation. In fact, below pH 7, the dynamics of the WT*/K73H/L85A variant were slower.

An A81H variant of yeast iso-1-cytochrome c is prepared to test the hypothesis that the steric size of the amino acid at sequence position 81 of cytochrome c, which has evolved from Ala in yeast to Ile in mammals, slows the dynamics of the opening of the heme crevice. The A81H mutation is used both to increase steric size and to provide a probe of the dynamics of the heme crevice through measurement of the thermodynamics and kinetics of the His81-mediated alkaline conformational transition of A81H iso-1-cytochrome c. Thermodynamic measurements show that the native conformer is more stable than the His81-heme alkaline conformer for A81H iso-1-cytochrome c. ΔGu°(H2O) is approximately 1.9 kcal/mol for formation of the His81-heme alkaline conformer. By contrast, for K79H iso-1-cytochrome c, the native conformer is less stable than the His79-heme alkaline conformer. ΔGu°(H2O) is approximately −0.34 kcal/mol for formation of the His79-heme alkaline conformer. pH jump and gated electron transfer kinetics demonstrate that this stabilization of the native conformer in A81H iso-1-cytochrome c arises primarily from a decrease in the rate constant for formation of the His81-heme alkaline conformer, kf,His81, relative to kf,His79 for formation of the His79-heme alkaline conformer, which forms by a mechanism similar to that observed for the His81-heme alkaline conformer. The result is discussed in terms of the effect of global protein stability on protein dynamics and in terms of optimization of the sequence of cytochrome c for its role as a peroxidase in the early stages of apoptosis in higher eukaryotes.

Trimethyllysine 72 (tmK72) has been suggested to play a role in sterically constraining the heme crevice dynamics of yeast iso-1-cytochrome c mediated by the Ω-loop D cooperative substructure (residues 70–85). A tmK72A mutation causes a gain in peroxidase activity, a function of cytochrome c that is important early in apoptosis. More than one higher energy state is accessible for the Ω-loop D substructure via tier 0 dynamics. Two of these are alkaline conformers mediated by Lys73 and Lys79. In the current work, the effect of the tmK72A mutation on the thermodynamic and kinetic properties of wild-type iso-1-cytochrome c (yWT versus WT*) and on variants carrying a K73H mutation (yWT/K73H versus WT*/K73H) is studied. Whereas the tmK72A mutation confers increased peroxidase activity in wild-type yeast iso-1-cytochrome c and increased dynamics for formation of a previously studied His79-heme alkaline conformer, the tmK72A mutation speeds return of the His73-heme alkaline conformer to the native state through destabilization of the His73-heme alkaline conformer relative to the native conformer. These opposing behaviors demonstrate that the response of the dynamics of a protein substructure to mutation depends on the nature of the perturbation to the substructure. For a protein substructure which mediates more than one function of a protein through multiple non-native structures, a mutation could change the partitioning between these functions. The current results suggest that the tier 0 dynamics of Ω-loop D that mediates peroxidase activity has similarities to the tier 0 dynamics required to form the His79-heme alkaline conformer.

At the onset of apoptosis, the peroxidation of cardiolipin at the inner mitochondrial membrane by cytochrome c requires an open coordination site on the heme. We report a 1.45-Å resolution structure of yeast iso-1-cytochrome c with the Met80 heme ligand swung out of the heme crevice and replaced by a water molecule. This conformational change requires modest adjustments to the main chain of the heme crevice loop and is facilitated by a trimethyllysine 72-to-alanine mutation. This mutation also enhances the peroxidase activity of iso-1-cytochrome c. The structure shows a buried water channel capable of facilitating peroxide access to the active site and of moving protons produced during peroxidase activity to the protein surface. Alternate positions of the side chain of Arg38 appear to mediate opening and closing of the buried water channel. In addition, two buried water molecules can adopt alternate positions that change the network of hydrogen bonds in the buried water channel. Taken together, these observations suggest that low and high proton conductivity states may mediate peroxidase function. Comparison of yeast and mammalian cytochrome c sequences, in the context of the steric factors that permit opening of the heme crevice, suggests that higher organisms have evolved to inhibit peroxidase activity, providing a more stringent barrier to the onset of apoptosis.

Alkaline conformers of cytochrome c may be involved in both its electron transport and apoptotic functions. We use cobalt(II)bis(terpyridine), Co(terpy)22+, as a reagent for conformationally gated electron-transfer (gated ET) experiments to study the alkaline conformational transition of K79H variants of yeast iso-1-cytochrome c expressed in Escherichia coli, WT*K79H, with alanine at position 72 and Saccharomyces cerevisiae, yK79H, with trimethyllysine (Tml) at position 72. Co(terpy)22+ is well-suited to the 100 ms to 1 s time scale of the His79-mediated alkaline conformational transition of these variants. Reduction of the His79-heme alkaline conformer by Co(terpy)22+ occurs primarily by gated ET, which involves conversion to the native state followed by reduction, with a small fraction of the His79-heme alkaline conformer directly reduced by Co(terpy)22+. The gated ET experiments show that the mechanism of formation of the His79-heme alkaline conformer involves only two ionizable groups. In previous work, we showed that the mechanism of the His73-mediated alkaline conformational transition requires three ionizable groups. Thus, the mechanism of heme crevice opening depends upon the position of the ligand mediating the process. The microscopic rate constants provided by gated ET studies show that mutation of Tml72 (yK79H variant) in the heme crevice loop to Ala72 (WT*K79H variant) affects the dynamics of heme crevice opening through a small destabilization of both the native conformer and the transition state relative to the His79-heme alkaline conformer. Previous pH jump data had indicated that the Tml72→Ala mutation primarily stabilized the transition state for the His79-mediated alkaline conformational transition.

Trimethyllysine 72 (Tml72) of yeast iso-1-cytochrome c lies across the surface of the heme crevice loop (Ω-loop D, residues 70–85) like a brace. Lys72 is oriented similarly in horse cytochrome c (Cytc). To determine whether this residue affects the dynamics of opening the heme crevice loop, we have studied the effect of a Tml72 to Ala substitution on the formation of the His79–heme alkaline conformer near neutral pH using a variant of iso-1-Cytc including K72A and K79H mutations. Guanidine hydrochloride denaturation shows that the Tml72 to Ala substitution within error does not affect the global stability of the protein. The effect of the Tml72 to Ala substitution on the thermodynamics of the His79–heme alkaline transition is also small. However, pH-jump kinetic studies of the His79–heme alkaline transition show that both the forward and backward rates of conformational change are increased by the Tml72 to Ala substitution. The barrier for opening the heme crevice is reduced by 0.5 kcal/mol and for closing the heme crevice by 0.3 kcal/mol. The ability of Tml72 to modulate the heme crevice dynamics may indicate a crucial role in regulating function, such as in the peroxidase activity seen in the early stages of apoptosis.

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, kb,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.

Protein dynamics is intimately linked to function. In metalloproteins, dynamics are often coupled to redox activity, ligand binding and enzyme function. We provide a concise overview of the field and then focus on the use of the alkaline conformer of cytochrome c as a model system to probe the factors that control the conformational dynamics of proteins in general and metalloproteins in particular. We consider the effects of ligands on metal-mediated dynamics, the interplay between intrinsic metal-ligand dynamics and barriers imposed by the protein scaffold itself, and the effects of local and overall protein stability on dynamics. Discussed within are the collected results from equilibrium thermodynamic methods, pH jump kinetics and conformationally gated redox reactions between small inorganic reagents and metalloproteins used as a means to probe conformational switching in metalloproteins.

To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine−heme loop formation equilibria for cytochrome c′ to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν3, of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine−heme loop breakage in the denatured state of cytochrome c′ show that some histidine−heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine−heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c′, the histidine−heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c′ at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c′ set up the topology of cytochrome c′ in the denatured state, predisposing the protein to fold efficiently to its native structure.

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 (pKHL ∼ 6.5). Furthermore, the forward and backward rate constants, kf and kb, respectively, for the conformational change are modulated by two auxiliary ionizations (pKH1 ∼ 5.5, and pKH2 ∼ 9). A possible candidate for pKH1 is His26, which has a strongly shifted pKa 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 kobs for the His73–heme alkaline transition from pH 5 to 8, suggesting that pKH1 has disappeared. However, direct measurement of kf and kb using conformationally gated electron transfer methods shows that the pH independence of kobs results from coincidental compensation between the decrease in kb due to pKH1 and the increase in kf due to pKHL. Thus, His26 is not the source of pKH1. 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.

How the primary sequence of a protein encodes conformational preferences that operate early in folding to promote efficient formation of the correct native topology is still poorly understood. To address this issue, we have prepared a set of yeast iso-1-cytochrome c variants that contain polyalanine inserts ranging from 6 to 30 residues in length near the N terminus of the protein. We study the thermodynamics and kinetics of His-heme loop formation in the denatured state at 3 and 6 M guanidine-HCl concentration. We find that polyalanine closely approximates a random coil with excluded volume giving scaling exponents, ν3, for equilibrium loop formation of 2.26 ± 0.13 and 1.97 ± 0.04 in 3 and 6 M guanidine-HCl, respectively. The rate of loop breakage initially decreases and then becomes independent of loop size as would be expected for a random coil. Comparison with previously reported data for denatured state His-heme loop formation for iso-1-cytochrome c and Rhodopseudomonas palustris cytochrome c′, shows that foldable sequences deviate significantly from random coil behavior and that the deviation is fold-dependent.

The competition between intramolecular histidine−heme loop formation and ligand-mediated oligomer formation in the denatured state is investigated for two yeast iso-1-cytochrome c variants, AcH26I52 and AcA25H26I52. Besides the native His 18 heme ligand, both variants contain a single His at position 26. The AcA25H26I52 variant has Pro 25 mutated to Ala. The concentration dependence of the apparent pKa for His 26−heme binding in 3 M guanidine hydrochloride indicates that the P25A mutation disfavors oligomerization mediated by intermolecular heme ligation by 10-fold. Single- and double-pH-jump stopped-flow experiments with the AcH26I52 variant show that fast phases for His−heme bond formation and breakage are due to intramolecular loop formation and slow phases for His−heme bond formation and breakage are due to intermolecular aggregation. The presence of two closely spaced slow phases in the kinetics of loop formation for both variants suggests that intermolecular His 26−heme ligation results in both dimers and higher-order aggregates. The P25A mutation slows formation and speeds breakdown of an initial dimer, demonstrating a strong effect of local sequence on aggregation. Analysis of the kinetic data yields equilibrium constants for intramolecular loop formation and intermolecular dimerization at pH 7.1 and indicates that the rate constant for intermolecular aggregation is very fast at this pH (107−108 M−1 s−1). In light of the very fast rates of aggregation in the denatured state, comparison of models involving reversible or irreversible oligomerization steps suggests that equilibrium control of the partitioning between folding and aggregation is advantageous for productive protein folding in vivo.

Recent work on the thermodynamics of protein denatured states is providing insight into the stability of residual structure and the conformational constraints that affect the disordered states of proteins. Current data from native state hydrogen exchange and the pH dependence of protein stability indicate that residual structure can modulate the stability of the denatured state by up to 4 kcal mol–1. NMR structural data have emphasized the role of hydrophobic clusters in stabilizing denatured state residual structures, however recent results indicate that electrostatic interactions, both favorable and unfavorable, are also important modulators of the stability of the denatured state. Thermodynamics methods that take advantage of histidine–heme ligation chemistry have also been developed to probe the conformational constraints that act on denatured states. These methods have provided insights into the role of excluded volume, chain stiffness, and loop persistence in modulating the conformational preferences of highly disordered proteins. New insights into protein folding and novel methods to manipulate protein stability are emerging from this work.

The observation that denatured proteins yield scaling exponents, ν, consistent with random-coil behavior and yet can also have pockets of residual or nonrandom structure has been termed the “reconciliation problem”. To provide greater insight into the denatured state of a foldable sequence, we have measured histidine–heme loop formation equilibria in the denatured state of a class II c-type cytochrome, cytochrome c′ from Rhodopseudomonas palustris. We have prepared a series of variants that provide His–heme loop stabilities, pKloop(His), for loop sizes ranging from 10 to 111 residues at intervals of 7 to 11 residues along the sequence of the protein. We observe a scaling exponent for loop formation, ν3, of 2.5 ± 0.3. Theoretical values for ν3 range from 1.8 to 2.4; thus, the observed ν3 is consistent with random-coil behavior. However, in contrast to data for loop formation as a function of loop size obtained with peptides of homogeneous sequence, we observe considerable scatter about the linear dependence of loop stability on loop size. Thus, foldable sequences behave very differently from homogeneous peptide sequences. The observed scatter suggests that there is considerable variation in the conformational properties along the backbone of a foldable sequence, consistent with alternating compact and extended regions. With regard to the reconciliation problem, it is evident that a scaling exponent consistent with a random coil is necessary but not sufficient to demonstrate random-coil behavior.

Two sets of iso-1-cytochrome c variants have been prepared with N-terminal insertions of pure polyglutamine, i.e., PolyQ variants, or polyglutamine interrupted with lysine every sixth residue, i.e., Gln-rich variants. The polymer properties of these pure polyGln or Gln-rich sequences have been evaluated using equilibrium and kinetic His-heme loop formation methods for loop sizes ranging from 22 to 46 in 1.5, 3.0, and 6.0 M guanidine hydrochloride (GdnHCl). In 6.0 M GdnHCl, the scaling exponent, ν3, for the pure polyGln sequences, is ∼1.7—significantly less than ν3 ≈ 2.15 for the Gln-rich sequences. The stability of the His-heme loops becomes progressively greater for the pure polyGln sequences relative to the Gln-rich sequences as GdnHCl concentration decreases from 6.0 to 1.5 M. Thus, the context of the sequence effects the polymer properties of Gln repeats even in denaturing concentrations of GdnHCl. Comparison of data for the Gln-rich variants with previous results for Gly-rich and Ala-rich variants shows that ν3 ∼ 2.2 for the Gln-rich, Gly-rich, and Ala-rich sequences in 6.0 M GdnHCl, whereas ν3 remains unchanged at 3.0 M GdnHCl concentration for the Gln-rich and Ala-rich sequences but decreases to ∼1.7 for the Gly-rich sequences. Thus, the polymer properties of Gln-rich and Ala-rich sequences are less sensitive to solvent quality in denaturing solutions of GdnHCl than Gly-rich sequences. Evaluation of Flory’s characteristic ratio, Cn, for the Gln-rich and Ala-rich sequences relative to the Gly-rich sequences shows that Gln-rich sequences are stiffer than Ala-rich sequences at both 3.0 and 6.0 M GdnHCl.

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.

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 pKa 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.

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, ν3, 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 ν3 = 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, Cn, for alanine-rich versus glycine-rich sequences. In 3.0 M GdnHCl, Cn(Ala)/Cn(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.

A novel approach to quantification of cardiolipin and DOPC lipid and vesicle concentration that is rapid and inexpensive is described. Traditional approaches to quantifying vesicle concentration destroy sample and are often time consuming. Using common laboratory equipment and software, lipid vesicles were reliably quantified allowing for immediate use without significant sample loss. Once calibrated, only absorbance measurements with a UV-Vis spectrophotometer are necessary as input into a Matlab program, which calculates the corresponding vesicle and lipid concentration. Fast and accurate concentration determination for preparations of vesicles is essential for analytical titration experiments necessary for protein/vesicle binding curves.