Temperature and driving force dependence of the folding rate of reduced horse heart cytochrome c

Utilizing the stability difference between the ferro and ferri forms of horse heart cytochrome c (cyt c), folding of reduced cyt c was triggered by laser-induced reduction of unfolded oxidized cyt c. Measurements were made of the kinetics of the main folding phase (1 ms-10 s) in which collapsed reduced cyt c transforms to the native conformation. The folding rates were studied extensively as a function of temperature (5-75 degrees C) and guanidine hydrochloride (GdnHCl) concentration (1.6-4.9 M). At constant [GdnHCl], the Arrhenius plot of the folding rate constant (k) is nonlinear. At temperatures above 40 degrees C, the decrease in protein stability counteracts the expected increase in folding rate. Introducing free energy (DeltaG), derived from protein stability data, into the Eyring and Arrhenius equations leads to: ln k = ln(k(b)T/h) + DeltaS()/R - DeltaH()/RT - theta(m)DeltaG/RT = ln A - E(a)/RT - theta(m)DeltaG/RT, where theta(m) is the ratio between the denaturant dependence of the folding rate and the stability. By using this equation at constant DeltaG [or constant equilibrium constant (K)], linear Arrhenius plots are obtained. For the main folding phase of reduced cyt c, a positive DeltaS() is obtained indicating that the transition state is less ordered than the reactant. A model is proposed in which reduced cyt c first collapses into a compact intermediate, which needs to expand to reach the transition state of the rate-limiting folding reaction.     

Analysis of the differences in the folding mechanisms of c-type lysozymes based on contact maps constructed with interresidue average distances

A method for analyzing differences in the folding mechanisms of proteins in the same family is presented. Using only information from the amino acid sequences, contact maps derived from the interresidue average distances are employed. These maps, referred to as average distance maps (ADM), are applied to the folding of c-type lysozymes. The results reveal that the ADMs of these lysozymes reflect the differences in the detailed folding mechanisms. Further possible applications of the present method are also discussed.     

A functioning complex of two cytochrome c fragments with deletion of the (39-58) eicosapeptide

Two major fragments of horse heart cytochrome c involving the sequences (1-38) and (60-104) were found to produce a stable complex. The two fragments were devoid of any cytochrome c activity. The complex exhibited a hardly measurable electron transfer capacity with respect to cytochrome c oxidase and missed the 695 nm absorption band. The introduction of tryptophan in position 59 restored the intrinsic activity of the complex to the level of native cytochrome c. This was concluded from the convergence of the Eady-Hofstee plots which extrapolate to the same Vmax at high substrate concentrations. The absorption spectrum of the complex in the ferriform contained a clear absorption band at 695 nm (84% of that found with native cytochrome c). The investigation proves the indispensability of tryptophan in position 59 for the transfer of an electron to cytochrome c oxidase and supports the conclusions of Parr et al. about the existence of two consecutive processes in the folding of the two fragments (vide infra).     

An obligatory intermediate in the folding pathway of cytochrome c552 from Hydrogenobacter thermophilus

The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c551, which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.     

The stability and folding process of amyloidogenic mutant human lysozymes.

Amyloid deposits are frequently formed by mutant proteins that have a lower stability than the wild-type proteins. Some reports, however, have shown that mutant-induced thermodynamic destabilization is not always a general mechanism of amyloid formation. To obtain a better understanding of the mechanism of amyloid fibril formation, we show in this study that equilibrium and kinetic refolding-unfolding reaction experiments with two amyloidogenic mutant human lysozymes (I56T and D67H) yield folding pathways that can be drawn as Gibbs energy diagrams. The equilibrium stabilities between the native and denatured states of both mutant proteins were decreased, but the degrees of instability were different. The Gibbs energy diagrams of the folding process reveal that the Gibbs energy change between the native and folding intermediate states was similar for both proteins, and also that the activation Gibbs energy change from the native state to the transition state decreased. Our results confirm that the tendency to favor the intermediate of denaturation facilitates amyloid formation by the mutant human lysozymes more than equilibrium destabilization between the native and completely denatured states does.     

Three-dimensional structure of goose-type lysozyme from the egg white of the Australian black swan, Cygnus atratus

The egg white of C. atratus contains two forms of lysozyme, a 'chick-type' which is similar to that found in the egg white of the domestic hen, and a 'goose-type' similar to that found in the egg white of the Embden goose. The molecular structure of the goose-type lysozyme has been determined at a resolution of a 2.8 A by X-ray crystallographic analysis. The structure consists of two domains linked by a long stretch of alpha-helix. In all, there are seven helical segments in the structure. While there is no amino acid sequence homology with either hen egg-white or bacteriophage T4 lysozymes, there are portions of the structure where the folding of the main chain is similar to that found in portions of either hen egg-white lysozyme or T4 lysozyme or both. In particular, there is a consistency of structure in the arrangement of acid groups in the catalytic site. G-o plots calculated for this structure and for the bacteriophage T4 lysozyme structure show that both have similar 'modules' of structure with boundaries occurring at structurally equivalent positions. Three of the common boundaries are equivalent structurally to three of the four module boundaries observed in G-o plots of hen egg-white lysozyme. The variation in the position of the remaining boundary may be related to differences in substrate binding.     

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.     

Effect of chemical modifications of tryptophan residues on the folding of reduced hen egg-white lysozyme

The effects of chemical modifications of Trp62 and Trp108 on the folding of hen egg-white lysozyme from the reduced form were investigated by means of the sulfhydryl-disulfide interchange reaction at pH 8 and 40 degrees C. The folding of reduced lysozyme was monitored by following the recovery of the original activity. Under the conditions employed, the apparent first-order rate constant for the folding of reduced lysozyme was not changed by the modifications of both Trp62 and Trp108 and the folding was completed within 30 min. However, the extent of the correct folding was changed by the modification of Trp62 but not by that of Trp108. Native and oxindolealanine108 lysozymes recovered 80 and 81% of their original activities after 30-min refolding, respectively, but Trp62-modified lysozymes recovered their activities to a lesser extent than native and oxindolealanine108 lysozymes. The recovered activities of Trp62-modified lysozymes after 30-min refolding were 63% for oxindolealanine62 lysozyme, 65% for delta 1-carboxamidomethylthiotryptophan62 lysozyme, and 52% for delta 1-carboxymethylthiotryptophan62 lysozyme. These results suggest that Trp62 is important for preventing the misfolding of reduced lysozyme, but that neither Trp62 nor Trp108 is involved in the rate-determining step (the slowest step) in the folding pathway. A decrease in the hydrophobic nature of Trp62 seems to increase the misfolding and thus to decrease the extent of the correct folding of reduced lysozyme. A mechanism for the involvement of Trp62 in the folding pathway of reduced lysozyme is proposed.     

Kinetics and mechanism of the folding of cytochrome c.

The reversible folding of cytochrome c in urea at pH 4.0 was investigated by repetitive pressure perturbation kinetics and by equilibrium spectroscopic methods. Two folding reactions were observed in the 1 ms to 10 s time range. The rates and amplitudes of these reactions depend on urea concentration in a complex manner, which is different for each process. The absorbance spectra of the kinetic amplitudes of the two reactions also differ from each other. A model with a three-state mechanism can quantitatively account for all of the kinetic and equilibrium data, and it enables us to determine the rate constants and volume changes of the two steps. If a rapid protonation step is added to the mechanism, the analysis can be extended to calculate the pH dependence of the rate and amplitude of the faster folding step. This pH dependence is in excellent agreement with previously published data [Tsong, T. Y. (1977) J. Biol. Chem. 252, 8778-8780]. Kinetic experiments in the 695-nm band show clearly that the axial ligand methionine-80 is involved in the slow folding process and the other axial ligand, histidine-18, is involved in the fast process. Additional experiments with a cyanogen bromide fragment of the protein, and fluorescence detection of the folding kinetics of the intact protein, support an interpretation of the model in terms of known structural elements of cytochrome c. This work provides new information about the mechanism of the folding of cytochrome c, resolves conflicts in earlier interpretations, and demonstrates the applicability of the repetitive pressure perturbation kinetics method to protein folding.     

Yeast tRNAPhe conformation wheels: a novel probe of the monoclinic and orthorhombic models.

A series of conformation wheels is constructed from the recently refined X-ray crystallographic data of monoclinic and orthorhombic yeast tRNAPhe. These circular plots relate the primary chemical structure (i.e., base sequence) directly to the secondary and tertiary structure of the molecule. The circular sequence of backbone torsion angles displays a unique pattern that is useful both in distinguishing the ordered and disordered regions of the molecule and in comparing the three sets of experimental data. Composite conformation wheels describe the fluctuations in the "fixed" parameters (phi', phi, chi) and independent conformation wheels reveal the changes in the "variable" parameters (omega', omega, psi, psi') of the three different yeast tRNAPhe models. Additional plots of base-stacking parameters help to visualize the intimate interrelationship between chemical sequence and three-dimensional folding of yeast tRNAPhe. The composite data illustrate several conformational schemes that position the bases of adjacent nucleosides in a parallel stacked array and reveal an even larger number of conformations that introduce bends or turns in the polynucleotide chain.     

Amino acid sequence and immunological properties of chachalaca egg white lysozyme

Summary The amino acid sequence of lysozyme c from chachalaca egg white was determined. Like other bird lysozymes c, that of the chachalaca has 129 amino acid residues. It differs from other avian lysozymes c by 27 to 31 amino acid substitutions as well as by being devoid of phenylalanine. It contains substitutions at 9 positions which are invariant in the other 7 bird lysozymes of known sequence. Although the chachalaca is classified zoologically in the order Galliformes, which includes chickens and other pheasant-like birds, its lysozyme differs more from those of pheasant-like birds than do the lysozymes c of ducks. Phylogenetic analysis of the sequence comparisons confirms that the lineage leading to chachalaca lysozyme c separated from that leading to other galliform lysozymes c before the duck lysozyme c lineage did. This indicates a contrast between protein evolution and evolution at the organismal level. Immunological comparison of chachalacalysozyme c with other lysozymes of known sequence provides further support for the proposal that immunological cross-reactivity is strongly dependent on degree of sequence resemblance among bird lysozymes.103rd communication on lysozymes from the Laboratory of P. Jollès. Supported in part by grants from C.N.R.S. (ER 102), I.N.S.E.R.M. (Groupe de recherche U-116), N.S.F. (GB-42028X), and N.I.H. (GM-21509).     

Mapping of four discontiguous antigenic determinants on horse cytochrome c

The epitopes (antigenic determinants) recognized by four different monoclonal antibodies on horse cytochrome c have been partially characterized by differential acetylation of lysine residues of free and antibody-bound cytochrome c. The degree of acetylation in the bound and free antigen molecule was assessed by a double-labeling procedure with [3H]acetic anhydride and [14C]acetic anhydride. Out of the 19 lysine residues of cytochrome c only very few were less reactive in the antigen-antibody complex, i.e. presumably located at the epitope for the antibody under study. The protection varied from 1.5-fold to over 20-fold lower reactivity in antibody-bound cytochrome c. The present results are complemented by previous data obtained by cross-reactivity analysis with cytochromes c from different species, with chemically modified cytochrome c derivatives, and by inhibition of proteolysis of cytochrome c in the presence of the antibodies. From the combined data we conclude that each of the four epitopes depends on the precise spatial folding of the antigen and contains residues which are brought together by the folding of the polypeptide chain. This work exemplifies that mapping of conformation-dependent epitopes can be achieved by applying a combination of mapping procedures of which each by itself provides partial information.     

Exploring the cytochrome c folding mechanism: cytochrome c552 from thermus thermophilus folds through an on-pathway intermediate

Understanding the role of partially folded intermediate states in the folding mechanism of a protein is a crucial yet very difficult problem. We exploited a kinetic approach to demonstrate that a transient intermediate of a thermostable member of the widely studied cytochrome c family (cytochrome c552 from Thermus thermophilus) is indeed on-pathway. This is the first clear indication of an obligatory intermediate in the folding mechanism of a cytochrome c. The fluorescence properties of this intermediate demonstrate that the relative position of the heme and of the only tryptophan residue cannot correspond to their native orientation. Based on an analysis of the three-dimensional structure of cytochrome c552, we propose an interpretation of the data which explains the residual fluorescence of the intermediate and is consistent with the established role played by some conserved interhelical interactions in the folding of other members of this family. A limited set of topologically conserved contacts may guide the folding of evolutionary distant cytochromes c through the same partially structured state, which, however, can play different kinetic roles, acting either as an intermediate or a transition state.     

Are the parameters of various stabilization factors estimated from mutant human lysozymes compatible with other proteins?

The various factors which contribute to protein stability have been extensively examined using mutant proteins, but the same kinds of substitutions have given different results depending on the substitution sites. Recently, the contributions of some stabilization factors have been quantitatively derived as parameters by a unique equation, considering the conformational changes due to the mutations using mutant human lysozymes [Funahashi et al. (1999) Protein ENG: 12, 841-850]. To evaluate these parameters estimated from the mutant human lysozymes, stability-structure datasets for the mutant T4 lysozymes were selected. The stabilities for the mutant T4 lysozymes could be roughly estimated using these parameters. Notable differences between the estimated and experimental stabilities were caused by the uncertainty in part of the structures due to some Arg and Lys residues fluctuating on the surface of the T4 lysozyme. Excluding these atoms from the estimation gave a good correlation between the estimated and experimental stabilities. These results suggest that the parameters of the various stabilization factors derived from the mutant human lysozymes are compatible with the mutant T4 lysozymes, although they should be improved with respect to some points using more information.     

Effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of glycosylated lysozymes.

Disruption of the calnexin gene in Saccharomyces cerevisiae did not lead to gross effects on the levels of cell growth and secretion of wild-type hen egg white lysozymes (HEWL). To investigate the function of calnexin in relation to the secretion of glycoproteins, we expressed both stable and unstable mutant glycosylated lysozymes in calnexin-disrupted S. cerevisiae. The secreted amounts of stable mutant glycosylated lysozymes (G49N and S91T/G49N) were almost the same in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of unstable mutant glycosylated lysozymes (K13D/G49N, C76A/G49N, and D66H/G49N) greatly increased in calnexin-disrupted S. cerevisiae, although their secretion was very low in the wild-type strain. This indicates that calnexin may act in the quality control of glycoproteins. We further investigated the expression level of the mRNA of the molecular chaperones BiP and PDI, which play a major role in the protein folding process in the ER, when glycosylated lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when the glycosylated lysozymes were expressed in calnexin-disrupted S. cerevisiae. This observation indicates that BiP and PDI may be induced by the accumulation of unfolded glycosylated lysozymes due to the deletion of calnexin.     

Equilibrium unfolding of a small low-potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris.

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.     

Kinetics of cytochrome C folding: atomically detailed simulations

The vast range of time scales (from nanoseconds to seconds) during protein folding is a challenge for experiments and computations. To make concrete predictions on folding mechanisms, atomically detailed simulations of protein folding, using potentials derived from chemical physics principles, are desired. However, due to their computational complexity, straightforward molecular dynamics simulations of protein folding are impossible today. An alternative algorithm is used that makes it possible to compute approximate atomically detailed long time trajectories (the Stochastic Difference Equation in Length). This algorithm is used to compute 26 atomically detailed folding trajectories of cytochrome c (a millisecond process). The early collapse of the protein chain (with marginal formation of secondary structure), and the earlier formation of the N and C helices (compare to the 60's helix) are consistent with the experiment. The existence of an energy barrier upon entry to the molten globule is examined as well. In addition to (favorable) comparison to experiments, we show that non-native contacts drive the formation of the molten globule. In contrast to popular folding models, the non-native contacts do not form off-pathway kinetic traps in cytochrome c.     

S-class cytochromes c have a variety of folding patterns: structure of cytochrome c-553 from Desulfovibrio vulgaris determined by the multi-wavelength anomalous dispersion method

The three-dimensional structure of cytochrome c-553 isolated from sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F strain, has been determined by the multi-wavelength anomalous dispersion technique with use of synchrotron radiation. The result shows that bacterial S-class cytochromes c have a variety of folding patterns. The relative location of two a-helices at amino- and carboxyl-terminals and the style of bonding to the heme group show "cytochrome c folding," but other regions of the structure are different from those of other cytochromes c previously reported. The results also give useful information about the location of sulfate-reducing bacterium on the phylogenetic tree of the bacterial cytochromes c superfamily.     

Formation of the molten globule-like state of cytochrome c induced by n-alkyl sulfates at low concentrations

The molten globule state of cytochrome c is the major intermediate of protein folding. Elucidation of the thermodynamic mechanism of conformational stability of the molten globule state would enhance our understanding of protein folding. The formation of the molten globule state of cytochrome c was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS, at low concentrations. The refolding states of the protein were monitored by spectroscopic techniques including circular dichroism (CD), visible absorbance and fluorescence. The effect of n-alkyl sulfates on the structure of acid-unfolded horse cytochrome c at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of the molten globule state. The addition of n-alkyl sulfates to the unfolded state of cytochrome c appears to support the stabilized form of the molten globule. The m-values of the refolded state of cytochrome c by SOS, SDeS, SDS, and STS showed substantial variation. The enhancement of m-values as the stability criterion of the molten globule state corresponded with increasing chain length of the cited n-alkyl sulfates. The compaction of the molten globule state induced by SDS, as a prototype for other n-alkyl sulfates, relative to the unfolded state of cytochrome c was confirmed by Stokes radius and thermal transition point (T(m)) measured by microviscometry and differential scanning calorimetry (DSC), respectively. Thus, hydrophobic interactions play an important role in stabilizing the molten globule state.