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The properties of the guanidine hydrochloride induced unfolding transition of iso-2 cytochrome c (iso-2) from Saccharomyces cerevisiae have been investigated by using kinetic and equilibrium techniques and have been compared with previously published studies of horse cytochrome c, which differs from iso-2 by 46% in amino acid sequence. Measurements of absorbance in the ultraviolet and visible spectral regions as a function of guanidine hydrochloride concentration give superimposable equilibrium transition curves with a midpoint of 1.15 M at pH 7.2 and 20 degrees C. A two-state analysis of the equilibrium data gives a Gibbs free energy of unfolding of 3.1 kcal/mol at 20 degrees C in the absence of denaturant. This agrees well with the predicted difference in stability between S. cerevisiae iso-2 and horse cytochrome c estimated from the free energies of transfer of buried hydrophobic groups. Three kinetic phases associated with folding can be detected throughout most of the transition zone. Two of the phases are detected by stopped-flow mixing experiments. The third phase is over within the mixing time of the flow experiments but is detectable by temperature jumps. At 20 degrees C, pH 7.2, the slowest phase (T1) is in the 20-100-s time range, the middle phase (T2) is in the 0.1-3-s range, and the fastest phase (T3) is on the order of 1 ms. For the reactions observed in the stopped flow (T1 and T2), a simplified three-state mechanism can be used to predict quantitatively the relative amplitudes of the phases and the equilibrium unfolding curve from the observed time constant data. Previously this same mechanism has been successful in describing the folding reactions of horse cytochrome c [Hagerman, P. J. (1977) Biopolymers 16, 731]. We suggest that the qualitative features of protein folding reactions may be conserved among homologous proteins.

Preferential interactions between chain segments are studied in unfolded cytochrome c. The method takes advantage of heme ligation in the unfolded protein, a feature unique to proteins with covalently attached heme. The approach allows estimation of the effective concentration of one polypeptide chain segment relative to another, and is successful in detecting differences for peptide chain segments separated by different numbers of residues in the linear sequence. The method uses proton NMR spectroscopy to monitor displacement of the histidine heme ligands by imidazole as guanidine hydrochloride unfolded cytochrome c is titrated with deuterated imidazole. When the imidazole concentration exceeds the effective (local) concentration of histidine ligands, the protein ligands are displaced by deuterated imidazole. On displacement, the histidine ring proton resonances move from the paramagnetic region of the spectrum to the diamagnetic region. Titrations have been carried out for members of the mitochondrial cytochrome c family that contain different numbers of histidine residues. These include cytochromes c from tuna (2), yeast iso-2 (3), and yeast iso-1-MS (4). At high imidazole concentration, the number of proton resonances that appear in the histidine ring C2H region of the NMR spectrum is one less than the number of histidine residues in the protein. So one histidine, probably His-18, remains as a heme ligand. The effective local concentrations of histidines-26, -33, and -39 relative to the heme (position 14-17) are estimated to be (3-16) X 10(-3) M.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetics of the refolding reaction of ribonuclease A from high concentrations of guanidine hydrochloride or urea are biphasic, and show two refolding reactions whose rates differ 450-fold at pH 5.8 and 250. Measurements of cytidine 2'-phosphate binding during refolding, after stopped-flow dilution of guanidine hydrochloride (Gdn-HCl) or urea, show that functional bovine pancreatic ribonuclease A (RNase A; ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) is formed in both the fast and slow phases of the refolding process. We conclude that the guanidine-unfolded state of RNase A is an equilibrium mixture of fast-and slow-refolding species, as was found previously for the heat-unfolded state at low pH. The fraction of the fast-refolding species in guanidine or ureaunfolded RNase A is the same as that in the heat-unfolded protein at pH 2.Previous work has shown that the fast-refolding species disappears as the pH is raised from 3 to 5 for heat-unfolded RNase A. This pH effect is not present in refolding from concentrated GdnHCl solutions: the same proportion of the fast-refolding species is found from pH 2 to pH 6, and also from 2 M to 6 M Gdn-HCI at pH 5.8. We conclude that the same proportion of the fast-refolding species is present at equilibrium whenever the residual structure in unfolded RNase A is reduced to a low level, and that the structural difference between the fast-refolding and slow-refolding species of RNase A lies in the configuration of the random coil polypeptide chain.The observed rates of protein folding reactions are many orders of magnitude faster than predicted from a purely random search of all possible configurations for a random coil polypeptide chain devoid of structuret. A possible explanation for this difference is that an unfolded protein is not a random coil. The elements of residual structure play a crucial role in directing the course of protein folding, and thus considerably restrict the possible pathways (1) There seems to be a contradiction between the two proposals: (a) that a guanidine-unfolded protein behaves as a homogeneous random chain and (b) that this same unfolded state still possesses some elements of residual structure which increase the rate and determine the pathway(s) of folding. However, these proposals are compatible if the residual structure, important for the folding process, is not present as such in the unfolded state, but is rapidly formed after refolding is initiated. For instance, formation of a-helical segments could take place within the dead time of stopped-flow measurements (6) and limit the possible pathways for refolding, if the a-helical segments are stable under the conditions in which refolding is initiated, without a requirement of prior slow steps in refolding to provide a stabilizing environment.Studies of the refolding of heat-unfolded RNase A (7-11) have shown that the heat-unfolded state does not behave as a single species in refolding. Instead, distinct fast-refolding and slow-refolding species of the heat-unfold...