Using deeply trapped intermediates to map the cytochrome c folding landscape

Abstract

Replacement of iron with cobalt(III) selectively introduces a deep trap in the folding-energy landscape of the heme protein cytochrome c. Remarkably, neither the protein structure nor the folding thermodynamics is perturbed by this metal-ion substitution, as shown by data from spectroscopic and x-ray diffraction experiments. Through kinetics measurements, we have found parallel folding pathways involving several different misligated Co(III) species, and, as these folding intermediates persist for several hours under certain conditions, we have been able to elucidate fully their spectroscopic properties. The results, along with an analysis of the fluorescence energy-transfer kinetics during refolding, show that rapidly equilibrating populations of compact and extended polypeptide conformations are present until all molecules have reached the native structure. These measurements provide direct evidence that collapsed denatured structures are not substantially more stable than extended conformations of cytochrome c. Significant advances in theory and experiment are producing an increasingly detailed description of how a polypeptide self-assembles into the native structure of a protein (1-11). Energy landscape theories (1, 12-15), in particular, have provided a framework for interpreting experimental investigations of protein folding kinetics. Despite this progress, many fundamental questions about protein folding dynamics remain to be answered.Years of experimental work on the energetics and dynamics of self-assembly of cytochrome c (Cyt c) have failed to resolve whether unfolded polypeptides undergo global collapse to compact conformations on dilution or laser triggering to solution conditions that strongly favor the native structure (11,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Indeed, recent kinetics experiments suggest that comparable populations of compact and extended polypeptides are formed rapidly (Ͻ1 ms), and these two populations disappear in parallel as the protein folds (27,28). To test this energy landscape model rigorously, we have isolated the structures that comprise the submillisecond (or ''burst'') phase by replacing the native iron center with cobalt(III) (29): because of very high ligand substitution barriers, the final step of cobalt(III)-Cyt c (Co-Cyt c) folding is well separated in time from all early events, and, in this kinetically stabilized system, the early folding intermediates can be examined carefully over a period of hours by an entire armamentarium of physical methods. Materials and MethodsUV-visible and CD and fluorescence spectra were collected with Hewlett-Packard 8452A, Aviv (Lakewood, NJ) 62A DS, and Hitachi (Tokyo) F-4500 spectrometers, respectively. Fluorescence decay kinetics were measured as described previously (27). Horse, tuna, and yeast Co-Cyt were prepared according to established protocols (30, 31) with minor modifications. For the modification of yeast-Co-Cyt c with a dansyl fluorophore at Cys-102 (DNS-Co-Cyt c), FPLC-purified yeast Co-Cyt c was treated with exce...

Phosphorylation-induced Conformational Changes in a Mitogen-activated Protein Kinase Substrate

“…By contrast, in this work we apply FRET to small peptides that can adopt a number of different conformations in aqueous solvent (25). Typically, FRET is used in conjunction with time-resolved donor decay measurements to obtain the distribution of distances between a given donor and acceptor within a flexible macromolecule (32)(33)(34)(35). This is possible because time-resolved decay spectra are a function, in part, of the donor-acceptor distance distribution (22).…”

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

“…Experimentally, one would expect an exponential response in the microsecond time range. Although barriers have been observed for cytochrome c 11,12 and some other proteins, 13,14 random searches of conformational space could also display exponential behavior. 15 Another, perhaps more robust test, involves the development of sufficient stability in the burst-phase species to provide protection against exchange with solvent for amide hydrogens involved in main-chain main-chain hydrogen bonds.…”

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