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What happens in the early stage of protein folding remains an interesting unsolved problem. Rapid kinetics measurements with cytochrome c using submillisecond continuous flow mixing devices suggest simultaneous formation of a compact collapsed state and secondary structure. These data seem to indicate that collapse formation is guided by specific short and long range interactions (heteropolymer collapse). A contrasting interpretation also has been proposed, which suggests that the collapse formation is rapid, nonspecific, and a trivial solvent related compaction, which could as well be observed by a homopolymer (homopolymer collapse). We address this controversy using fluorescence correlation spectroscopy (FCS), which enables us to monitor the salt-induced compaction accompanying collapse formation and the associated time constant directly at single molecule resolution. In addition, we follow the formation of secondary structure using far UV CD. The data presented here suggest that both these models (homopolymer and heteropolymer) could be applicable depending on the solution conditions. For example, the formation of secondary structure and compact state is not simultaneous in aqueous buffer. In aqueous buffer, formation of the compact state occurs through a two-state co-operative transition following heteropolymer formalism, whereas secondary structure formation takes place gradually. In contrast, in the presence of urea, a compaction of the protein radius occurs gradually over an extended range of salt concentration following homopolymer formalism. The salt-induced compaction and the formation of secondary structure take place simultaneously in the presence of urea. [ABSTRACT FROM AUTHOR]

Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c. [ABSTRACT FROM AUTHOR]