cytochrome-c-t and sodium-perchlorate

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.

Topics: Cytochromes c; Dose-Response Relationship, Drug; Entropy; Hydrodynamics; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Perchlorates; Protein Folding; Protein Multimerization; Protein Structure, Secondary; Saccharomyces cerevisiae; Salts; Sodium Compounds; Solutions; Time Factors; Urea

Chicken apocytochrome c has been shown to possess a much stronger tendency to fold spontaneously in aqueous solution than the equivalent enzyme from other species. In the present work, the amino acid that determines its folding ability was elucidated by site-directed mutagenesis. Wild-type chicken apocytochrome c and three mutants V92A, S103A, and V92A/S103A were expressed in Escherichia coli. The wild-type apoprotein and S103A exhibited the same folding property during dialysis renaturation processes as that chemically prepared from chicken cytochrome c, while those containing V92A mutation did not. Quantitative studies by 2,2,2-trifluoroethanol (TFE) and sodium perchlorate (NaClO4) titration demonstrated that the V92A mutation decreased the helix content that could be induced and confirmed that valine 92 is the major determinant of the folding propensity of chicken apocytochrome c. Furthermore, CD spectra, turbidity measurements, and a translocation assay on a model membrane system showed that the V92A mutation also drastically altered the conformation of apocytochrome c after being incorporated into lipid bilayer and decreased the aggregation of phospholipid vesicles after association of the apoprotein, thus rendering the molecule more competent for translocation across the membrane. Our results showed that a single amino acid substitution could radically alter the folding propensity of an unfolded polypeptide chain and thus influence the conformation following its insertion into phospholipid bilayer.

Topics: Animals; Apoproteins; Base Sequence; Chickens; Circular Dichroism; Cytochrome c Group; Cytochromes c; DNA Primers; Escherichia coli; Gene Expression; Lipoproteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Perchlorates; Phospholipids; Protein Conformation; Protein Denaturation; Protein Folding; Sodium Compounds; Spectrophotometry; Titrimetry; Trifluoroethanol; Trypsin