Rapid collapse precedes the fast two-state folding of the cold shock protein.
The cold shock protein Bc-Csp folds very rapidly in a reaction that is well described by a kinetic two-state mechanism without intermediates. We measured the shortening of six intra-protein distances during folding by Förster resonance energy transfer (FRET) in combination with stopped-flow experiments. Single tryptophan residues were engineered into the protein as the donors, and single 5-(((acetylamino)ethyl)amino)naphthalene-1-sulfonate (AEDANS) residues were placed as the acceptors at solvent-exposed sites of Bc-Csp. Their R0 value of about 22 A was well suited for following distance changes during the folding of this protein with a high sensitivity. The mutagenesis and the labeling did not alter the refolding kinetics. The changes in energy transfer during folding were monitored by both donor and acceptor emission and reciprocal effects were found. In two cases the donor-acceptor distances were similar in the unfolded and the folded state and, as a consequence, the kinetic changes in energy transfer upon folding were very small. For four donor/acceptor pairs we found that > or =50% of the increase in energy transfer upon folding occurred prior to the rate-limiting step of folding. This reveals that about half of the shortening of the intra-molecular distances upon folding has occurred already before the rate-limiting step and suggests that the fast two-state folding reaction of Bc-Csp is preceded by a very rapid collapse.
Magg
Christine
C
Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany.
Schmid
Franz X
FX
D019943:Amino Acid Substitution / Q000235:genetics; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004735:Energy Transfer; D019791:Guanidine / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; C407094:cold shock protein, Bacillus; D014364:Tryptophan; D019791:Guanidine