Rapid collapse precedes the fast two-state folding of the cold shock protein.


Abstract

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. Study holds ProTherm entries: 16883, 16884, 16885, 16886, 16887, 16888, 16889, 16890, 16891, 16892, 16893, 16894, 16895, 16896, 16897, 16898, 16899, 16900, 16901, 16902, 16903, 16904 Extra Details: The probe for the measurements was fluorescence at 305 nm (Tyr) after excitation at 280 nm. protein folding; fluorescence energy transfer; folding intermediates; folding kinetics; intra-molecular distances

Submission Details

ID: J3h3V2HA4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Magg C;Schmid FX,J. Mol. Biol. (2004) Rapid collapse precedes the fast two-state folding of the cold shock protein. PMID:14729346
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