title: Early turn formation and chain collapse drive fast folding of the major cold shock protein CspA of Escherichia coli.
abstract: The folding mechanism of the β-sheet protein CspA, the major cold shock protein of Escherichia coli, was previously reported to be a concerted, two-state process. We have reexamined the folding of CspA using multiple spectroscopic probes of the equilibrium transition and laser-induced temperature jump (T-jump) to achieve better time resolution of the kinetics. Equilibrium temperature-dependent Fourier transform infrared (1634 cm(-1)) and tryptophan fluorescence measurements reveal probe-dependent thermal transitions with midpoints (T(m)) of 66 ± 1 and 61 ± 1 °C, respectively. Singular-value decomposition analysis with global fitting of the temperature-dependent infrared (IR) difference spectra reveals two spectral components with distinct melting transitions with different midpoints. T-jump relaxation measurements of CspA probed by IR and fluorescence spectroscopy show probe-dependent multiexponential kinetics characteristic of non-two-state folding. The frequency-dependent IR transients all show biphasic relaxation with average time constants of 50 ± 7 and 225 ± 25 μs at a T(f) of 77 °C and almost equal amplitudes. Similar biphasic kinetics are observed using Trp fluorescence of the wild-type protein and the Y42W and T68W mutants, with comparable lifetimes. All of these observations support a model for the folding of CspA through a compact intermediate state. The transient IR and fluorescence spectra are consistent with a diffuse intermediate having β-turns and substantial β-sheet structure. The loop β3-β4 structure is likely not folded in the intermediate state, allowing substantial solvent penetration into the barrel structure.
authors.affiliation: Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States.
authors.forename: Dung M
authors.initials: DM
authors.lastname: Vu
authors.forename: Scott H
authors.initials: SH
authors.lastname: Brewer
authors.forename: R Brian
authors.initials: RB
authors.lastname: Dyer
mesh_terms: D057773:Cold Shock Proteins and Peptides; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006360:Heat-Shock Proteins / Q000737:chemistry*; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan / Q000737:chemistry
chemical_list: D057773:Cold Shock Proteins and Peptides; C498192:CspA protein, E coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D014364:Tryptophan