Early Turn Formation and Chain Collapse Drive Fast Folding of the Major Cold Shock Protein CspA of Escherichia coli
详细信息 查看全文
- 作者：Dung M. Vu ; Scott H. Brewer ; R. Brian Dyer
- 刊名：Biochemistry
- 出版年：2012
- 出版时间：November 13, 2012
- 年：2012
- 卷：51
- 期：45
- 页码：9104-9111
- 全文大小：334K
- 年卷期：v.51,no.45(November 13, 2012)
- ISSN：1520-4995
文摘
The folding mechanism of the 尾-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鈥?) and tryptophan fluorescence measurements reveal probe-dependent thermal transitions with midpoints (Tm) of 66 卤 1 and 61 卤 1 掳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 卤 7 and 225 卤 25 渭s at a Tf of 77 掳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 尾-turns and substantial 尾-sheet structure. The loop 尾3鈭捨? structure is likely not folded in the intermediate state, allowing substantial solvent penetration into the barrel structure.