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首页> 外文期刊>Biochemistry >Early turn formation and chain collapse drive fast folding of the major cold shock protein CspA of Escherichia coli
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Early turn formation and chain collapse drive fast folding of the major cold shock protein CspA of Escherichia coli

机译：早期转弯形成和链折叠驱动大肠杆菌主要冷激蛋白CspA的快速折叠

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摘要

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.
机译：先前报道，β-折叠蛋白CspA（大肠杆菌的主要冷休克蛋白）的折叠机制是一致的两个状态的过程。我们已经使用平衡转移和激光诱导的温度跃迁（T跃迁）的多个光谱探针重新检查了CspA的折叠，以实现更好的动力学时间分辨。平衡温度依赖性傅立叶变换红外光谱（1634 cm〜（-1））和色氨酸荧光测量显示，探针依赖性热转变的中点（T_m）分别为66±1和61±1°C。全局拟合温度依赖的红外（IR）差异光谱的奇异值分解分析显示了两个具有不同中点的不同熔融转变的光谱成分。通过红外光谱和荧光光谱探测的CspA的T跃迁弛豫测量结果显示了非二态折叠的探针依赖性多指数动力学特征。频率相关的IR瞬变都显示出双相弛豫，在77°C的T _f下，平均时间常数分别为50±7和225±25μs，并且振幅几乎相等。使用野生型蛋白和Y42W和T68W突变体的Trp荧光观察到相似的双相动力学，具有相当的寿命。所有这些观察结果都支持通过紧凑的中间状态折叠CspA的模型。瞬态IR和荧光光谱与具有β-转角和基本β-折叠结构的扩散中间体一致。环状β3-β4结构在中间状态下可能不会折叠，从而使大量溶剂渗透到桶状结构中。

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