Distinguishing between cooperative and unimodal downhill protein folding
Distinguishing between cooperative and unimodal downhill protein folding
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DOI：
10.1073/pnas.0609717104
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发表时间：
2007-01-02
影响因子：
11.1
通讯作者：
Fersht, Alan R.
中科院分区：
文献类型：
--
作者：
Huang, Fang;Sato, Satoshi;Fersht, Alan R.
传统的协同蛋白质折叠在单独的自由能威尔斯阱中调用天然和变性状态结构的离散集合。然而，单峰非合作（“下坡”）折叠提出了一个占据单个自由能阱的状态系综，蛋白质在298 K下以>= 4 × 10（4）s（-1）折叠。通过标准平衡实验很难证伪这种快速折叠蛋白质的单峰机制，因为当用于观察的时间尺度比用于平衡的时间尺度长时，协同机制和单峰机制都可以呈现相同的时间平均结构、光谱和热力学性质。然而，动力学可以提供必要的证据。具有强烈倾斜的线性重折叠臂的雪佛龙图很难用下坡折叠来解释，并且是通过过渡态系综进行合作折叠的签名。外周亚基结合结构域POB及其突变体的折叠动力学符合强烈倾斜的V形曲线，在变性剂溶液中观察到的速率常数> 6 x 10（4）s（-1），在水中外推至2 x 10（5）s（-1）。蛋白质A在298 K下以105 s-1折叠，也具有明确的V形。单分子荧光能量转移实验表明，在变性剂的存在下，标记的蛋白A的天然和变性状态的直接双峰分布。
Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate free-energy wells. Unimodal noncooperative ("downhill") folding, however, proposes an ensemble of states occupying a single free-energy well for proteins folding at >= 4 x 10(4) s(-1) at 298 K. It is difficult to falsify unimodal mechanisms for such fast folding proteins by standard equilibrium experiments because both cooperative and unimodal mechanisms can present the same time-averaged structural, spectroscopic, and thermodynamic properties when the time scale used for observation is longer than for equilibration. However, kinetics can provide the necessary evidence. Chevron plots with strongly sloping linear refolding arms are very difficult to explain by downhill folding and are a signature for cooperative folding via a transition state ensemble. The folding kinetics of the peripheral subunit binding domain POB and its mutants fit to strongly sloping chevrons at observed rate constants of > 6 x 10(4) s(-1) in denaturant solution, extrapolating to 2 x 10(5) s(-1) in water. Protein A, which folds at 105 s-1 at 298 K, also has a well-defined chevron. Single-molecule fluorescence energy transfer experiments on labeled Protein A in the presence of denaturant demonstrated directly bimodal distributions of native and denatured states.