Baumketner A, Jewett A, Shea J E
Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA.
J Mol Biol. 2003 Sep 19;332(3):701-13. doi: 10.1016/s0022-2836(03)00929-x.
Chaperonins, such as the GroE complex of the bacteria Escherichia coli, assist the folding of proteins under non-permissive folding conditions by providing a cavity in which the newly translated or translocated protein can be encapsulated. Whether the chaperonin cage plays a passive role in protecting the protein from aggregation, or an active role in accelerating folding rates, remains a matter of debate. Here, we investigate the role of confinement in chaperonin mediated folding through molecular dynamics simulations. We designed a substrate protein with an alpha/beta sandwich fold, a common structural motif found in GroE substrate proteins and confined it to a spherical hydrophilic cage which mimicked the interior of the GroEL/ES cavity. The thermodynamics and kinetics of folding were studied over a wide range of temperature and cage radii. Confinement was seen to significantly raise the collapse temperature, T(c), as a result of the associated entropy loss of the unfolded state. The folding temperature, T(f), on the other hand, remained unaffected by encapsulation, a consequence of the folding mechanism of this protein that involves an initial collapse to a compact misfolded state prior to rearranging to the native state. Folding rates were observed to be either accelerated or retarded compared to bulk folding rates, depending on the temperature of the simulation. Rate enhancements due to confinement were observed only at temperatures above the temperature T(m), which corresponds to the temperature at which the protein folds fastest. For this protein, T(m) lies above the folding temperature, T(f), implying that encapsulation alone will not lead to a rate enhancement under conditions where the native state is stable (T<T(f)). For confinement to positively impact folding rates under physiological conditions, it is hence necessary for the protein to exhibit a folding transition above the temperature at which it exhibits its fastest folding rate (T(m)<T(f)). We designed a protein with this property by reducing the energetic frustration in the original alpha/beta sandwich substrate protein. The modified protein exhibited a twofold acceleration in folding rates upon encapsulation. This rate enhancement is due to a mechanistic change in folding involving the elimination, upon encapsulation, of accessible local energy minima corresponding to structures with large radii of gyration. For this protein, confinement hence plays more than the role of a passive cage, but rather adopts an active role, accelerating folding rates by decreasing the roughness of the energy landscape of the protein.
伴侣蛋白，比如大肠杆菌的GroE复合体，通过提供一个腔室来辅助蛋白质在非允许折叠条件下的折叠，新翻译或转运的蛋白质可以被包裹在这个腔室中。伴侣蛋白笼是在保护蛋白质不发生聚集方面起被动作用，还是在加速折叠速率方面起主动作用，仍然存在争议。在这里，我们通过分子动力学模拟研究了限制作用在伴侣蛋白介导的折叠过程中的作用。我们设计了一种具有α/β三明治折叠结构的底物蛋白，这是在GroE底物蛋白中常见的结构基序，并将其限制在一个模拟GroEL/ES腔室内的球形亲水笼中。在广泛的温度和笼半径范围内研究了折叠的热力学和动力学。由于未折叠状态相关的熵损失，限制作用被发现显著提高了塌缩温度T(c)。另一方面，折叠温度T(f)不受包裹的影响，这是该蛋白质折叠机制的结果，该机制涉及在重排到天然状态之前先初始塌缩到紧密的错误折叠状态。与本体折叠速率相比，观察到折叠速率要么加快要么减慢，这取决于模拟温度。仅在高于温度T(m)的温度下观察到由于限制作用导致的速率增强，T(m)对应于蛋白质折叠最快的温度。对于这种蛋白质，T(m)高于折叠温度T(f)，这意味着在天然状态稳定的条件下（T<T(f)），仅包裹不会导致速率增强。因此，为了使限制作用在生理条件下对折叠速率产生积极影响，蛋白质必须在其折叠最快的温度以上表现出折叠转变（T(m)<T(f)）。我们通过减少原始α/β三明治底物蛋白中的能量挫折设计了一种具有这种特性的蛋白质。修饰后的蛋白质在包裹后折叠速率加快了两倍。这种速率增强是由于折叠机制的改变，即在包裹时消除了与具有大回转半径的结构相对应的可及局部能量最小值。因此，对于这种蛋白质，限制作用不仅仅起到被动笼的作用，而是发挥了主动作用，通过降低蛋白质能量景观的粗糙度来加速折叠速率。
