Osváth Szabolcs, Herényi Levente, Závodszky Péter, Fidy Judit, Köhler Gottfried
Department of Biophysics and Radiation Biology, Semmelweis University, Puskin u. 9, H-1088 Budapest, Hungary.
J Biol Chem. 2006 Aug 25;281(34):24375-80. doi: 10.1074/jbc.M601915200. Epub 2006 Jun 28.
One of the most intriguing predictions of energy landscape models is the existence of non-exponential protein folding kinetics caused by hierarchical structures in the landscapes. Here we provide the strongest evidence so far of such hierarchy and determine the time constants and weights of the kinetic components of the suggested hierarchic energy landscape. To our knowledge, the idea of hierarchical folding energy barriers has never been tested over such a broad timescale. Refolding of yeast phosphoglycerate kinase was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 ms to 15 min. The strategy to build a model that describes folding of yeast phosphoglycerate kinase was to start from the simplest paradigm and modify it stepwise to the necessary minimal extent after repeated comparisons with the experiments. We made no a priori assumptions about the folding landscape. The result was a hierarchic finite level landscape model that quantitatively describes the refolding of yeast phosphoglycerate kinase from 1 ms to 15 min. The early steps of the folding process happen in the upper region of the landscape, where the surface has a hierarchic structure. This leads to stretched kinetics in the early phase of the folding. The lower region of the energy landscape is dominated by a trap that reflects the accumulation of molten globule intermediate state. From this intermediate, the protein can reach the global energy minimum corresponding to the native state through a cross-barrier folding step.
能量景观模型最引人入胜的预测之一是，景观中的层次结构会导致蛋白质折叠动力学呈现非指数形式。在此，我们提供了迄今为止关于这种层次结构的最有力证据，并确定了所提出的层次化能量景观中动力学成分的时间常数和权重。据我们所知，层次化折叠能垒的概念从未在如此广泛的时间尺度上得到验证。通过停流法或手动混合，从胍变性状态引发酵母磷酸甘油酸激酶的复性，并通过色氨酸荧光监测1毫秒至15分钟内的复性过程。构建一个描述酵母磷酸甘油酸激酶折叠的模型的策略是，从最简单的范式开始，在与实验反复比较后，逐步将其修改到必要的最小程度。我们没有对折叠景观做先验假设。结果得到了一个层次化有限能级景观模型，该模型定量描述了酵母磷酸甘油酸激酶在1毫秒至15分钟内的复性过程。折叠过程的早期步骤发生在景观的上部区域，该区域的表面具有层次结构。这导致折叠早期阶段出现伸展动力学。能量景观的下部区域由一个陷阱主导，该陷阱反映了熔球态中间态的积累。从这个中间态开始，蛋白质可以通过一个跨能垒折叠步骤达到对应于天然态的全局能量最小值。
