Chiti F, Taddei N, van Nuland N A, Magherini F, Stefani M, Ramponi G, Dobson C M
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QT, UK.
J Mol Biol. 1998 Nov 6;283(4):893-903. doi: 10.1006/jmbi.1998.2010.
The transition state for folding of a small protein, muscle acylphosphatase, has been studied by measuring the rates of folding and unfolding under a variety of solvent conditions. A strong dependence of the folding rate on the concentration of urea suggests the occurrence in the transition state of a large shielding of those groups that are exposed to interaction with the denaturant in the unfolded state (mainly hydrophobic moieties and groups located on the polypeptide backbone). The heat capacity change upon moving from the unfolded state to the transition state is small and is indicative of a substantial solvent exposure of hydrophobic groups. The solvent-accessibility of such groups in the transition state has also been found to be significant by measuring the rates of folding and unfolding in the presence of sugars. These rates have also been found to be accelerated by the addition of small quantities of alcohols. Trifluoroethanol and hexafluoroisopropanol were particularly effective, suggesting that stabilisation of local hydrogen bonds lowers the energy of the transition state relative to the folded and unfolded states. Finally, a study with a competitive inhibitor of acylphosphatase has provided evidence for the complete loss of ligand binding affinity in the transition state, indicating that specific long-range interactions at the level of the active site are not yet formed at this stage of the folding reaction. A model of the transition state for acylphosphatase folding, in which beta-turns and one or both alpha-helices are formed to a significant extent but in which the persistent long-range interactions characteristic of the folded state are largely absent, accounts for all our data. These results are broadly consistent with models of the transition states for folding of other small proteins derived from mutagenesis studies, and suggest that solvent perturbation methods can provide complementary information about the transition region of the energy surfaces for protein folding.
通过测量在各种溶剂条件下的折叠和去折叠速率，对一种小蛋白质——肌肉酰基磷酸酶的折叠过渡态进行了研究。折叠速率对尿素浓度有强烈依赖性，这表明在过渡态中，那些在未折叠状态下暴露于与变性剂相互作用的基团（主要是疏水部分和位于多肽主链上的基团）有大量屏蔽。从未折叠状态转变到过渡态时的热容变化很小，这表明疏水基团有大量溶剂暴露。通过测量在糖存在下的折叠和去折叠速率，还发现这些基团在过渡态中的溶剂可及性也很显著。还发现添加少量醇会加速这些速率。三氟乙醇和六氟异丙醇特别有效，这表明局部氢键的稳定降低了过渡态相对于折叠态和未折叠态的能量。最后，一项关于酰基磷酸酶竞争性抑制剂的研究提供了证据，表明在过渡态中配体结合亲和力完全丧失，这表明在折叠反应的这个阶段，活性位点水平的特定长程相互作用尚未形成。酰基磷酸酶折叠过渡态的模型，其中β-转角和一个或两个α-螺旋在很大程度上形成，但折叠态特有的持久长程相互作用在很大程度上不存在，解释了我们所有的数据。这些结果与来自诱变研究的其他小蛋白质折叠过渡态模型大致一致，并表明溶剂扰动方法可以提供关于蛋白质折叠能量表面过渡区域的补充信息。
