Morris Elizabeth R, Searle Mark S
Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, Nottingham, United Kingdom.
Curr Protoc Protein Sci. 2012 Apr;Chapter 28:28.2.1-28.2.22. doi: 10.1002/0471140864.ps2802s68.
We present an overview of the current experimental and theoretical approaches to studying protein folding mechanisms, set against current models of the folding energy landscape. We describe how stability and folding kinetics can be determined experimentally and how this data can be interpreted in terms of the characteristic features of various models from the simplest two-state pathway to a multi-state mechanism. We summarize the pros and cons of a range of spectroscopic methods for measuring folding rates and present a theoretical framework, coupled with protein engineering approaches, for elucidating folding mechanisms and structural features of folding transition states. A series of case studies are used to show how experimental kinetic data can be interpreted in the context of non-native interactions, populated intermediates, parallel folding pathways, and sequential transition states. We also show how computational methods now allow transient species of high energy, such as folding transition states, to be modeled on the basis of experimental Φ-value analysis derived from the effects of point mutations on folding kinetics.
我们概述了当前研究蛋白质折叠机制的实验和理论方法，并对照当前的折叠能量景观模型进行阐述。我们描述了如何通过实验确定稳定性和折叠动力学，以及如何根据从最简单的两态途径到多态机制的各种模型的特征来解释这些数据。我们总结了一系列用于测量折叠速率的光谱方法的优缺点，并提出了一个理论框架，结合蛋白质工程方法，以阐明折叠机制和折叠过渡态的结构特征。通过一系列案例研究，展示了如何在非天然相互作用、大量中间体、平行折叠途径和连续过渡态的背景下解释实验动力学数据。我们还展示了计算方法如何基于从点突变对折叠动力学的影响得出的实验Φ值分析，对高能量的瞬态物种（如折叠过渡态）进行建模。
