Walters Jad, Milam Sara L, Clark A Clay
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina, USA.
Methods Enzymol. 2009;455:1-39. doi: 10.1016/S0076-6879(08)04201-8.
We describe here the use of several spectroscopies, such as fluorescence emission, circular dichroism, and differential quenching by acrylamide, in examining the equilibrium and kinetic folding of proteins. The first section regarding equilibrium techniques provides practical information for determining the conformational stability of a protein. In addition, several equilibrium-folding models are discussed, from two-state monomer to four-state homodimer, providing a comprehensive protocol for interpretation of folding curves. The second section focuses on the experimental design and interpretation of kinetic data, such as burst-phase analysis and exponential fits, used in elucidating kinetic folding pathways. In addition, simulation programs are used routinely to support folding models generated by kinetic experiments, and the fundamentals of simulations are covered.
我们在此描述了几种光谱学方法的应用，如荧光发射、圆二色性以及丙烯酰胺的差异猝灭，用于研究蛋白质的平衡折叠和动力学折叠。关于平衡技术的第一部分提供了测定蛋白质构象稳定性的实用信息。此外，还讨论了几种平衡折叠模型，从两态单体到四态同二聚体，为解释折叠曲线提供了全面的方案。第二部分重点关注用于阐明动力学折叠途径的动力学数据的实验设计和解释，如爆发相分析和指数拟合。此外，常规使用模拟程序来支持动力学实验产生的折叠模型，并涵盖了模拟的基本原理。
