Miklos Andrew C, Li Conggang, Pielak Gary J
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Methods Enzymol. 2009;466:1-18. doi: 10.1016/S0076-6879(09)66001-8. Epub 2009 Nov 13.
The biophysical properties of proteins in the crowded intracellular environment are expected to differ from those for proteins in dilute solution. Crowding can be studied in vitro through addition of polymers at high concentrations. NMR-detected amide (1)H exchange is the only technique that provides equilibrium stability data for proteins on a per-residue basis under crowded conditions. We describe the theory behind amide (1)H exchange and provide a detailed description of the experiments used to quantify globular protein stability at the residue level under crowded conditions. We also discuss the detection of weak interactions between the test protein and the crowding molecules.
在拥挤的细胞内环境中，蛋白质的生物物理特性预计会与稀溶液中的蛋白质有所不同。可以通过添加高浓度聚合物在体外研究拥挤现象。核磁共振检测的酰胺（1）H交换是唯一一种在拥挤条件下按每个残基提供蛋白质平衡稳定性数据的技术。我们描述了酰胺（1）H交换背后的理论，并详细介绍了用于在拥挤条件下在残基水平量化球状蛋白质稳定性的实验。我们还讨论了测试蛋白质与拥挤分子之间弱相互作用的检测。
