Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Tokyo 113-0033, Japan.
Molecular Profiling Research Center, National Institute of Advanced Industrial Science and Technology, Aomi, Koto-ku, Tokyo 135-0064, Japan.
Q Rev Biophys. 2014 May;47(2):143-87. doi: 10.1017/S0033583514000043. Epub 2014 Apr 29.
Structural analyses of protein-protein interactions are required to reveal their functional mechanisms, and accurate protein-protein complex models, based on experimental results, are the starting points for drug development. In addition, structural information about proteins under physiologically relevant conditions is crucially important for understanding biological events. However, for proteins such as those embedded in lipid bilayers and transiently complexed with their effectors under physiological conditions, structural analyses by conventional methods are generally difficult, due to their large molecular weights and inhomogeneity. We have developed the cross-saturation (CS) method, which is an nuclear magnetic resonance measurement technique for the precise identification of the interfaces of protein-protein complexes. In addition, we have developed an extended version of the CS method, termed transferred cross-saturation (TCS), which enables the identification of the residues of protein ligands in close proximity to huge (>150 kDa) and heterogeneous complexes under fast exchange conditions (>0.1 s(-1)). Here, we discuss the outline, basic theory, and practical considerations of the CS and TCS methods. In addition, we will review the recent progress in the construction of models of protein-protein complexes, based on CS and TCS experiments, and applications of TCS to in situ analyses of biologically and medically important proteins in physiologically relevant states.
蛋白质-蛋白质相互作用的结构分析对于揭示其功能机制是必需的，并且基于实验结果的准确蛋白质-蛋白质复合物模型是药物开发的起点。此外，在生理相关条件下关于蛋白质的结构信息对于理解生物事件至关重要。然而，对于那些嵌入在脂质双层中和在生理条件下与效应物瞬时复性的蛋白质，由于其分子量较大且不均匀，传统方法的结构分析通常较为困难。我们已经开发了交叉饱和（CS）方法，这是一种用于精确鉴定蛋白质-蛋白质复合物界面的核磁共振测量技术。此外，我们还开发了 CS 方法的扩展版本，称为转移交叉饱和（TCS），它能够在快速交换条件（>0.1 s(-1))下识别接近巨大 (>150 kDa)和异质复合物的蛋白质配体的残基。在这里，我们将讨论 CS 和 TCS 方法的概述、基本理论和实际考虑因素。此外，我们将回顾基于 CS 和 TCS 实验构建蛋白质-蛋白质复合物模型的最新进展，以及 TCS 在生理相关状态下原位分析生物学和医学上重要蛋白质的应用。
