Vorup-Jensen Thomas
Biophysical Immunology Laboratory, Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus C, Denmark.
Methods Mol Biol. 2012;757:55-71. doi: 10.1007/978-1-61779-166-6_5.
Measurements on the kinetic aspects of binding between macromolecular species such as proteins have been greatly advanced by the application of surface plasmon resonance (SPR) biosensors. In studies of ligand binding by integrin I domains, technologies such as the BIAcore instruments have provided important insights into the role of conformational regulation. This chapter describes a protocol for studying the binding between the I domain from integrin α(X)β(2) and its ligand iC3b. Also included are topics on the interpretation of data. Integrin I domains appear to support heterogeneous interactions with ligands, which pose significant challenges in deriving valid information on the binding kinetics from the SPR measurements. Fortunately, new algorithms are available that may resolve even complex ligand-binding reactions; with the application to data on the binding between the α(X) I domain, a more consistent and unambiguous result is obtained compared to those obtained by classical approaches for analyzing SPR biosensor data.
表面等离子体共振（SPR）生物传感器的应用极大地推动了对蛋白质等大分子物种之间结合动力学方面的测量。在整合素I结构域的配体结合研究中，诸如BIAcore仪器等技术为构象调节的作用提供了重要见解。本章描述了一种研究整合素α(X)β(2)的I结构域与其配体iC3b之间结合的实验方案。还包括数据解读的相关主题。整合素I结构域似乎支持与配体的异质相互作用，这在从SPR测量中获取有关结合动力学的有效信息方面带来了重大挑战。幸运的是，有可用的新算法，甚至可以解析复杂的配体结合反应；将其应用于α(X) I结构域结合的数据时，与通过经典方法分析SPR生物传感器数据所获得的结果相比，能得到更一致且明确的结果。
