Abstract：
Fluorescence polarization (FP) assays can be used to identify small-molecule inhibitors that bind to SH2 domain-containing proteins. We have developed FP assays by which to identify inhibitors of the SH2 domains of the two closely-related transcription factors STAT5a and STAT5b. Point mutation of selected amino acids in the putative binding site of the protein is a valuable tool by which to gain insight into the molecular mechanism of binding. In this chapter, we describe the cloning and application of point mutant proteins in order to transfer the binding preference of selected SH2 domain-binding STAT5b inhibitors to STAT5a, with results that highlight the importance of considering a role for residues outside the SH2 domain in contributing to the binding interactions of SH2 domain inhibitors.
摘要：
荧光偏振(FP)测定可用于鉴定与含SH2结构域的蛋白质结合的小分子抑制剂。我们已经开发了FP测定法,通过其鉴定两个密切相关的转录因子STAT5a和STAT5b的SH2结构域的抑制剂。在蛋白质的推定结合位点中选择的氨基酸的点突变是获得对结合的分子机制的洞察的有价值的工具。在这一章中,我们描述了点突变蛋白的克隆和应用，以便将选定的SH2结构域结合STAT5b抑制剂的结合偏好转移到STAT5a，结果突出了考虑SH2结构域外的残基在促进SH2结构域抑制剂的结合相互作用中的作用的重要性。
