Wavreille Anne-Sophie, Garaud Mathieu, Zhang Yanyan, Pei Dehua
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA.
Methods. 2007 Jul;42(3):207-19. doi: 10.1016/j.ymeth.2007.02.010.
Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing short phosphotyrosyl (pY) peptide motifs in their partner proteins. Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of pY proteins, counteracting the protein tyrosine kinases. Both types of proteins exhibit primary sequence specificity, which plays at least a partial role in dictating their physiological interacting partners or substrates. A combinatorial peptide library method has been developed to systematically assess the sequence specificity of SH2 domains and PTPs. A "one-bead-one-compound" pY peptide library is synthesized on 90-microm TentaGel beads and screened against an SH2 domain or PTP of interest for binding or catalysis. The beads that carry the tightest binding sequences against the SH2 domain or the most efficient substrates of the PTP are selected by an enzyme-linked assay and individually sequenced by a partial Edman degradation/mass spectrometry technique. The combinatorial method has been applied to determine the sequence specificity of 8 SH2 domains from Src and Csk kinases, adaptor protein Grb2, and phosphatases SHP-1, SHP-2, and SHIP1 and a prototypical PTP, PTP1B.
Src同源2（SH2）结构域通过识别其伴侣蛋白中的短磷酸酪氨酸（pY）肽基序来介导蛋白质-蛋白质相互作用。蛋白质酪氨酸磷酸酶（PTP）催化pY蛋白的去磷酸化，与蛋白质酪氨酸激酶起拮抗作用。这两种类型的蛋白质都表现出一级序列特异性，这在决定它们的生理相互作用伴侣或底物方面至少起部分作用。已经开发出一种组合肽库方法来系统地评估SH2结构域和PTP的序列特异性。在90微米的TentaGel珠上合成“一珠一化合物”pY肽库，并针对感兴趣的SH2结构域或PTP进行结合或催化筛选。通过酶联测定法选择对SH2结构域具有最紧密结合序列或对PTP具有最有效底物的珠子，并通过部分埃德曼降解/质谱技术对其进行单独测序。该组合方法已用于确定来自Src和Csk激酶、衔接蛋白Grb2以及磷酸酶SHP-1、SHP-2和SHIP1的8个SH2结构域以及典型PTP即PTP1B的序列特异性。
