Adey N B, Kay B K
Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280, USA.
Biochem J. 1997 Jun 1;324 ( Pt 2)(Pt 2):523-8. doi: 10.1042/bj3240523.
Peptides isolated from combinatorial libraries typically interact with, and thus help to characterize, biologically relevant binding domains of target proteins. To characterize the binding domains of the focal adhesion protein vinculin, vinculin-binding peptides were isolated from two phage-displayed random peptide libraries. Altogether, five non-similar vinculin-binding peptides were identified. Despite the lack of obvious sequence similarity between the peptides, binding and competition studies indicated that all five interact with the talin-binding domain of vinculin and do not disrupt the binding of alpha-actinin or paxillin to vinculin. The identified peptides and talin bind to vinculin in a comparable manner; both bind to immobilized vinculin, but neither binds to soluble vinculin unless the C-terminus of vinculin has been deleted. An analysis of amino acid variants of one of the peptides has revealed three non-contiguous motifs that also occur in the region of talin previously demonstrated to bind vinculin. Amino acid substitutions within a 127-residue segment of talin capable of binding vinculin confirmed the importance of two of the motifs and suggest that residues critical for binding are within a 16-residue region. This study demonstrates that the vinculin-binding peptides interact with vinculin in a biologically relevant manner and represent an excellent tool for further study of the biochemistry of vinculin.
从组合文库中分离出的肽通常与靶蛋白的生物学相关结合域相互作用，从而有助于对其进行表征。为了表征粘着斑蛋白纽蛋白的结合域，从两个噬菌体展示随机肽文库中分离出纽蛋白结合肽。总共鉴定出五个不相似的纽蛋白结合肽。尽管这些肽之间缺乏明显的序列相似性，但结合和竞争研究表明，所有这五个肽都与纽蛋白的踝蛋白结合域相互作用，并且不会破坏α-辅肌动蛋白或桩蛋白与纽蛋白的结合。所鉴定的肽和踝蛋白以类似的方式与纽蛋白结合；两者都与固定化的纽蛋白结合，但除非纽蛋白的C末端已被删除，否则两者都不与可溶性纽蛋白结合。对其中一个肽的氨基酸变体的分析揭示了三个不连续的基序，这些基序也出现在先前已证明与纽蛋白结合的踝蛋白区域中。能够结合纽蛋白的踝蛋白127个残基片段内的氨基酸取代证实了其中两个基序的重要性，并表明对结合至关重要的残基位于一个16个残基的区域内。这项研究表明，纽蛋白结合肽以生物学相关的方式与纽蛋白相互作用，是进一步研究纽蛋白生物化学的极佳工具。
