Becker Jeffrey M, Naider Fred
Microbiology Department, University of Tennessee.
Chemistry Department, College of Staten Island, City University of New York.
Methods Enzymol. 2015;556:527-47. doi: 10.1016/bs.mie.2014.12.001. Epub 2015 Mar 20.
Experiments are described that allowed cross-linking of analogs of a 13-amino acid peptide into the binding site of a model G protein-coupled receptor. Syntheses of peptide analogs that were used for photochemical or chemical cross-linking were carried out using solid-phase peptide synthesis. Chemical cross-linking utilized 3,4-dihydroxy-l-phenylalanine-incorporated peptides and subsequent periodate-mediated activation, whereas photochemical cross-linking was mediated by p-benzoyl-l-phenylalanine (Bpa)-labeled peptides and UV-initiated activation. Mass spectrometry was employed to locate the site(s) in the receptor that formed the cross-links to the ligand. We also describe a method called unnatural amino acid replacement that allowed capture of a peptide ligand into the receptor. In this method, the receptor was genetically modified by replacement of a natural amino acid with Bpa. The modified receptor was UV-irradiated to capture the ligand. The approaches described are applicable to other peptide-binding proteins and can reveal the ligand-binding site in atomic detail.
本文描述了一些实验，这些实验能够将一种13个氨基酸的肽类似物交联到一种模型G蛋白偶联受体的结合位点。用于光化学或化学交联的肽类似物的合成是通过固相肽合成进行的。化学交联利用了掺入3,4-二羟基-L-苯丙氨酸的肽以及随后的高碘酸盐介导的活化，而光化学交联则由对苯甲酰-L-苯丙氨酸（Bpa）标记的肽和紫外线引发的活化介导。采用质谱法来定位受体中与配体形成交联的位点。我们还描述了一种称为非天然氨基酸置换的方法，该方法能够将肽配体捕获到受体中。在这种方法中，通过用Bpa替换天然氨基酸对受体进行基因改造。对改造后的受体进行紫外线照射以捕获配体。所描述的方法适用于其他肽结合蛋白，并且能够在原子细节上揭示配体结合位点。
