McMahon Heather A, Alfieri Katherine N, Clark Katherine A A, Londergan Casey H
Department of Chemistry, Haverford College, 370 Lancaster Avenue, Haverford, Pennsylvania 19041.
J Phys Chem Lett. 2010 Mar 4;1(5):850-855. doi: 10.1021/jz1000177. Epub 2010 Feb 12.
Cyanylated cysteine, or beta-thiocyanatoalanine, is an artificial amino acid that can be introduced into peptides and proteins by post-translational chemical modification of solvent-exposed cysteine side chains, and thus it can be used in any protein with a suitable expression and mutagenesis system. In this study, cyanylated cysteine is introduced at selected sites in two model peptides that have been shown to bind to membrane interfaces: a membrane-binding sequence of the human myelin basic protein and the antimicrobial peptide CM15. Far-UV circular dichroism indicates that the secondary structures of the bound peptides are not influenced by introduction of the artificial side chain. Infrared spectra of both systems in buffer and exposed to dodecylphosphocholine micelles indicate that the CN stretching absorption band of cyanylated cysteine can clearly distinguish between membrane burial and solvent exposure of the artificial side chain. Since infrared spectroscopy can be applied in a wide variety of lipid systems, and since cyanylated cysteine can be introduced into proteins of arbitrary size via mutagenesis and post-translational modification, this new probe could see wide use in characterizing the protein-lipid interactions of membrane proteins.
氰基化半胱氨酸，即β-硫氰酸丙氨酸，是一种人工合成氨基酸，可通过对暴露于溶剂中的半胱氨酸侧链进行翻译后化学修饰引入到肽和蛋白质中，因此可用于任何具有合适表达和诱变系统的蛋白质。在本研究中，氰基化半胱氨酸被引入到已证明能与膜界面结合的两种模型肽的选定位点：人髓鞘碱性蛋白的膜结合序列和抗菌肽CM15。远紫外圆二色性表明，结合肽的二级结构不受人工侧链引入的影响。两种体系在缓冲液中以及暴露于十二烷基磷酸胆碱胶束中的红外光谱表明，氰基化半胱氨酸的CN伸缩吸收带能够清晰区分人工侧链的膜埋入和溶剂暴露情况。由于红外光谱可应用于多种脂质体系，且氰基化半胱氨酸可通过诱变和翻译后修饰引入任意大小的蛋白质中，这种新型探针在表征膜蛋白的蛋白质-脂质相互作用方面可能会得到广泛应用。
