Arkin I T, Adams P D, Brünger A T, Aimoto S, Engelman D M, Smith S O
Department of Cell Biology, Yale University, New Haven, CT 06520, USA.
J Membr Biol. 1997 Feb 1;155(3):199-206. doi: 10.1007/s002329900172.
Phospholamban, a 52-residue membrane protein, associates to form a pentameric complex of five long alpha-helices traversing the sarcoplasmic reticulum membrane of cardiac muscle cells. The transmembrane domain of the protein is largely hydrophobic, with only three cysteine residues having polar side chains, yet it functions as a Ca2+-selective ion channel. In this report, infrared spectroscopy is used to probe the conformation of the three cysteine side chains and to establish whether the free S-H groups form intrahelical hydrogen bonds in the pentameric complex. Vibrational spectra of a transmembrane peptide were obtained which corresponded to the transmembrane domain of wild-type phospholamban and three peptides each containing a cysteine --> alanine substitution. The observed S-H frequencies argue that each of the sulfhydryl groups is hydrogen-bonded to an i-4 backbone carbonyl oxygen. Electrostatic calculations on a model of phospholamban based on molecular dynamics and mutagenesis studies, show that the sulfhydryl groups may significantly contribute to the electrostatic potential field of the protein.
受磷蛋白是一种由52个氨基酸残基组成的膜蛋白，它结合形成一个由五个长α螺旋组成的五聚体复合物，这些螺旋贯穿心肌细胞的肌浆网膜。该蛋白的跨膜结构域主要是疏水的，只有三个半胱氨酸残基具有极性侧链，但它却起着Ca2+选择性离子通道的作用。在本报告中，红外光谱被用于探测这三个半胱氨酸侧链的构象，并确定游离的S-H基团在五聚体复合物中是否形成螺旋内氢键。获得了一个跨膜肽的振动光谱，它对应于野生型受磷蛋白的跨膜结构域以及三个分别含有半胱氨酸→丙氨酸取代的肽。观察到的S-H频率表明，每个巯基都与一个i-4主链羰基氧形成了氢键。基于分子动力学和诱变研究对受磷蛋白模型进行的静电计算表明，巯基可能对该蛋白的静电势场有显著贡献。
