STRUCTURE OF THE TRANSMEMBRANE CYSTEINE RESIDUES IN PHOSPHOLAMBAN

Phospholamban, a 52-residue membrane protein, associates to form a pen tameric 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 residu es having polar side chains, yet it functions as a Ca2+-selective ion channel. In this report, infrared spectroscopy is used to probe the co nformation 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 phosphola mban and three peptides each containing a cysteine double right arrow alanine substitution. The observed S-H frequencies argue that each of the sulfhydryl groups is hydrogen-bonded to an i-4 backbone carbonyl o xygen. 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 fie ld of the protein.