Role of cysteine residues in structural stability and function of a transmembrane helix bundle

To study the structural and functional roles of the cysteine residues at po sitions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synt hesis to prepare alpha -amino-n-butyric acid (Abu)-PLB, the analogue in whi ch all three cysteine residues are replaced by Abu. Whereas previous studie s have shown that replacement of the three Cys residues by Ala (producing A la-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (fro m SDS-polyacrylamide gel electrophoresis) and function (inhibition of the C a-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wil d-type PLB and Ala-PLB. Molecular modeling provides structural and energeti c insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer for mation or inhibitory function; 2) the steric properties of cysteine residue s in the PLB transmembrane domain contribute substantially to pentameric st ability, whereas the polar or chemical properties of the sulfhydryl group p lay only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-termi nal methionine has neither a functional nor a structural effect in full-len gth PLB.