Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: Application to phospholamban

We have used synthetic lipidated peptides (“peptide‐amphiphiles”) to study the structure and function of isolated domains of integral transmembrane proteins. We used 9‐fluorenylmethyloxycarbonyl (Fmoc) solid‐phase peptide synthesis to prepare full‐length phospholamban (PLB1–52) and its cytoplasmic (PLB1–25K: phospholamban residues 1–25 plus a C‐terminal lysine), and transmembrane (PLB26–52) domains, and a 38‐residue model α‐helical sequence as a control. We created peptide‐amphiphiles by linking the C‐terminus of either the isolated cytoplasmic domain or the model peptide to a membrane‐anchoring, lipid‐like hydrocarbon tail. Circular dichroism measurements showed that the model peptide‐amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of α‐helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide‐amphiphile system would allow us to study the function and structure of the PLB1–25K cytoplasmic domain in a native‐like configuration. We compared the function (inhibition of the Ca‐ATPase in reconstituted membranes) and structure (via CD) of the PLB1–25 amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB1–25K has no effect on Ca‐ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca‐ATPase. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 283–292, 2003