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Structure and function of integral membrane protein domains resolved by peptide‐amphiphiles: Application to phospholamban
Author(s) -
Lockwood Nathan A.,
Tu Raymond S.,
Zhang Zhiwen,
Tirrell Matthew V.,
Thomas David D.,
Karim Christine B.
Publication year - 2003
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.10365
Subject(s) - phospholamban , chemistry , peptide , transmembrane domain , transmembrane protein , amphiphile , circular dichroism , protein secondary structure , cytoplasm , biophysics , membrane , biochemistry , endoplasmic reticulum , receptor , biology , organic chemistry , copolymer , polymer
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 (PLB 1–52 ) and its cytoplasmic (PLB 1–25 K: phospholamban residues 1–25 plus a C‐terminal lysine), and transmembrane (PLB 26–52 ) 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 PLB 1–25 K 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 PLB 1–25 amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB 1–25 K 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