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 (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 alpha-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 alpha-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.
Lockwood
Nathan A
NA
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA.
Tu
Raymond S
RS
Zhang
Zhiwen
Z
Tirrell
Matthew V
MV
Thomas
David D
DD
Karim
Christine B
CB
D002021:Buffers; D002135:Calcium-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000737:chemistry; D008081:Liposomes; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D000099211:Phospholamban
D002021:Buffers; D002135:Calcium-Binding Proteins; D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins; D010455:Peptides; D000099211:Phospholamban