A solid-state NMR study of the phospholamban transmembrane domain: local structure and interactions with Ca(2+)-ATPase.
The structure and dynamics of a double (13)C-labelled 24-residue synthetic peptide ([(13)C(2)]CAPLB(29-52)), corresponding to the membrane-spanning sequence of phospholamban (PLB), were examined using (13)C cross-polarisation magic-angle spinning (CP-MAS) NMR spectroscopy. CP-MAS spectra of [(13)C(2)]CAPLB(29-52) reconstituted into unsaturated lipid membranes indicated that the peptide was mobile at temperatures down to -50 degrees C. The NMR spectra showed that peptide motion became constrained in the presence of the SERCA1 isoform of Ca(2+)-ATPase, and chemical cross-linking experiments indicated that [(13)C(2)]CAPLB(29-52) and Ca(2+)-ATPase came into close contact with one another. These results together suggested that the peptide and the 110-kDa calcium pump were interacting in the membrane. Rotational resonance CP-MAS (13)C-(13)C distance measurements on [(13)C(2)]CAPLB(29-52) reconstituted into lipid bilayers confirmed that the sequence spanning Phe-32 and Ala-36 was alpha-helical, and that this structure was not disrupted by interaction with Ca(2+)-ATPase. These results support the finding that the transmembrane domain of PLB is partially responsible for regulation of Ca(2+) transport through interactions with cardiac muscle Ca(2+)-ATPase in the lipid bilayer, and also demonstrate the feasibility of performing structural measurements on PLB peptides when bound to their physiological target.
Ahmed
Z
Z
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Reid
D G
DG
Watts
A
A
Middleton
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D002942:Circular Dichroism; D003593:Cytoplasm / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013696:Temperature; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D008051:Lipid Bilayers; D010455:Peptides; D000099211:Phospholamban