Structure and orientation of sarcolipin in lipid environments.
Sarcolipin (SLN) is a 31 amino acid integral membrane protein that regulates Ca-ATPase activity in skeletal muscle. Here, we report the three-dimensional structure and topology of synthetic SLN in lipid environments, as determined by solution and solid-state NMR spectroscopy. 2D solution NMR experiments were performed on SLN solubilized in sodium dodecyl sulfate (SDS) micelles. We found that SLN adopts a highly defined alpha-helical conformation from F9 through R27, with a backbone RMSD of 0.65 A and a side chain RMSD of 1.66 A. The N-terminus (M1 through L8) and the C-terminus (S28 through Y31) are mostly unstructured. The orientation of the SLN was determined using one-dimensional (15)N NMR solid-state spectroscopy. The protein was incorporated into phospholipid bilayers prepared from a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. The (15)N chemical shift solid-state spectra from selectively labeled SLN samples indicate that SLN orients perpendicularly to the plane of the membrane bilayers. These results support the proposed mechanism of Ca-ATPase regulation of SLN via protein-protein intramembranous interactions between the highly conserved transmembrane domains of the Ca-ATPase and the conserved transmembrane domain of SLN.
Mascioni
Alessandro
A
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Karim
Christine
C
Barany
George
G
Thomas
David D
DD
Veglia
Gianluigi
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D004983:Ethanolamines / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010767:Phosphorylcholine / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013053:Spectrophotometry
D000596:Amino Acids; D004983:Ethanolamines; D008051:Lipid Bilayers; D008055:Lipids; D008823:Micelles; D009124:Muscle Proteins; D010455:Peptides; D011510:Proteolipids; D010767:Phosphorylcholine; C077258:sarcolipin; D012967:Sodium Dodecyl Sulfate; C005448:phosphorylethanolamine; D000252:Calcium-Transporting ATPases; D002118:Calcium