Structural studies on phospholamban and implications for regulation of the Ca(2+)-ATPase.
The cardiac sarcoplasmic reticulum (SR) protein phospholamban (PLB) is an endogenous inhibitor of the SR Ca(2+)-ATPase. Phosphorylation of PLB relieves this inhibition and up-regulates calcium transport. PLB has proved remarkably difficult to study by conventional solution-state nuclear magnetic resonance (NMR) methods, due primarily to the extreme hydrophobic nature of the protein and its propensity to form pentamers. That the C-terminal domain of PLB is helical and membrane spanning is now well established; the structure of the cytoplasmic domain is relatively ill defined. In order to discern the effect of phosphorylation on the structure of the cytoplasmic domain, we have characterized a variety of model peptides in several structure-inducing and/or lipid-mimicking environments using circular dichroism and solution-state NMR. The resolution of peptide structures obtained in aqueous trifluoroethanol was markedly improved by the incorporation of 15N labels into the peptide backbone, allowing a variety of isotope edited, filtered, and resolved techniques to be applied. Molecular dynamics simulations on the full-length protein were combined with an analysis of published data to suggest a revised model for the structure of PLB.
Mortishire-Smith
R J
RJ
Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, United Kingdom. russell_mortishire-smith@merck.com
Broughton
H
H
Garsky
V M
VM
Mayer
E J
EJ
Johnson
R G
RG
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003198:Computer Simulation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012984:Software; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D010446:Peptide Fragments; D000099211:Phospholamban