title: Co-reconstitution and co-crystallization of phospholamban and Ca(2+)-ATPase.
abstract: Significant advances have recently been made in understanding the regulation of Ca(2+)-ATPase by phospholamban and in modeling their structures. However, these insights would be furthered by determining the 3-D structure of both proteins within the membrane, thus revealing the structural basis for their interaction. To this end, we have developed methods for reconstituting purified Ca(2+)-ATPase with recombinant phospholamban. After reconstitution at high lipid-to-protein ratios, we have verified their functional association by measuring calcium transport and ATPase activity. Furthermore, we have grown co-crystals after reconstitution at low lipid-to-protein ratios. The structure of Ca(2+)-ATPase has recently been solved by cryoelectron microscopy at 8-A resolution, thus revealing transmembrane alpha-helices. Using a variety of constraints, we have associated these helices with the predicted transmembrane sequences to produce a detailed model for the packing of transmembrane helices. Structure determination of the co-crystals is currently underway, which we hope will eventually reveal the interaction of phospholamban with Ca(2+)-ATPase at a similar level of detail.
authors.affiliation: Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York 10012, USA.
authors.forename: H S
authors.initials: HS
authors.lastname: Young
authors.forename: L G
authors.initials: LG
authors.lastname: Reddy
authors.forename: L R
authors.initials: LR
authors.lastname: Jones
authors.forename: D L
authors.initials: DL
authors.lastname: Stokes
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D003460:Crystallization; D007700:Kinetics; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D011994:Recombinant Proteins; D000099211:Phospholamban