title: Ca(2+) ATPase Conformational Transitions in Lipid Bilayers Mapped by Site-directed Ethylation and Solid-State NMR.
abstract: To transmit signals across cellular compartments, many membrane-embedded enzymes undergo extensive conformational rearrangements. Monitoring these events in lipid bilayers by NMR at atomic resolution has been challenging due to the large size of these systems. It is further exacerbated for large mammalian proteins that are difficult to express and label with NMR-active isotopes. Here, we synthesized and engineered (13)C ethyl groups on native cysteines to map the structural transitions of the sarcoplasmic reticulum Ca(2+)-ATPase, a 110 kDa transmembrane enzyme that transports Ca(2+) into the sarcoplasmic reticulum. Using magic angle spinning NMR, we monitored the chemical shifts of the methylene and methyl groups of the derivatized cysteine residues along the major steps of the enzymatic cycle. The methylene chemical shifts are sensitive to the ATPase conformational changes induced upon nucleotide and Ca(2+) ion binding and are ideal probes for active and inactive states of the enzyme. This new approach is extendable to large mammalian enzymes and signaling proteins with native or engineered cysteine residues in their amino acid sequence.
authors.affiliation: Department of Biochemistry, Biophysics & Molecular Biology, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Vitaly V
authors.initials: VV
authors.lastname: Vostrikov
authors.affiliation: Department of Biochemistry, Biophysics & Molecular Biology, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Martin
authors.initials: M
authors.lastname: Gustavsson
authors.affiliation: Department of Biochemistry, Biophysics & Molecular Biology, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Tata
authors.initials: T
authors.lastname: Gopinath
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Dan
authors.initials: D
authors.lastname: Mullen
authors.affiliation: Department of Biochemistry, Biophysics & Molecular Biology, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Alysha A
authors.initials: AA
authors.lastname: Dicke
authors.affiliation: Department of Biochemistry, Biophysics & Molecular Biology, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Vincent
authors.initials: V
authors.lastname: Truong
authors.affiliation: Department of Biochemistry, Biophysics & Molecular Biology, University of Minnesota , Minneapolis, Minnesota 55455, United States.|Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Veglia
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003545:Cysteine / Q000032:analysis; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D003545:Cysteine; D002118:Calcium