Ca(2+) ATPase Conformational Transitions in Lipid Bilayers Mapped by Site-directed Ethylation and Solid-State NMR. ACS Chem Biol 2016 Feb 19;11(2):329-34
Date
12/10/2015Pubmed ID
26650884Pubmed Central ID
PMC4993155DOI
10.1021/acschembio.5b00953Scopus ID
2-s2.0-84959343457 (requires institutional sign-in at Scopus site) 5 CitationsAbstract
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.
Author List
Vostrikov VV, Gustavsson M, Gopinath T, Mullen D, Dicke AA, Truong V, Veglia GAuthor
Gopinath Tata PhD Assistant Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsBinding Sites
Calcium
Cysteine
Lipid Bilayers
Models, Molecular
Nuclear Magnetic Resonance, Biomolecular
Protein Conformation
Rabbits
Sarcoplasmic Reticulum Calcium-Transporting ATPases
