Cysteine-ethylation of tissue-extracted membrane proteins as a tool to detect conformational states by solid-state NMR spectroscopy. Methods Enzymol 2019;621:281-304
Date
05/28/2019Pubmed ID
31128784Pubmed Central ID
PMC7418180DOI
10.1016/bs.mie.2019.02.001Scopus ID
2-s2.0-85062440806 (requires institutional sign-in at Scopus site) 3 CitationsAbstract
Solid-state NMR (ssNMR) is an ideal tool to study structure and dynamics of membrane proteins in their native lipid environment. In principle, ssNMR has no size limitations. However, this feature is rarely exploited as large membrane proteins display severe resonance overlap. In addition, dismal yields from recombinant bacterial expression systems limit severely spectroscopic characterization of membrane proteins. For very large mammalian membrane proteins, extraction from the original organism remains the most viable approach. In this case, NMR-observable nuclei must be introduced post-translationally, but the approaches developed so far are rather scarce. Here, we detail the synthesis and engineering of a reactive 13C-ethylmethanethiosulfonate (13C-EMTS) reagent for the post-translational alkylation of cysteine sidechains of a 110kDa sarcoplasmic reticulum Ca2+-ATPase (SERCA) extracted from rabbit skeletal muscle tissue. When reconstituted into liposomes, it is possible to resolve the resonances of the engineered ethyl groups by magic-angle spinning (MAS) 2D [13C,13C]-DARR experiments. Notably, the ethyl-group modification does not perturb the function of SERCA, yielding well-resolved 13C-13C fingerprints that are used to image its structural states in the catalytic cycle and filtering out overwhelming naturally-abundant 13C nuclei signals arising from the enzyme and lipids. We anticipate that this approach will be used together with 19F NMR to monitor conformational transitions of enzymes and proteins that are difficult to express recombinantly.
Author List
Weber DK, Bader T, Larsen EK, Wang S, Gopinath T, Distefano M, 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
AnimalsCalcium-Binding Proteins
Cysteine
Humans
Isotope Labeling
Membrane Proteins
Models, Molecular
Muscle Proteins
Nuclear Magnetic Resonance, Biomolecular
Protein Conformation
Proteolipids
Sarcoplasmic Reticulum Calcium-Transporting ATPases
