Cysteine-ethylation of tissue-extracted membrane proteins as a tool to detect conformational states by solid-state NMR spectroscopy

Abstract

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.

Keywords: Conformational states; Cysteine ethylation; Isotopic labeling; Mammalian proteins; Phospholamban; SERCA; Sarcolipin; Solid-state NMR.