Chapter Sixteen - Cysteine-ethylation of tissue-extracted membrane proteins as a tool to detect conformational states by solid-state NMR spectroscopy
Introduction
Solid-state NMR (ssNMR) is a powerful technique for investigating structure and dynamics of membrane proteins, which unlike solution NMR, does not rely on perturbing detergents for their functional reconstitution (Chipot et al., 2018). With no upper size limit, ssNMR is accessible to the largest membrane proteins embedded in their native lipid-bilayer environment, but poorer resolution, combined with unique signal arising from hundreds to thousands of amino acids, produce spectra far too overlapped for meaningful interpretation. Furthermore, large membrane proteins may be extraordinarily difficult to obtain by heterologous expression from Escherichia coli, or other common hosts, in a correctly folded state and in milligram quantities required for ssNMR. Although a few successful attempts have been reported for cell-free expression and yeast expression systems, the extraction from the host organism often remains the only viable option. In such cases, sparse, selective and segmental labeling methods built on recombinant expression to reduce spectral overlap (Göbl, Madl, Simon, & Sattler, 2014; Iwai & Züger, 2007; Larsen et al., 2018; Prestegard et al., 2014; Verardi, Traaseth, Masterson, Vostrikov, & Veglia, 2012; Walters et al., 2001; Xu, Ayers, Cowburn, & Muir, 1999) are inapplicable and NMR-observable nuclei must be introduced post-translationally. Fortunately, there is an extensive toolbox for post-translational modification of proteins, exploiting the chemistry of residues such as cysteine, lysine, tyrosine, arginine, glutamate, aspartate, serine, threonine, methionine, histidine, tryptophan as well as N- and C-termini (Boutureira & Bernardes, 2015; Debelouchina & Muir, 2017). However, only a few to date have been utilized by the ssNMR community.
In this chapter, we detail protocols for the synthesis of a 13C-ethylmethanethiosulfonate (13C-EMTS) reagent (Vostrikov et al., 2016) and its use for ethylating cysteines of a 110 kDa sarcoplasmic reticulum Ca2 +-ATPase (SERCA) transmembrane enzyme isolated from rabbit skeletal muscle tissue. The 13C-EMTS reagent is analogous to the methylating reagent methylmethanethiosulfonate (13C-MMTS) introduced by Kay and co-workers (Religa, Ruschak, Rosenzweig, & Kay, 2011), but does not require deuterated recombinant proteins to increase signal intensity. Importantly, this approach eliminates the background from lipids and naturally-abundant 13C-nuclei that plague 1H-13C correlation spectra. Instead, the 13C-13C dipolar coupling of ethyl-tagged cysteines (Cys-S-13CH2-13CH3) provides a double-quantum filter previously demonstrated to yield background-free and well-resolved solid-state 13C-13C Dipolar-Assisted Rotational Resonance (DARR) experiments (Takegoshi, Nakamura, & Terao, 2001; Vostrikov et al., 2016). This reagent reacts with cysteine (Fig. 1A) by well-known chemistry between nucleophilic thiols and alkyl alkanethiosulfonates, which is appealing for its applicability under mild reaction conditions, i.e., aqueous solutions and physiological pH. EMTS reagent enables fast reaction times, stoichiometric conversion of cysteine without requiring excess reagent, and reversibility upon addition of common thiol reagents such as dithiothreitol and β-mercaptoethanol (Kenyon & Bruice, 1977). More importantly, it does not perturb the enzyme's function.
SERCA has 24 cysteines, of which 6 solvent-exposed residues are preferentially labeled throughout both the cytoplasmic nucleotide-binding (N) and phosphorylation (P) domains (Fig. 1B). When using a fivefold molar excess of 13C-EMTS, the ATPase activity is fully retained (Vostrikov et al., 2016) and the 13C-13C DARR spectra give compelling fingerprints of the different structural states throughout the enzymatic cycle (Toyoshima, 2008).
Section snippets
Synthesis and purification of the 13C-EMTS reagent
The synthesis of 13C-EMTS is described at 9.2 mmol scale, which accounting for a total yield of 75%, produces 35 mL of 200 mM stock and enough for over 18,000 NMR sample preparations using the protocol described in the following section. The scale was chosen to match the quantities of 13C2-bromoethane available commercially, but can be significantly reduced. In our hands, we have successfully used this scheme at 0.7, 2, and 3.5 mmol scales to produce sufficient amounts for ssNMR studies. 13C-EMTS
Hardware requirements
To further demonstrate the use of 13C-EMTS labeling, we acquired MAS ssNMR spectra of tissue-extracted SERCA in the calcium-bound E1 state (E1·2Ca2 +) prepared according to Section 2. Detection of 13C-ethylated protein is achievable without the need of highly-specialized ssNMR equipment. For example, all spectra presented in this review were acquired on a simple setup including a 700 MHz Varian spectrometer equipped with a low-E Black Fox 3.2 mm HXY MAS probe (designed by Peter Gor'kov, National
Conclusions and perspectives
In this chapter, we reported the protocols for utilizing 13C-EMTS to post-translationally introduce 13C-ethyl tags to the 110 kDa SERCA Ca2 +-pump, extracted from the skeletal muscle of rabbits, for ssNMR characterization in lipid membranes. Similar post-translational methods based on 13C-methyl labeling have enabled solution NMR characterization of large systems including the 180 kDa α7 ring of the 20S proteasome (Religa et al., 2011), the 300 kDa ClpP protease (Religa et al., 2011), the 100 kDa
Acknowledgment
This work was supported by the National Institute of Health (GM 64742 and HL 144130).
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