Cysteine methanethiosulfonate

Methyl methanethiosulfonate (MMTS) is a small reversible blocking agent for sulfhydryl groups (Thermo Fisher, Toronto Research). It reacts with free thiols to form a dithiomethane modification with release of sulfinic acid (Figure 1.122). The sulfinic acid component decomposes into volatile products, which don t affect the disulfide formed from the MMTS reaction Alkylthiosulfonates react rapidly with thiols under mild conditions at physiological pH. The MMTS compound is a liquid at 10.6 M concentration and is conveniently added to a reaction medium by pipette. Complete thiol modifications of available cysteine residues in proteins can... 

Stauffer, D.A., and Karlin, A. (1994) Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reaction of binding-site cysteines with charged methanethiosulfonates. Biochemistry 33, 6840-6849. 

Roberts, D. D., Lewis, S. D Ballou, D. P., Olson, S. T and Shafer, J. A. (1986) Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. Biochemistry 25, 5595-5601. 

Fig. 24. Methanethiosulfonate (MTS) reagent reacting with a thiol (sulfhy-dryl), typically of a cysteine residue in a protein, forming a disulfide bond and thus introducing the dye moiety (label)—SR.

The binding site crevice was probed by cysteine accessibility mapping, where each amino acid from TM6 was replaced by a Cys residue and tested for reactivity to methanethiosulfonate ethylammonium [38]. The comparative study accross mu, delta, and kappa receptors showed a water-accessible surface on the extracellular face of the helix for all opioid receptors and located above the Pro kink (Ile277 and Phe280 to Leu286 in the delta receptor). The data were consistent with the notion of an opioid binding pocket penetrating the upper half of the helical bundle (see Fig. 1A). 

Cysteine lodoacetamide, iodoacetate p -Hydro gmaercuribenzoate Methyl methanethiosulfonate Ellman s reagent (DTNB) N-Ethylmaleimide lodoacetamide has the potential to modify histidines and lysines... 

To date, the most commonly used spin labels for membrane proteins are pyrroline-type nitroxide radicals. Several labeling strategies and spin labels are available at present. However, the extreme specificity for the free thiol of cysteines, a stoichiometric reaction at most sites, and the relatively small size and flexibility of the modified side chain make the (l-oxyl-2,2,5,5-tetramethyl-A3-p3Troline-3-methyl) methanethiosulfonate spin label (MTSSL) the most popular choice for SDSL applications. 3 -(2-lodoacetamido)-2,2,5,5-tetramethyl-1 -pyrrolidinyloxy radical labels (lAP) are also used in combination with cysteines. The advantage of LAP vs MTSSL is that the covalent bond with the cysteine is irreversibly formed and this may be more suitable for particular situations in which the MTSSL tends to be released from the labeled sites. 

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