Site-directed methyl group labeling as an NMR probe of structure and dynamics in supramolecular protein systems: Applications to the proteasome and to the ClpP protease

Methyl groups are powerful reporters of structure, motion, and function in NMR studies of supramolecular protein assemblies. Their utility can be hindered, however, by spectral overlap, difficulties with assignment or lack of probes in biologically important regions of the molecule studied. Here we show that ¹³CH₃-S- labeling of Cys side chains using ¹³C-methyl-methanethiosulfonate (¹³C-MMTS) (IUPAC name: methylsulfonylsulfanylmethane) provides a convenient probe of molecular structure and dynamics. The methodology is demonstrated with an application focusing on the gating residues of the Thermoplasma acidophilum proteasome, where it is shown that the ¹³CH₃-S- label reports faithfully on the conformational heterogeneity and dynamics in this region of the complex. A second and related application involves labeling with ¹³C-MMTS at the N-termini of the subunits comprising the E. coli ClpP protease that reveals multiple conformations of gating residues in this complex as well. These N-terminal residues adopt a single conformation upon gate opening.