A simple strategy for ¹³C, ¹H labeling at the Ile-γ2 methyl position in highly deuterated proteins

A straightforward approach for the production of highly deuterated proteins labeled with ¹³C and ¹H at Ile-γ2 methyl positions is described. The utility of the methodology is illustrated with an application involving the half proteasome (360 kDa). High quality 2D Ile ¹³C(γ)²,¹H(γ)² HMQC data sets, exploiting the methyl-TROSY principle, are recorded with excellent sensitivity and resolution, that compare favorably with Ile ¹³C(δ)¹,¹H(δ)¹ spectra. This labeling scheme adds to a growing list of different approaches that are significantly impacting the utility of solution NMR spectroscopy in studies of supra-molecular systems.