An isotope labeling strategy for methyl TROSY spectroscopy

Abstract

Recently we have shown that HMQC spectra of protonated methyl groups in high molecular weight, highly deuterated proteins have large enhancements in sensitivity and resolution relative to HSQC-generated data sets. These enhancements derive from a TROSY effect in which complete cancellation of intra-methyl (1)H-(1)H and (1)H-(13)C dipolar interactions occurs for 50% of the signal in the case of HMQC, so long as the methyl is attached to a molecule tumbling in the macromolecular limit (Tugarinov, V., Hwang, P.M., Ollerenshaw, J.E., Kay, L.E. J. Am. Chem. Soc. (2003) 125, 10420-10428; Ollerenshaw, J.E., Tugarinov, V. and Kay, L.E. Magn. Reson. Chem. (2003) 41, 843-852. The first demonstration of this effect was made for isoleucine delta1 methyl groups in a highly deuterated 82 kDa protein, malate synthase G. As with (1)H-(15)N TROSY spectroscopy high levels of deuteration are critical for maximizing the TROSY effect. Here we show that excellent quality methyl TROSY spectra can be recorded on U-[(2)H] Iledelta1-[(13)CH(3)] Leu,Val-[(13)CH(3)/(12)CD(3)] protein samples, significantly extending the number of probes available for structural and dynamic studies of high molecular weight systems.