High resolution measurement of methyl 13C(m)-13C and 1H(m)-13C(m) residual dipolar couplings in large proteins.

Abstract

NMR methodology is developed for high-resolution, accurate measurements of methyl (1)H(m)-(13)C(m) ((1)D(CH)) and (13)C(m)-(13)C ((1)D(CC)) residual dipolar couplings (RDCs) in ILV-methyl-protonated high-molecular-weight proteins. Both types of RDCs are measured in a three-dimensional (3D) mode that allows dispersion of correlations to the third ((13)C(β/γ)) dimension, alleviating the problem of overlap of methyl resonances in highly complex and methyl-abundant protein structures. The methodology is applied to selectively ILV-protonated 82-kDa monomeric enzyme malate synthase G (MSG) that contains 273 ILV methyl groups with substantial overlap of methyl resonances in 2D methyl (1)H-(13)C correlation maps. A good agreement is observed between the measured RDCs of both types and those calculated from the crystallographic coordinates of MSG for the residues with low-amplitude internal dynamics. Although the measurement of (1)D(CH) RDCs from the acquisition dimension of NMR spectra imposes certain limitations on the accuracy of obtained (1)D(CH) values, (1)D(CH) couplings can be approximately corrected for cross-correlated relaxation effects. The ratios of (1)D(CH) and (1)D(CC) couplings ((1)D(CH)/(1)D(CC)) are independent of methyl axis dynamics and the details of residual alignment [Ottiger, M.; Bax, A. J. Am. Chem. Soc. 1999, 121, 4690.]. The (1)D(CH)/(1)D(CC) ratios obtained in MSG can therefore validate the employed correction scheme.