High Resolution Measurement of Methyl 13Cm−13C and 1Hm−13Cm Residual Dipolar Couplings in Large Proteins

摘要

NMR methodology is developed for high-resolution, accurate measurements of methyl 1H_m−13C_m (1D_CH) and 13C_m−13C (1D_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 (13Cβ/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 1H−13C 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 1D_CH RDCs from the acquisition dimension of NMR spectra imposes certain limitations on the accuracy of obtained 1D_CH values, 1D_CH couplings can be approximately corrected for cross-correlated relaxation effects. The ratios of 1D_CH and 1D_CC couplings (1D_CH/1D_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 1D_CH/1D_CC ratios obtained in MSG can therefore validate the employed correction scheme.