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

• DOI: 10.1021/ja1041435
• Publication Date: 2010-09-22T14:17:05Z
• Authors: Chenyun Guo; Raquel Godoy-Ruiz; Vitali Tugarinov
• Keywords: Oxygen-18

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