How Tetrahedral Are Methyl Groups in Proteins? A Liquid Crystal NMR Study

A small degree of protein alignment with an external magnetic field can be obtained in a dilute aqueous liquid crystalline solution of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC). It is demonstrated that residual one-bond 13C−13C and 13C−1H dipolar couplings of methyl groups in weakly aligned human ubiquitin can be measured with high accuracy. Experimentally, the ratio between 13C−1H and 13C−13C dipolar couplings is found to be −3.17 ± 0.03. Assuming a static conformation of the methyl group, rapidly spinning about its 3-fold symmetry axis, this ratio corresponds to an average C−C−H bond angle of 110.9 ± 1°, which is larger than the ideal tetrahedral value of 109.5°. Data indicate that the geometry of the various methyl groups is quite uniform, but that small (≤1°) deviations between the C−C vector and the axis connecting the methyl carbon to the geometric center of the three methyl protons may occur. The largest outlier is found for Ala46, which has a positive φ backbone angle, causing its methyl group to be within van der Waals contact of the preceding carbonyl oxygen.