Journal or Book Title
Journal of the American Chemical Society
The use of side chain methyl 13C chemical shifts for the determination of the rotameric conformation of Val and Leu residues in proteins by solid-state NMR spectroscopy is described. Examination of the solution NMR stereospecifically assigned methyl groups shows significant correlation between the difference in the two methyl carbons’ chemical shifts and the side chain conformation. It is found that α-helical and β-sheet backbones cause different side chain methyl chemical shift trends. In α-helical Leu’s, a relatively large absolute methyl 13C shift difference of 2.89 ppm is found for the most populated mt rotamer (χ1 = −60°, χ2 = 180°), while a much smaller value of 0.73 ppm is found for the next populated tp rotamer (χ1 = 180°, χ2 = 60°). For α-helical Val residues, the dominant t rotamer (χ1 = 180°) has more downfield Cγ2 chemical shifts than Cγ1 by 1.71 ppm, while the next populated m rotamer (χ1 = −60°) shows the opposite trend of more downfield Cγ1 chemical shift by 1.23 ppm. These significantly different methyl 13C chemical shifts exist despite the likelihood of partial rotameric averaging at ambient temperature. We show that these conformation-dependent methyl 13C chemical shifts can be utilized for side chain structure determination once the methyl 13C resonances are accurately measured by double-quantum (DQ) filtered 2D correlation experiments, most notably the dipolar DQ to single-quantum (SQ) correlation technique. The advantage of the DQ−SQ correlation experiment over simple 2D SQ−SQ correlation experiments is demonstrated on the transmembrane peptide of the influenza A M2 proton channel. The methyl chemical shifts led to predictions of the side chain rotameric states for several Val and Leu residues in this tetrameric helical bundle. The predicted Val rotamers were further verified by dipolar correlation experiments that directly measure the χ1 torsion angles. It was found that the chemical-shift-predicted side chain conformations are fully consistent with the direct torsion angle results; moreover, the methyl 13C chemical shifts are sensitive to ∼5° changes in the χ1 torsion angle due to drug binding.
American Chemical Society
Hong, Mei; Mishanina, Tatiana V.; and Cady, Sarah D., "Accurate Measurement of Methyl 13C Chemical Shifts by Solid-State NMR for the Determination of Protein Side Chain Conformation: The Influenza A M2 Transmembrane Peptide as an Example" (2009). Chemistry Publications. 1151.