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Comparison of Methyl Rotation Axis Order Parameters Derived from Model-Free Analyses of 2H and 13C Longitudinal and Transverse Relaxation Rates Measured in the Same Protein Sample

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journal contribution
posted on 30.05.2001 by Rieko Ishima, Aneta P. Petkova, John M. Louis, Dennis A. Torchia
Recombinant HIV-1 protease was obtained from bacteria grown on a 98% D2O medium containing 3-13C pyruvic acid as the sole source of 13C and 1H. The purified protein is highly deuterated at non-methyl carbons, but contains significant populations of 13CHD2 and 13CH2D methyl isotopomers. This pattern of isotope labeling permitted measurements of 1H and 13C relaxation rates of 13CHD2 isotopomers and 2H (D) relaxation rates of 13CH2D isotopomers using a single sample. The order parameters Saxis2, which characterize the motions of the methyl rotation axes, were derived from model-free analyses of R1 and R2 data sets measured for 13C and 2H spins. Our primary goal was to compare the Saxis2 values derived from the two independent types of data sets to test our understanding of the relaxation mechanisms involved. However, Saxis2 values derived from the analyses depend strongly on the geometry of the methyl group, the sizes of the quadrupolar and dipolar couplings, and the effects of bond vibrations and librations on these couplings. Therefore uncertainties in these basic physical parameters complicate comparison of the order parameters. This problem was circumvented by using an experimental relationship, between the methyl quadrupolar, 13C−13C and 13C−1H dipolar couplings, derived from independent measurements of residual static couplings of weakly aligned proteins by Ottiger and Bax (J. Am. Chem. Soc. 1999, 121, 4690−4695) and Mittermaier and Kay (J. Am. Chem. Soc. 1999, 121, 10608−10613). This approach placed a tight experimental restraint on the values of the 2H quadrupolar and 13C−1H dipolar interactions and greatly facilitated the accurate comparison of the relative values of the order parameters. When applied to our data this approach yielded satisfactory agreement between the Saxis2 values derived from the 13C and 2H data sets.