Relaxation Rates of Degenerate ¹H Transitions in Methyl Groups of Proteins as Reporters of Side-Chain Dynamics

Abstract

Experiments for quantifying the amplitudes of motion of methyl-containing side chains are presented that exploit the rich network of cross-correlated spin relaxation interactions between intra-methyl dipoles in highly deuterated, selectively 13CH2D- or 13CH3-labeled proteins. In particular, the experiments measure spin relaxation rates of degenerate 1H transitions in methyl groups that, for high-molecular-weight proteins, are very simply related to methyl three-fold symmetry axis order parameters. The methodology presented is applied to studies of dynamics in a pair of systems, including the 7.5-kDa protein L and the 82-kDa enzyme malate synthase G. Good agreement between 1H- and 2H-derived measures of side-chain order are obtained on highly deuterated proteins with correlation times exceeding approximately 10 ns (correlation coefficients greater than 0.95). Although 2H- and 13C-derived measures of side-chain dynamics are still preferred, the present work underscores the potential of using 1H relaxation for semiquantitative estimates of methyl side-chain flexibility, while the high level of consistency between the different spin probes of motion establishes the reliability of the dynamics parameters.

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

“…We also measured methyl 1 H transverse relaxation (R 2,S ) rates (49) (Fig. 4E); because the rate is biexponential for a methyl group attached to a protein (35), we selected the slowly relaxing component of magnetization (49). As expected, R 2,S rates increase much more rapidly as a function of Gd(III)DTPA-BMA concentration for peak A than for peak B.…”

Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy

“…R S MQ % 0 for an isolated methyl group , and the line-widths of central MQ transitions are largely determined by dipolar interactions with external protons and deuterons). Of note, the same value of r HH = 1.813 Å has been used in earlier methyl 1 H relaxation studies (Sun et al 2011;Tugarinov and Kay 2006b;Tugarinov et al 2007) and is in reasonable agreement with the value of 1.807 ± 0.012 Å reported for methyl iodide based on the ratios of methyl 1 H-1 H and 1 H-13 C dipolar couplings (Wooten et al 1979), while the chosen value of r CH corresponds to P 2 ðcos h axis;CH Þr À3 CH = -0.228 Å -3 assuming ideal methyl tetrahedrality (h axis,CH = 109.5°) as derived from the ratios 13 C-13 C m and 1 H-13 C dipolar couplings in a pair of previous studies (Mittermaier and Kay 2002;Ottiger and Bax 1999).…”

“…The values of the diffusion tensor for MSG (D 2 O; 37°C) were estimated from diffusion measurements as described previously (Tugarinov and Kay 2006b;Tugarinov et al 2005) resulting in s C,eff = (2D || ? 4D \ ) -1 = 49 ns, diffusion anisotropy D || /D \ = 1.21 and the polar angles h = 13°, / = 48°d efining the orientation of the unique diffusion axis relative to the inertia frame (Tugarinov and Kay 2006b).…”

“…(1) the measurement of transverse relaxation rates of individual 1 H transitions and extraction of methyl three-fold axis order parameters, S 2 axis , that report on the amplitudes of sidechain motions, from the difference in the measured rates (Tugarinov and Kay 2006b), and (2) the more versatile scheme that relies upon the measurement of 1 H-1 H dipolar cross-correlation rates, g HH , via what was termed as ''forbidden fruits of NMR spectroscopy'' by Ernst et al (1987)namely, the use of relaxation violated coherence transfer (Kay and Prestegard 1987;Muller et al 1987) whereby methyl proton multiple-quantum (MQ)-so-called 'forbidden'-coherences are created despite that all 1 H transitions in a 13 CH 3 methyl are magnetically equivalent. It has been demonstrated that the time dependence of the preparation of 1 H MQ states is a sensitive probe of side-chain dynamics, even in systems with molecular weights of hundreds of kilodaltons, and that robust measures of motion can be obtained (Tugarinov et al 2007).…”

1H and 19F spin-lattice relaxation and CH3 or CF3 reorientation in molecular solids containing both H and F atoms

“…Finally, as an aside, we note for completeness that because the reorientational properties of methyl groups in the solid state provide information concerning their environment, spin-lattice relaxation experiments are becoming very helpful in investigating the intramolecular and intermolecular interactions in biologically relevant molecules. [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55] …”