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

Abstract

Relaxation violated coherence transfer NMR spectroscopy (Tugarinov et al. in J Am Chem Soc 129:1743-1750, 2007) is an established experimental tool for quantitative estimation of the amplitudes of side-chain motions in methyl-protonated, highly deuterated proteins. Relaxation violated coherence transfer experiments monitor the buildup of methyl proton multiple-quantum coherences that can be created in magnetically equivalent spin-systems as long as their transverse magnetization components relax with substantially different rates. The rate of this build-up is a reporter of the methyl-bearing side-chain mobility. Although the build-up of multiple-quantum (1)H coherences is monitored in these experiments, the decay of the methyl signal during relaxation delays occurs when methyl proton magnetization is in a single-quantum state. We describe a relaxation violated coherence transfer approach where the relaxation of multiple-quantum (1)H-(13)C methyl coherences during the relaxation delay period is quantified. The NMR experiment and the associated fitting procedure that models the time-dependence of the signal build-up, are applicable to the characterization of side-chain order in [(13)CH(3)]-methyl-labeled, highly deuterated protein systems up to ~100 kDa in molecular weight. The feasibility of extracting reliable measures of side-chain order is experimentally verified on methyl-protonated, perdeuterated samples of an 8.5-kDa ubiquitin at 10°C and an 82-kDa Malate Synthase G at 37°C.

Estimating Side-Chain Order in [U-2H;13CH3]-Labeled High Molecular Weight Proteins from Analysis of HMQC/HSQC Spectra

“…Of note, these correlations are only slightly inferior to those obtained upon comparison of S axis 2 values derived from different nuclear spin probes in a number of previous studies. 13,16,17,[19][20][21]36 Mean values of the distributions of r HHext calculated from R MQ S rates for α 7 α 7 (3.7 ± 0.8 Å) and α 7 β 7 β 7 α 7 (3.3 ± 0.4 Å) are in reasonable agreement with 3.5 ± 1.0 Å calculated from the X-ray structure of the 20S proteasome CP (PDB access code 1pma 31 ). The corresponding values for MSG are significantly different (3.7 ± 0.8 Å from R MQ S vs 5.5 Å from the X-ray structure, PDB access code 1d8c 30 ), which reflects minor levels of protonation at additional sites (∼3%) in the Ile δ1 -labeled sample.…”

Measuring Entropy in Molecular Recognition by Proteins

“…Notable contributions for implementing 13 C relaxation in the context of proteins came from Torchia and colleagues (36) in their unraveling of the complexity of this particular mechanism of relaxation in the context of proteins. More recently, cross-correlated relaxation in 13 CH 3 groups has emerged as a highly sensitive though perhaps not as accurate route to measures of protein motion in even very large protein systems (99,100,105). Finally, computational strategies were needed to confidently extract the desired model-free parameters (12,72,103).…”

Optimal design of adaptively sampled NMR experiments for measurement of methyl group dynamics with application to a ribosome-nascent chain complex

“…3 reveals the impact of both HH/HH and HH/HC cross-correlated relaxation during the initial INEPT transfer on the initial amplitudes of inner and outer lines in the 13 C quartet. This builds on earlier analyses of the fully decoupled HSQC experiment, which derived the impact of HH/HH cross-correlations on the observed signal [17], as well as on the analysis of HH/HC and HH/HH cross-correlation relaxation for the measurement of side chain order [25]. For rapid tumbling, Δ is close to one (Eq.…”

“…A variety of experiments have been reported for the measurement of rotational diffusion via cross-correlated relaxation in 13 CH 3 spin systems [18][19][20][21][22][23][24][25][26][27]. The majority of these fall into two categories.…”