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

Abstract

A simple approach for quantification of methyl-containing side-chain mobility in high molecular weight methyl-protonated, uniformly deuterated proteins is described, based on the measurement of peak intensities in methyl (1)H-(13)C HMQC and HSQC correlation maps and relaxation rates of slowly decaying components of methyl (1)H-(13)C multiple-quantum coherences. A strength of the method is that [U-(2)H;(13)CH3]-labeled protein samples are required that are typically available at an early stage of any analysis. The utility of the methodology is demonstrated with applications to three protein systems ranging in molecular weight from 82 to 670 kDa. Although the approach is only semiquantitative, a high correlation between order parameters extracted via this scheme and other more established methods is nevertheless demonstrated.

Structural mechanism underlying G protein family-specific regulation of G protein-gated inwardly rectifying potassium channel

“…where R 2 diaH and R 2 diaHC are the transverse relaxation rates of the 1 H single quantum coherence and the 1 H- 13 C multiple quantum coherence of the side chain methyl groups in the diamagnetic state, respectively. The R 2 diaH and R 2 diaHC rates were measured using NMR samples containing 200 μM Gαi3[ILVA]βγ or 250 μM Gαiqi[ILVA]βγ, with the pulse sequences, which create 1 H single quantum or 1 H– 13 C multiple quantum coherences during the relaxation periods 53,54 . The magnetization transfer time in HMQC, t HMQC, was set to 6.9 ms.…”

Histone chaperone exploits intrinsic disorder to switch acetylation specificity

“…Diamagnetic transverse relaxation rates for the 1 H single-quantum coherence ( R 2 diaH ) and the 1 H- 13 C double-quantum coherence ( R 2 diaHC ) were quantified using the pulse schemes reported in Korzhnev et al 55 and Tugarinov et al 56,57 . Relaxation delays for the quantification of R 2 diaHC of both Vps75 and Rtt109 methyl resonances were 0, 2, 4, 7, 10, 13 & 20 ms.…”

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

“…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.…”

“…As 1 H transitions are degenerate, multiple experiments must be acquired to isolate the various coherences being observed in these approaches. In a related approach, cross-correlated relaxation may be estimated as a function of S 2 τ c through the relative resonance intensities in HSQC and HMQC experiments [27]. Second, measurements of differential relaxation in the 13 C multiplet, which is dominated by CH/CH DD/DD cross-correlated relaxation, with additional contributions from CH/C DD/CSA (chemical shift anisotropy) cross-correlations.…”