A ²H NMR Relaxation Experiment for the Measurement of the Time Scale of Methyl Side-Chain Dynamics in Large Proteins

An NMR experiment is presented for the measurement of the time scale of methyl side-chain dynamics in proteins that are labeled with methyl groups of the 13CHD2 variety. The measurement is accomplished by selecting a magnetization mode that to excellent approximation relaxes in a single-exponential manner with a T1-like rate. The combination of R1(13CHD2) and R2(13CHD2) 2H relaxation rates facilitates the extraction of motional parameters from 13CHD2-labeled proteins exclusively. The utility of the methodology is demonstrated with applications to proteins with tumbling times ranging from 2 ns (protein L, 7.5 kDa, 45 °C) to 54 ns (malate synthase G, 82 kDa, 37 °C); dynamics parameters are shown to be in excellent agreement with those obtained in 2H NMR studies of other methyl isotopomers. A consistency relationship is found to exist between R1(13CHD2) and the relaxation rates of pure longitudinal and quadrupolar order modes in 13CH2D-labeled methyl groups, and experimental rates measured for a number of proteins are shown to be in excellent agreement with expectations based on theory. The present methodology extends the applicability of 2H relaxation methods for the quantification of side-chain dynamics in high molecular weight proteins

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

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 ¹H–¹³C HMQC and HSQC correlation maps and relaxation rates of slowly decaying components of methyl ¹H–¹³C multiple-quantum coherences. A strength of the method is that [U-²H;¹³CH₃]-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

Precision Measurements of Deuterium Isotope Effects on the Chemical Shifts of Backbone Nuclei in Proteins: Correlations with Secondary Structure

Precision NMR measurements of deuterium isotope effects on the chemical shifts of backbone nuclei in proteins (¹⁵N, ¹³CO, ¹³C_α, and ¹HN) arising from ¹H-to-²H substitutions at aliphatic carbon sites. Isolation of molecular species with a defined protonation/deuteration pattern at carbon-α/β positions allows distinguishing and accurately quantifying different isotope effects within the protein backbone. The isotope shifts measured in the partially deuterated protein ubiquitin are interpreted in terms of backbone geometry via empirical relationships describing the dependence of isotope shifts on (φ; ψ) backbone dihedral angles. Because of their relatively large magnitude and clear dependence on the protein secondary structure, the two- and three-bond backbone amide ¹⁵N isotope shifts, ²ΔN­(C_α,iD) and ³ΔN­(C_α,i‑1D), can find utility for NMR structural refinement of small-to-medium size proteins

Ile, Leu, and Val Methyl Assignments of the 723-Residue Malate Synthase G Using a New Labeling Strategy and Novel NMR Methods

New NMR experiments are presented for the assignment of methyl 13C and 1H chemical shifts from Ile, Leu, and Val residues in high molecular weight proteins. The first class of pulse schemes transfers magnetization from the methyl group to the backbone amide spins for detection, while the second more sensitive class uses an “out-and-back” transfer scheme in which side-chain carbons or backbone carbonyls are correlated with methyl 13C and 1H spins. Both groups of experiments benefit from a new isotopic labeling scheme for protonation of Leu and Val methyl groups in large deuterated proteins. The approach makes use of α-ketoisovalerate that is 13C-labeled and protonated in one of its methyl groups (13CH3), while the other methyl is 12CD3. The use of this biosynthetic precursor leads to production of Leu and Val residues that are 13CH3-labeled at only a single methyl position. Although this labeling pattern effectively reduces by 2-fold the concentration of Leu and Val methyls in NMR samples, it ensures linearity of Val and Leu side-chain 13C spin-systems, leading to higher sensitivity and, for certain classes of experiments, substantial simplification of NMR spectra. Very near complete assignments of the 276 Ile (δ1 only), Leu, and Val methyl groups in the single-chain 723-residue enzyme malate synthase G (MSG, molecular tumbling time 37 ± 2 ns at 37 °C) have been obtained using the proposed isotopic labeling strategy in combination with the new NMR experiments

Quantitative ¹³C and ²H NMR Relaxation Studies of the 723-Residue Enzyme Malate Synthase G Reveal a Dynamic Binding Interface^†

A detailed understanding of molecular recognition is predicated not only on high-resolution static structures of the free and bound states but also on information about how these structures change with time, that is, molecular dynamics. Here we present a deuterium (2H) and carbon (13C) NMR relaxation study of methyl side chain dynamics in the 82 kDa enzyme malate synthase G (MSG) that is a promising target for the development of new antibiotic agents. It is shown that excellent agreement between 2H- and 13C-derived measures of dynamics is obtained, with correlation coefficients exceeding 0.95. The binding interface formed by MSG and its substrates is found to be highly dynamic in the ligand-free state of the enzyme with rigidification upon binding substrate. This study establishes that detailed, quantitative information about methyl side chain dynamics can be obtained by NMR on proteins with molecular masses on the order of 100 kDa and opens up the possibilities for studies of motion in a large number of important systems

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

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

Stereospecific NMR Assignments of Prochiral Methyls, Rotameric States and Dynamics of Valine Residues in Malate Synthase G

Near complete stereospecific assignments of the prochiral methyl carbons of Leu and Val residues in malate synthase G, a 723 residue enzyme, are reported. Assignments were obtained on the basis of a 10% fractional 13C-labeling strategy developed by Wüthrich and co-workers [Neri, D; Szyperski, T; Otting, G; Senn, H; Wüthrich, K. Biochemistry 1989, 28, 7510−7516] and, in the case of Val residues, supplemented with results from a series of new methyl-TROSY quantitative J experiments for measuring 3JCγN and 3JCγC‘ couplings. The measured 3J couplings were also used to probe Val side chain dynamics. A strong correlation is observed between rotamer averaging established on the basis of the couplings and side chain millisecond time scale dynamics measured using methyl-TROSY based 1H−13C multiple quantum relaxation dispersion experiments

4D ¹H−¹³C NMR Spectroscopy for Assignments of Alanine Methyls in Large and Complex Protein Structures

4D 1H−13C NMR Spectroscopy for Assignments of Alanine Methyls in Large and Complex Protein Structure

High Resolution Measurement of Methyl ¹³C_m−¹³C and ¹H_m−¹³C_m Residual Dipolar Couplings in Large Proteins

NMR methodology is developed for high-resolution, accurate measurements of methyl 1Hm−13Cm (1DCH) and 13Cm−13C (1DCC) residual dipolar couplings (RDCs) in ILV-methyl-protonated high-molecular-weight proteins. Both types of RDCs are measured in a three-dimensional (3D) mode that allows dispersion of correlations to the third (13Cβ/γ) dimension, alleviating the problem of overlap of methyl resonances in highly complex and methyl-abundant protein structures. The methodology is applied to selectively ILV-protonated 82-kDa monomeric enzyme malate synthase G (MSG) that contains 273 ILV methyl groups with substantial overlap of methyl resonances in 2D methyl 1H−13C correlation maps. A good agreement is observed between the measured RDCs of both types and those calculated from the crystallographic coordinates of MSG for the residues with low-amplitude internal dynamics. Although the measurement of 1DCH RDCs from the acquisition dimension of NMR spectra imposes certain limitations on the accuracy of obtained 1DCH values, 1DCH couplings can be approximately corrected for cross-correlated relaxation effects. The ratios of 1DCH and 1DCC couplings (1DCH/1DCC) are independent of methyl axis dynamics and the details of residual alignment [Ottiger, M.; Bax, A. J. Am. Chem. Soc. 1999, 121, 4690.]. The 1DCH/1DCC ratios obtained in MSG can therefore validate the employed correction scheme