A comparison of HSQC and HMQC pulse schemes for recording (1)H[bond](13)C correlation maps of protonated methyl groups in highly deuterated proteins is presented. It is shown that HMQC correlation maps can be as much as a factor of 3 more sensitive than their HSQC counterparts and that the sensitivity gains result from a TROSY effect that involves cancellation of intra-methyl dipolar relaxation interactions. (1)H[bond](13)C correlation spectra are recorded on U-[(15)N,(2)H], Ile delta 1-[(13)C,(1)H] samples of (i) malate synthase G, a 723 residue protein, at 37 and 5 degrees C, and of (ii) the protease ClpP, comprising 14 identical subunits, each with 193 residues (305 kDa), at 5 degrees C. The high quality of HMQC spectra obtained in short measuring times strongly suggests that methyl groups will be useful probes of structure and dynamics in supramolecular complexes.

The two-body Slowly Relaxing Local Structure (SRLS) model was applied to (15)N NMR spin relaxation in proteins and compared with the commonly used original and extended model-free (MF) approaches. In MF, the dynamic modes are assumed to be decoupled, local ordering at the N-H sites is represented by generalized order parameters, and internal motions are described by effective correlation times. SRLS accounts for dynamical coupling between the global diffusion of the protein and the internal motion of the N-H bond vector. The local ordering associated with the coupling potential and the internal N-H diffusion are tensors with orientations that may be tilted relative to the global diffusion and magnetic frames. SRLS generates spectral density functions that differ from the MF formulas. The MF spectral densities can be regarded as limiting cases of the SRLS spectral density. SRLS-based model-fitting and model-selection schemes similar to the currently used MF-based ones were devised, and a correspondence between analogous SRLS and model-free parameters was established. It was found that experimental NMR data are sensitive to the presence of mixed modes. Our results showed that MF can significantly overestimate order parameters and underestimate local motion correlation times in proteins. The extent of these digressions in the derived microdynamic parameters is estimated in the various parameter ranges, and correlated with the time scale separation between local and global motions. The SRLS-based analysis was tested extensively on (15)N relaxation data from several isotropically tumbling proteins. The results of SRLS-based fitting are illustrated with RNase H from E. coli, a protein extensively studied previously with MF.

New NMR experiments are presented for the assignment of methyl (13)C and (1)H 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 (13)C and (1)H 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 alpha-ketoisovalerate that is (13)C-labeled and protonated in one of its methyl groups ((13)CH(3)), while the other methyl is (12)CD(3). The use of this biosynthetic precursor leads to production of Leu and Val residues that are (13)CH(3)-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 (13)C 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 (delta 1 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 degrees C) have been obtained using the proposed isotopic labeling strategy in combination with the new NMR experiments.

The development of isotope labeling methodology has had a significant impact on NMR studies of high-molecular-weight proteins and macromolecular complexes. Here we review some of this methodology that has been developed and used in our laboratory. In particular, experimental protocols are described for the production of highly deuterated, uniformly 15N- and 13C-labeled samples of large proteins, with optional incorporation of selective isotope labels into methyl groups of isoleucine, leucine and valine residues. Various types of methyl labeling schemes are assessed, and the utility of different methyl labeling strategies is highlighted for studies ranging from protein structure determination to the investigation of side-chain dynamics. In the case of malate synthase G (MSG), the time frame of the whole preparation, including the protein refolding step, is about 70 h.