Methyl-TROSY

Tugarinov V, Kay LE, Ibraghimov 1, Orekhov VY (2005) High-resolution four-dimensional 1H-13C NOE spectroscopy using methyl-TROSY, sparse data acquisition, and multidimensional decomposition. J Am Chem Soc 127 2767-2775... 

Kay LE (2011) Solution NMR spectroscopy of supra-molecular systems, why bother A methyl-TROSY view. J Magn Reson 210 159-170... 

Ollerenshaw JE, Tugarinov V, Kay LE (2003) Methyl TROSY explanation and experimental verification. Magn Reson Chem 41 843-852... 

Tugarinov V, Kay LE (2004) An isotope labeling strategy for methyl TROSY spectroscopy. J Biomol NMR 28 165-172... 

D.M. Korzhnev, K. Kloiber, V. Kanelis, V. Tugarinov, L.E. Kay, Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy application to a 723-residue enzyme, J. Am. Chem. Soc. 126 (2004) 3964—3973. 

A particularly powerful approach for large proteins has resulted from the combined selective methyl protonation of aliphatic residues, particularly He, Leu and Val but more recently Ala and Met, in a perdeuterated background with optimised H, heteronuclear multiple quantum coherence (HMQC) pulse sequences, the methyl TROSY approach. Ayala et al. demonstrate labelling with methyl-protonated alanine using minimal media... 

Application of RDCs derived from methyl-TROSY experiment to test models for nucleotide binding to aspartate trancarbamoylase. ... 

NOESY-HMQC spectrum for a uniformly deuterated C-labelled protein with protonated Me-groups. The HMQC sequence utilises methyl-TROSY evolution, reducing the relaxation rates, allowing to extend the acquisition times. The modified pulse sequence of the experiment was presented that makes use of the INEPT period to record evolution. The experiment took 6.5 days to measure, that constituted 30% of the experiment time based on the uniform data sampling, resulting in a 4D spectrum with a resolution similar to that of a 3D spectrum. The data were processed using multidimensional decomposition. Both of the above processing methods produce data formats compatible with currently used spectra analysis software. 

I wish to quote some more examples of applications of transverse relaxation dispersion data. Li and co-workers (with erratum ) used the H R2 dispersion experiments to directly observe and characterize intermediate states in the dimerization of the protein E-cadherin. Sanchez-Medina et a/. used relaxation dispersion spectroscopy (for H, N and C) to probe the energy landscape and to characterize intermediates for the fast-folding protein denoted gpW. They identified a sparsely populated state with the lifetime of 250 ps, interconverting with the native state. In another paper from the same group, Shi and Kay studied the allosteric pathway regulating the activity of the HsIV protease, explored through methyl-TROSY C relaxation dispersion experiments. Libich... 

Lapinaite and colleagues have published a particularly impressive study of the box C/D ribonucleoprotein (RNP) from the archaeon Pyrococcus furiosus, a complex of 390 kDa responsible for methylating ribosomal RNA. ° By I, L, V-methyl labelling the three protein components of the complex, methyl TROSY spectra were used to monitor chemical shifts and PREs. Models for the entire complex could thus be calculated both with and without substrate RNA present using previously-determined structures of protein and RNA sub-units. Domain boundaries were allowed to be flexible during docking using NMR and SANS data, with... 

Fig. 4 Alanine labelling and 2D methyl-TROSY spectra of (a) CH3-Ala,U- H-labelled...

When a suitable sample has been prepared, a 2D methyl-TROSY can be recorded to see whether the peaks have enough sensitivity and resolution. If the TROSY spectrum looks promising, it can be used to probe the interactions and dynamics of the protein. However, such details can only be fully explained after the peaks have been assigned to specific methyl groups in the protein sequence and mapped onto the structure. 

With methyl-labelled samples, methyl-TROSY can provide detailed information regarding the structure, site-specific interaction, and dynamics of many important molecules that used to be too large for conventional NMR approaches. Spectra of... 

In addition to the examples mentioned in Sect. 4, methyl-TROSY has also been used to explore co-translational folding of a ribosome-bound nascent chain [56] as well as the structural basis for ion channel gating [57]. It is worth mentioning that most membrane proteins fall into the size range of TROSY experiments, and the methods reviewed here are relevant to their study. 

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