A methyl-TROSY based 13C relaxation dispersion NMR experiment for studies of chemical exchange in proteins

• Published in: Journal of biomolecular NMR 2023-06, Vol.77 (3), p.83-91
• Main Authors: Tugarinov, Vitali, Baber, James L., Clore, G. Marius
• Format: Article
• Language: English
• Subjects: Biochemistry; Biological and Medical Physics; Biophysics; Chemical equilibrium; Decoupling; Dispersion; Exchanging; Excitation; Experiments; Fyn protein; Malate synthase; Molecular weight; mutants; NMR; Nuclear magnetic resonance; Physics; Physics and Astronomy; Proteins; Spectroscopy; Spectroscopy/Spectrometry
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-023-00413-8

A methyl Transverse Relaxation Optimized Spectroscopy (methyl-TROSY) based, multiple quantum (MQ) 13 C Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiment is described. The experiment is derived from the previously developed MQ 13 C– 1 H CPMG scheme (Korzhnev in J Am Chem Soc 126: 3964–73, 2004) supplemented with a CPMG train of refocusing 1 H pulses applied with constant frequency and synchronized with the 13 C CPMG pulse train. The optimal 1 H ‘decoupling’ scheme that minimizes the amount of fast-relaxing methyl MQ magnetization present during CPMG intervals, makes use of an XY-4 phase cycling of the refocusing composite 1 H pulses. For small-to-medium sized proteins, the MQ 13 C CPMG experiment has the advantage over its single quantum (SQ) 13 C counterpart of significantly reducing intrinsic, exchange-free relaxation rates of methyl coherences. For high molecular weight proteins, the MQ 13 C CPMG experiment eliminates complications in the interpretation of MQ 13 C– 1 H CPMG relaxation dispersion profiles arising from contributions to exchange from differences in methyl 1 H chemical shifts between ground and excited states. The MQ 13 C CPMG experiment is tested on two protein systems: (1) a triple mutant of the Fyn SH3 domain that interconverts slowly on the chemical shift time scale between the major folded state and an excited state folding intermediate; and (2) the 82-kDa enzyme Malate Synthase G (MSG), where chemical exchange at individual Ile δ1 methyl positions occurs on a much faster time-scale.