Abstract
Optimized selection of the slow-relaxing components of single-quantum 13C magnetization in 13CH3 methyl groups of proteins using acute (< 90°) angle 1H radio-frequency pulses, is described. The optimal selection scheme is more relaxation-tolerant and provides sensitivity gains in comparison to the experiment where the undesired (fast-relaxing) components of 13C magnetization are simply ‘filtered-out’ and only 90° 1H pulses are employed for magnetization transfer to and from 13C nuclei. When applied to methyl 13C single-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments for studies of chemical exchange, the selection of the slow-relaxing 13C transitions results in a significant decrease in intrinsic (exchange-free) transverse spin relaxation rates of all exchanging species. For exchanging systems involving high-molecular-weight species, the lower transverse relaxation rates translate into an increase in the information content of the resulting relaxation dispersion profiles.
This is a preview of subscription content, log in via an institution to check access.
References
Baldwin AJ, Walsh P, Hansen DF, Hilton GR, Benesch JL, Sharpe S, Kay LE (2012) Probing dynamic conformations of the high-molecular-weight aB-crystallin heat shock protein ensemble by NMR spectroscopy. J Am Chem Soc 134:15343–15350
Bodenhausen G, Ruben DJ (1980) Natural abundance 15N NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189
Carr HY, Purcell EM (1954) Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys Rev 4:630–638
Karamanos TK, Tugarinov V, Clore GM (2019) Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis. Proc Natl Acad Sci USA 116:21529–21538
Karamanos TK, Tugarinov V, Clore GM (2020) Determining methyl sidechain conformations in a CS-ROSETTA model using methyl 1H–13C residual dipolar couplings. J Biomol NMR 74:111–118
Kontaxis G, Bax A (2001) Multiplet component separation for measurement of methyl 13C–1H dipolar couplings in weakly aligned media. J Biomol NMR 20:77–82
Lundström P, Vallurupalli P, Religa TL, Dahlquist FW, Kay LE (2007) A single-quantum methyl 13C-relaxation dispersion experiment with improved sensitivity. J Biomol NMR 38:79–88
Marion D, Ikura M, Tschudin R, Bax A (1989) Rapid recording of 2D NMR spectra without phase cycling: application to the study of hydrogen exchange in proteins. J Magn Reson 85:393–399
Massi F, Grey MJ, Palmer AG (2005) Microsecond timescale backbone conformational dynamics in ubiquitin studied with NMR R1r relaxation experiments. Protein Sci 14:735–742
Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688–691
Morris GA, Freeman R (1979) Enhancement of nuclear magnetic resonance signals by polarization transfer. J Am Chem Soc 101:760–762
Ottiger M, Delaglio F, Marquardt JL, Tjandra N, Bax A (1998) Measurement of dipolar couplings for methylene and methyl sites in weakly oriented macromolecules and their use in structure determination. J Magn Reson 134:365–369
Rosenzweig R, Kay LE (2014) Bringing dynamic molecular machines into focus by methyl-TROSY NMR. Annu Rev Biochem 83:291–315
Schütz S, Sprangers R (2020) Methyl TROSYspectroscopy: a versatile NMR approach to study challenging biological systems. Prog Nucl Magn Reson Spectrosc 116:56–84
Shaka AJ, Keeler T, Frenkiel T, Freeman R (1983) An improved sequence for broadband decoupling: Waltz-16. J Magn Reson 52:335–338
Tugarinov V, Hwang PM, Kay LE (2004) Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins. Annu Rev Biochem 73:107–146
Tugarinov V, Hwang PM, Ollerenshaw JE, Kay LE (2003) Cross-correlated relaxation enhanced 1H–13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. J Am Chem Soc 125:10420–10428
Tugarinov V, Karamanos TK, Ceccon A, Clore GM (2020a) Optimized NMR experiments for the isolation of I=1/2 manifold transitions in methyl groups of proteins. ChemPhysChem 21:13–19
Tugarinov V, Karamanos TK, Clore GM (2020b) Magic-angle-pulse driven separation of degenerate 1H transitions in methyl groups of proteins: Application to studies of methyl axis dynamics. ChemPhysChem 21:1087–1091
Tugarinov V, Kay LE (2007) Separating degenerate 1H transitions in methyl group probes for single-quantum 1H-CPMG relaxation dispersion NMR spectroscopy. J Am Chem Soc 129:9514–9521
Tugarinov V, Kay LE (2013) Estimating side-chain order in [U-2H;13CH3]-labeled high molecular weight proteins from analysis of HMQC/HSQC spectra. J Phys Chem B 117:3571–3577
Acknowledgement
We thank Drs. James Baber, Jinfa Ying and Dan Garrett for technical support. This work was supported by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK029023 to G.M.C.).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
10858_2020_349_MOESM1_ESM.pdf
Supplementary file1Density matrix analysis of the scheme in Figure 3A leading tothe derivation of optimal values of αβ1H pulse angles. ‘Materials and Methods’ describingNMR sample conditions and acquisition parameters of NMR experiments (PDF 476 kb)
Rights and permissions
About this article
Cite this article
Tugarinov, V., Karamanos, T.K. & Clore, G.M. Optimized selection of slow-relaxing 13C transitions in methyl groups of proteins: application to relaxation dispersion. J Biomol NMR 74, 673–680 (2020). https://doi.org/10.1007/s10858-020-00349-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10858-020-00349-3