Abstract
Although 15N- and 13C-based chemical exchange saturation transfer (CEST) experiments have assumed an important role in studies of biomolecular conformational exchange, 1H CEST experiments are only beginning to emerge. We present a methyl-TROSY 1H CEST experiment that eliminates deleterious 1H–1H NOE dips so that CEST profiles can be analyzed robustly to extract methyl proton chemical shifts of rare protein conformers. The utility of the experiment, along with a version that is optimized for 13CHD2 labeled proteins, is established through studies of exchanging protein systems. A comparison between methyl 1H CEST and methyl 1H CPMG approaches is presented to highlight the complementarity of the two experiments.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Boehr DD, McElheny D, Dyson HJ, Wright PE (2006) The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313:1638–1642. doi:10.1126/science.1130258
Bouvignies G, Kay LE (2012a) A 2D 13C-CEST experiment for studying slowly exchanging protein systems using methyl probes: an application to protein folding. J Biomol NMR 53:303–310. doi:10.1007/s10858-012-9640-7
Bouvignies G, Kay LE (2012b) Measurement of proton chemical shifts in invisible states of slowly exchanging protein systems by chemical exchange saturation transfer. J Phys Chem B 116:14311–14317. doi:10.1021/jp311109u
Bouvignies G et al (2011) Solution structure of a minor and transiently formed state of a T4 lysozyme mutant. Nature 477:111–114. doi:10.1038/nature10349
Bouvignies G, Vallurupalli P, Kay LE (2014) Visualizing side chains of invisible protein conformers by solution NMR. J Mol Biol 426:763–774. doi:10.1016/j.jmb.2013.10.041
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on Unix pipes. J Biomol NMR 6:277–293. doi:10.1007/Bf00197809
Di Nardo AA, Korzhnev DM, Stogios PJ, Zarrine-Afsar A, Kay LE, Davidson AR (2004) Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation. Proc Natl Acad Sci USA 101:7954–7959. doi:10.1073/pnas.0400550101
Eriksson AE, Baase WA, Wozniak JA, Matthews BW (1992) A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene. Nature 355:371–373. doi:10.1038/355371a0
Felli IC, Pierattelli R (2015) Spin-state-selective methods in solution- and solid-state biomolecular 13C NMR. Prog Nucl Magn Reson Spectrosc 84:1–13. doi:10.1016/j.pnmrs.2014.10.001
Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141. doi:10.1016/0022-2364(91)90034-Q
Gelis I et al (2007) Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 131:756–769. doi:10.1016/j.cell.2007.09.039
Guenneugues M, Berthault P, Desvaux H (1999) A method for determining B1 field inhomogeneity. Are the biases assumed in heteronuclear relaxation experiments usually underestimated? J Magn Reson 136:118–126. doi:10.1006/jmre.1998.1590
Henzler-Wildman K, Kern D (2007) Dynamic personalities of proteins. Nature 450:964–972. doi:10.1038/nature06522
Ishima R, Louis JM, Torchia DA (1999) Transverse 13C relaxation of CHD2 methyl isotopmers to detect slow conformational changes of protein side chains. J Am Chem Soc 121:11589–11590. doi:10.1021/ja992836b
Janin J, Miller S, Chothia C (1988) Surface, subunit interfaces and interior of oligomeric proteins. J Mol Biol 204:155–164. doi:10.1016/0022-2836(88)90606-7
Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665. doi:10.1021/ja00052a088
Kerfah R, Plevin MJ, Sounier R, Gans P, Boisbouvier J (2015) Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins. Curr Opin Struct Biol 32:113–122. doi:10.1016/j.sbi.2015.03.009
Korzhnev DM, Kloiber K, Kay LE (2004) Multiple-quantum relaxation dispersion NMR spectroscopy probing millisecond time-scale dynamics in proteins: theory and application. J Am Chem Soc 126:7320–7329. doi:10.1021/ja049968b
Levitt MH, Freeman R (1979) NMR population-inversion using a composite pulse. J Magn Reson 33:473–476. doi:10.1016/0022-2364(79)90265-8
Lundstrom 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. doi:10.1007/s10858-007-9149-7
Luy B (2004) Spin state selectivity and heteronuclear Hartmann-Hahn transfer. J Magn Reson 168:210–216. doi:10.1016/j.jmr.2004.03.005
Millet O, Loria JP, Kroenke CD, Pons M, Palmer AG (2000) The static magnetic field dependence of chemical exchange linebroadening defines the NMR chemical shift time scale. J Am Chem Soc 122:2867–2877. doi:10.1021/ja993511y
Mulder FAA, Skrynnikov NR, Hon B, Dahlquist FW, Kay LE (2001) Measurement of slow (μs-ms) time scale dynamics in protein side chains by 15N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme. J Am Chem Soc 123:967–975. doi:10.1021/ja003447g
Neudecker P et al (2012) Structure of an intermediate state in protein folding and aggregation. Science 336:362–366. doi:10.1126/science.1214203
Ottiger M, Delaglio F, Bax A (1998) Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. J Magn Reson 131:373–378. doi:10.1006/jmre.1998.1361
Palmer AG, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol 339:204–238. doi:10.1016/S0076-6879(01)39315-1
Religa TL, Kay LE (2010) Optimal methyl labeling for studies of supra-molecular systems. J Biomol NMR 47:163–169. doi:10.1007/s10858-010-9419-7
Rennella E, Huang R, Velyvis A, Kay LE (2015) 13CHD2-CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins. J Biomol NMR 63:187–199. doi:10.1007/s10858-015-9974-z
Rosenzweig R, Kay LE (2014) Bringing dynamic molecular machines into focus by methyl-TROSY NMR. Annu Rev Biochem 83:291–315. doi:10.1146/annurev-biochem-060713-035829
Schleucher J, Sattler M, Griesinger C (1993) Coherence selection by gradients without signal attenuation: application to the 3-dimensional HNCO experiment. Angew Chem Int Ed 32:1489–1491. doi:10.1002/anie.199314891
Sekhar A, Kay LE (2013) NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers. Proc Natl Acad Sci USA 110:12867–12874. doi:10.1073/pnas.1305688110
Sekhar A, Rumfeldt JAO, Broom HR, Doyle CM, Bouvignies G, Meiering EM, Kay LE (2015) Thermal fluctuations of immature SOD1 lead to separate folding and misfolding pathways. Elife 4:e07296. doi:10.7554/eLife.07296
Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broadband decoupling: WALTZ-16. J Magn Reson 52:335–338. doi:10.1016/0022-2364(83)90207-X
Shaka AJ, Lee CJ, Pines A (1988) Iterative schemes for bilinear operators: application to spin decoupling. J Magn Reson 77:274–293. doi:10.1016/0022-2364(88)90178-3
Sheppard D, Sprangers R, Tugarinov V (2010) Experimental approaches for NMR studies of side-chain dynamics in high-molecular-weight proteins. Prog Nucl Magn Reson Spectrosc 56:1–45. doi:10.1016/j.pnmrs.2009.07.004
Sklenar V, Piotto M, Leppik R, Saudek V (1993) Gradient-tailored water suppression for 1H–15N HSQC experiments optimized to retain full sensitivity. J Magn Reson Ser A 102:241–245. doi:10.1006/jmra.1993.1098
Skrynnikov NR, Dahlquist FW, Kay LE (2002) Reconstructing NMR spectra of “invisible” excited protein states using HSQC and HMQC experiments. J Am Chem Soc 124:12352–12360. doi:10.1021/ja0207089
Sprangers R, Kay LE (2007) Quantitative dynamics and binding studies of the 20 S proteasome by NMR. Nature 445:618–622. doi:10.1038/nature05512
Tugarinov V, Kay LE (2005) Methyl groups as probes of structure and dynamics in NMR studies of high-molecular-weight proteins. Chembiochem 6:1567–1577. doi:10.1002/cbic.200500110
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. doi:10.1021/ja0726456
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. doi:10.1021/ja030153x
Vallurupalli P, Bouvignies G, Kay LE (2012) Studying “invisible” excited protein states in slow exchange with a major state conformation. J Am Chem Soc 134:8148–8161. doi:10.1021/ja3001419
Wuthrich K (1986) NMR of proteins and nucleic acids. Wiley, Hoboken
Yang DW, Kay LE (1999) Improved 1HN-detected triple resonance TROSY-based experiments. J Biomol NMR 13:3–10. doi:10.1023/A:1008329230975
Yang DW, Nagayama K (1996) A sensitivity-enhanced method for measuring heteronuclear long-range coupling constants from the displacement of signals in two 1D subspectra. J Magn Reson Ser A 118:117–121. doi:10.1006/jmra.1996.0017
Yuwen T, Vallurupalli P, Kay LE (2016) Enhancing the sensitivity of CPMG relaxation dispersion to conformational exchange processes by multiple-quantum spectroscopy. Angew Chem Int Ed 55:11490–11494. doi:10.1002/anie.201605843
Yuwen T, Sekhar A, Kay LE (2017) Separating dipolar and chemical exchange magnetization transfer processes in 1H-CEST. Angew Chem Int Ed 56:6122–6125.doi:10.1002/anie.201610759
Acknowledgements
This work was supported by Grants from the Canadian Institutes of Health Research and the Natural Sciences and Research Council of Canada. L.E.K holds a Canada Research Chair in Biochemistry.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yuwen, T., Huang, R. & Kay, L.E. Probing slow timescale dynamics in proteins using methyl 1H CEST. J Biomol NMR 68, 215–224 (2017). https://doi.org/10.1007/s10858-017-0121-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10858-017-0121-x