Advertisement

Determining methyl sidechain conformations in a CS-ROSETTA model using methyl 1H-13C residual dipolar couplings

Abstract

Modelling of protein structures based on backbone chemical shifts, using programs such as CS-ROSETTA, is becoming increasingly popular, especially for systems where few restraints are available or where homologous structures are already known. While the reliability of CS-ROSETTA calculations can be improved by incorporation of some additional backbone NMR data such as those afforded by residual dipolar couplings or minimal NOE data sets involving backbone amide protons, the sidechain conformations are largely modelled by statistical energy terms. Here, we present a simple method based on methyl residual dipolar couplings that can be used to determine the rotameric state of the threefold symmetry axis of methyl groups that occupy a single rotamer, determine rotameric distributions, and identify regions of high flexibility. The method is demonstrated for methyl side chains of a deletion variant of the human chaperone DNAJB6b.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Anthis NJ, Clore GM (2015) Visualizing transient dark states by NMR spectroscopy. Quart Rev Biophys 48:1–82

  2. Chou JJ, Case DA, Bax A (2003) Insights into the mobility of methyl-bearing side chains in proteins from 3JCC and 3JCN couplings. J Am Chem Soc 125:8959–8966

  3. Clore GM, Garrett DS (1999) R-factor, free R, and complete cross-validation for dipolar coupling refinement of NMR structures. J Am Chem Soc 121:9008–9012

  4. Clore GM, Kuszewski J (2002) χ1 rotamer populations and angles of mobile surface side chains are accurately predicted by a torsion angle database potential of mean force. J Am Chem Soc 124:2866–2867

  5. Hansen DF, Kay LE (2011) Determining valine sidechain rotamer conformations in proteins from methyl 13C chemical shifts: application to the 360 kda half-proteasome. J Am Chem Soc 133:8272–8281

  6. Hansen DF, Neudecker P, Kay LE (2010a) Determination of isoleucine sidechain conformations in ground and excited states of proteins from chemical shifts. J Am Chem Soc 132:7589–7591

  7. Hansen DF, Neudecker P, Vallurupalli P, Mulder FAA, Kay LE (2010b) Determination of Leu sidechain conformations in excited protein states by NMR relaxation dispersion. J Am Chem Soc 132:42–43

  8. 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 43:21529–21538

  9. Kuszewski J, Gronenborn AM, Clore GM (1996) Improving the quality of NMR and crystallographic protein structures by means of a conformational database potential derived from structure databases. Protein Sci 5:1067–1080

  10. Leaver-Fay A et al (2011) Rosetta3: An object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol 487:545–574

  11. Lovell SC, Word JM, Richardson JS, Richardson DC (2000) The penultimate rotamer library. Proteins 40:389–408

  12. Mittermaier A, Kay LE (2001) χ1 torsion angle dynamics in proteins from dipolar couplings. J Am Chem Soc 123:6892–6903

  13. Mittermaier A, Kay LE (2002) Effect of deuteration on some structural parameters of methyl groups in proteins as evaluated by residual dipolar couplings. J Biomol NMR 23:35–45

  14. Neri D, Szyperski T, Otting G, Senn H, Wüthrich K (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional carbon-13 labeling. Biochemistry 28:7510–7516

  15. Nerli S, McShan AC, Sgourakis NG (2018) Chemical shift-based methods in NMR structure determination. Progr Nucl Magn Res Spec 106–107:1–25

  16. Ollerenshaw JE, Tugarinov V, Kay LE (2003) Methyl TROSY: Explanation and experimental verification. Magn Reson Chem 41:843–852

  17. Ottiger M, Bax A (1999) How tetrahedral are methyl groups in proteins? A liquid crystal NMR study. J Am Chem Soc 121:4690–4695

  18. Ottiger M, Delaglio F, Bax A (1998a) Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. J Magn Reson 131:373–378

  19. Ottiger M, Delaglio F, Marquardt JL, Tjandra N, Bax A (1998b) 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

  20. Raman S et al (2010) NMR structure determination for larger proteins using backbone-only data. Science 327:1014–1018

  21. Rosenzweig R, Kay LE (2014) Bringing dynamic molecular machines into focus by methyl-TROSY NMR. Annu Rev Biochem 83:291–315

  22. Rückert M, Otting G (2000) Alignment of biological macromolecules in novel nonionic liquid crystalline media for NMR experiments. J Am Chem Soc 122:7793–7797

  23. Schwieters CD, Bermejo GA, Clore GM (2018) Xplor-NIH for molecular structure determination from NMR and other data sources. Protein Sci 27:26–40

  24. Sgourakis NG et al (2014) The structure of mouse cytomegalovirus M04 protein obtained from sparse NMR data reveals a conserved fold of the M02–M06 viral immune modulator family. Structure 22:1263–1273

  25. Shen Y et al (2008) Consistent blind protein structure generation from NMR chemical shift data. Proc Natl Acad Sci USA 105:4685–4690

  26. Sprangers R, Kay LE (2007) Probing supramolecular structure from measurement of methyl 1H–13C residual dipolar couplings. J Am Chem Soc 129:12668–12669

  27. Tang C, Iwahara J, Clore GM (2005) Accurate determination of leucine and valine sidechain conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cγ–Hγ/Cβ–Hβ residual dipolar couplings: Application to the 34-kDa enzyme IIAchitobiose. J Biomol NMR 33:105–121

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

  29. 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

  30. Tugarinov V, Kay LE (2013) Estimating sidechain order in [U-2H; 13CH3]-labeled high molecular weight proteins from analysis of HMQC/HSQC spectra. J Phys Chem B 117:3571–3577

  31. 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

  32. Tugarinov V, Karamanos TK, Ceccon A, Clore GM (2020) Optimized NMR experiments for the isolation of I = 1/2 manifold transitions in methyl groups of proteins. ChemPhysChem. https://doi.org/10.1002/cphc.201900959

  33. Yang D, 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

Download references

Acknowledgements

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

Correspondence to Vitali Tugarinov or G. Marius Clore.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Karamanos, T.K., Tugarinov, V. & Clore, G.M. Determining methyl sidechain conformations in a CS-ROSETTA model using methyl 1H-13C residual dipolar couplings. J Biomol NMR (2020). https://doi.org/10.1007/s10858-019-00294-w

Download citation

Keywords

  • Sidechain conformation
  • Residual dipolar couplings
  • Methyl NMR
  • Protein structure refinement
  • CS-ROSETTA