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Simultaneous determination of protein structure and dynamics

Abstract

We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy—for obtaining experimental information at the atomic level about the structural and dynamical features of proteins—with the ability of molecular dynamics simulations to explore a wide range of protein conformations. We illustrate the method for human ubiquitin in solution and find that there is considerable conformational heterogeneity throughout the protein structure. The interior atoms of the protein are tightly packed in each individual conformation that contributes to the ensemble but their overall behaviour can be described as having a significant degree of liquid-like character. The protocol is completely general and should lead to significant advances in our ability to understand and utilize the structures of native proteins.

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Figure 1: Cross-validation of ubiquitin structures by comparison with independently determined NMR data.
Figure 2: Variability in structural ensembles of ubiquitin.
Figure 3: Examples of the liquid-like mobility of side chains in the DER ensemble.
Figure 4: Quantifying backbone and side-chain variability in ubiquitin ensembles by comparison of experimental and back-calculated order parameters.

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References

  1. Frauenfelder, H., Sligar, S. G. & Wolynes, P. G. The energy landscapes and motions on proteins. Science 254, 1598–1603 (1991)

    Article  ADS  CAS  Google Scholar 

  2. Rasmussen, B. F., Stock, A. M., Ringe, D. & Petsko, G. A. Crystalline ribonuclease A loses function below the dynamical transition at 220 K. Nature 357, 423–424 (1992)

    Article  ADS  CAS  Google Scholar 

  3. Karplus, M. & McCammon, J. A. Molecular dynamics simulations of biomolecules. Nature Struct. Biol. 9, 646–652 (2002)

    Article  CAS  Google Scholar 

  4. Eisenmesser, E. Z., Bosco, D. A., Akke, M. & Kern, D. Enzyme dynamics during catalysis. Science 295, 1520–1523 (2002)

    Article  ADS  CAS  Google Scholar 

  5. Wong, C. F. & McCammon, J. A. Protein flexibility and computer-aided drug design. Annu. Rev. Pharmacol. Toxicol. 43, 31–45 (2003)

    Article  CAS  Google Scholar 

  6. Benkovic, S. J. & Hammes-Schiffer, S. A perspective on enzyme catalysis. Science 301, 1196–1202 (2003)

    Article  ADS  CAS  Google Scholar 

  7. Kay, L. E. Protein dynamics from NMR. Nature Struct. Biol. 5, 513–517 (1998)

    Article  CAS  Google Scholar 

  8. Best, R. B. & Vendruscolo, M. Determination of ensembles of structures consistent with NMR order parameters. J. Am. Chem. Soc. 126, 8090–8091 (2004)

    Article  CAS  Google Scholar 

  9. Tjandra, N., Feller, S. E., Pastor, R. W. & Bax, A. Rotational diffusion anisotropy of human ubiquitin from 15N NMR relaxation. J. Am. Chem. Soc. 117, 12562–12566 (1995)

    Article  CAS  Google Scholar 

  10. Tjandra, N. & Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278, 1111–1114 (1997)

    Article  ADS  CAS  Google Scholar 

  11. Cornilescu, G., Marquardt, J. L., Ottiger, M. & Bax, A. Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J. Am. Chem. Soc. 120, 6836–6837 (1998)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Lee, A. L., Flynn, P. F. & Wand, A. J. Comparison of 2H and 13C NMR relaxation techniques for the study of protein methyl group dynamics in solution. J. Am. Chem. Soc. 121, 2891–2902 (1999)

    Article  CAS  Google Scholar 

  14. Peti, W., Meiler, J., Brüschweiler, R. & Griesinger, C. Model-free analysis of protein backbone motion from residual dipolar couplings. J. Am. Chem. Soc. 124, 5822–5833 (2002)

    Article  CAS  Google Scholar 

  15. Chou, J. J., Case, D. A. & Bax, A. Insights into the mobility of methyl-bearing side chains in proteins from 3J CC and 3J CN couplings. J. Am. Chem. Soc. 125, 8959–8966 (2003)

    Article  CAS  Google Scholar 

  16. Clore, G. M. & Schwieters, C. D. How much backbone motion in ubiquitin is required to account for dipolar coupling data measured in multiple alignment media as assessed by independent cross-validation? J. Am. Chem. Soc. 126, 2923–2938 (2004)

    Article  CAS  Google Scholar 

  17. Kitao, A. & Wagner, G. A space-time structure determination of human CD2 reveals the CD58-binding mode. Proc. Natl Acad. Sci. USA 97, 2064–2068 (2000)

    Article  ADS  CAS  Google Scholar 

  18. Scheek, R. M., Torda, A. E., Kemmink, J. & van Gunsteren, W. F. in Computational Aspects of the Study of Biological Macromolecules by Nuclear Magnetic Resonance Spectroscopy (eds Hoch, J. C., Redfield, C. & Poulsen, F. M.) 209–217 (Plenum, New York, 1991)

    Book  Google Scholar 

  19. Bonvin, A. M. J. J., Rullmann, J. A. C., Lamerichs, R. M. J. N., Boelens, R. & Kaptein, R. ‘Ensemble’ iterative relaxation matrix approach: A new NMR refinement protocol applied to the solution structure of crambin. Proteins 15, 385–400 (1993)

    Article  CAS  Google Scholar 

  20. Choy, W. Y. & Forman-Kay, J. D. Calculation of ensembles of structures representing the unfolded state of an SH3 domain. J. Mol. Biol. 308, 1011–1032 (2001)

    Article  CAS  Google Scholar 

  21. Vijay-Kumar, S., Bugg, C. E. & Cook, W. J. Structure of ubiquitin refined at 1.8 Å resolution. J. Mol. Biol. 194, 531–544 (1987)

    Article  CAS  Google Scholar 

  22. Mierke, D. F., Scheek, R. M. & Kessler, H. Coupling constants as restraints in ensemble driven dynamics. Biopolymers 34, 559–563 (1994)

    Article  CAS  Google Scholar 

  23. Stillinger, F. H. & Stillinger, D. K. Computational study of transition dynamics in 55-atom clusters. J. Chem. Phys. 93, 6013–6024 (1990)

    Article  ADS  CAS  Google Scholar 

  24. Zhou, Y., Vitkup, D. & Karplus, M. Native proteins are surface-molten solids: Application of the Lindemann criterion for the solid versus liquid state. J. Mol. Biol. 285, 1371–1375 (1999)

    Article  CAS  Google Scholar 

  25. DePristo, M. A., de Bakker, P. I. & Blundell, T. L. Heterogeneity and inaccuracy in protein structures solved by X-ray crystallography. Structure 12, 831–838 (2004)

    Article  CAS  Google Scholar 

  26. Hoch, J. C., Dobson, C. M. & Karplus, M. Vicinal coupling constants and protein dynamics. Biochemistry 24, 3831–3841 (1984)

    Article  Google Scholar 

  27. Best, R. B., Clarke, J. & Karplus, M. The origin of protein sidechain order parameter distributions. J. Am. Chem. Soc. 126, 7734–7735 (2004)

    Article  CAS  Google Scholar 

  28. Markley, J. L. et al. Recommendations for the presentation of NMR structures of proteins and nucleic acids. J. Mol. Biol. 280, 933–952 (1998)

    Article  CAS  Google Scholar 

  29. Richards, F. M. The interpretation of protein structures: Total volume, group volume distribution and packing density. J. Mol. Biol. 82, 1–14 (1974)

    Article  CAS  Google Scholar 

  30. Pontius, J., Richelle, J. & Wodak, S. J. Deviation from standard atomic volumes as a quality measure for protein crystal structure. J. Mol. Biol. 264, 121–136 (1996)

    Article  CAS  Google Scholar 

  31. Guerois, R., Nielsen, J. E. & Serrano, L. Predicting changes in the stability of proteins and protein complexes: A study of more than 1000 mutations. J. Mol. Biol. 320, 369–387 (2002)

    Article  CAS  Google Scholar 

  32. Buck, M. & Karplus, M. Internal and overall peptide group motion in proteins: Molecular dynamics simulations for lysozyme compared with results from X-ray and NMR spectroscopy. J. Am. Chem. Soc. 121, 9645–9658 (1999)

    Article  CAS  Google Scholar 

  33. Vendruscolo, M., Paci, E., Dobson, C. M. & Karplus, M. Rare fluctuations of native proteins sampled by equilibrium hydrogen exchange. J. Am. Chem. Soc. 125, 15686–15687 (2003)

    Article  CAS  Google Scholar 

  34. Lee, A. L. & Wand, A. J. Microscopic origins of entropy, heat capacity and the glass transition in proteins. Nature 411, 501–504 (2001)

    Article  ADS  CAS  Google Scholar 

  35. Yang, D. & Kay, L. E. Contributions to conformational entropy arising from bond vector fluctuations measured from NMR-derived order parameters: Application to protein folding. J. Mol. Biol. 263, 369–382 (1996)

    Article  CAS  Google Scholar 

  36. Johnson, E. C., Lazar, G. A., Desjarlais, J. R. & Handel, T. M. Solution structure and dynamics of a designed hydrophobic core variant of ubiquitin. Struct. Fold. Des. 7, 967–976 (1999)

    Article  CAS  Google Scholar 

  37. Benítez-Cardoza, C. G. et al. Exploring sequence/folding space: Folding studies on multiple hydrophobic core mutants of ubiquitin. Biochemistry 43, 5195–5203 (2004)

    Article  Google Scholar 

  38. Lindorff-Larsen, K. et al. Determination of a broad structural ensemble representing the denatured state of the bovine acyl-coenzyme A binding protein. J. Am. Chem. Soc. 126, 3291–3299 (2004)

    Article  CAS  Google Scholar 

  39. Korzhnev, D. M. et al. Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430, 586–590 (2004)

    Article  ADS  CAS  Google Scholar 

  40. Vendruscolo, M., Paci, E., Dobson, C. M. & Karplus, M. Three key residues form a critical contact network in a protein folding transition state. Nature 409, 641–645 (2001)

    Article  ADS  CAS  Google Scholar 

  41. Lindorff-Larsen, K., Paci, E., Vendruscolo, M. & Dobson, C. M. Transition states for protein folding have native topologies despite high structural variability. Nature Struct. Mol. Biol. 11, 443–449 (2004)

    Article  CAS  Google Scholar 

  42. Schwieters, C. D. & Clore, G. M. Reweighted atomic densities to represent ensembles of NMR structures. J. Biomol. NMR 23, 221–225 (2002)

    Article  CAS  Google Scholar 

  43. Jorgensen, W. J., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983)

    Article  ADS  CAS  Google Scholar 

  44. Brooks, B. R. et al. CHARMM: A program for macromolecular energy, minimization and dynamics calculations. J. Comput. Chem. 4, 187–217 (1983)

    Article  CAS  Google Scholar 

  45. Paci, E. & Karplus, M. Forced unfolding of fibronectin type 3 modules: An analysis by biased molecular dynamics simulations. J. Mol. Biol. 288, 441–459 (1999)

    Article  CAS  Google Scholar 

  46. Fox, T. & Kollman, P. A. The application of different solvation and electrostatic models in molecular dynamics simulations of ubiquitin: How well is the X-ray structure ‘maintained’? Proteins 25, 315–334 (1995)

    Article  Google Scholar 

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Acknowledgements

We are very grateful to P. I. de Bakker and T. L. Blundell for assistance in determining the X-ray rapper ensemble of ubiquitin. We thank S. E. Jackson for sharing the experimental data on ubiquitin stability changes before publication. K.L.L. is supported by the Danish Research Agency. M.A.D. was funded by the Marshall Aid Commemoration Commission, US National Science Foundation, and Cambridge Overseas Trust. M.V. is a Royal Society University Research Fellow. The research of M.V. and C.M.D. is supported in part by Programme Grants from the Wellcome and Leverhulme Trusts.

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Correspondence to Christopher M. Dobson or Michele Vendruscolo.

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Details on all methods for structure determination and analysis. (PDF 62 kb)

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Lindorff-Larsen, K., Best, R., DePristo, M. et al. Simultaneous determination of protein structure and dynamics. Nature 433, 128–132 (2005). https://doi.org/10.1038/nature03199

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