Abstract
Motions are essential for protein function, and knowledge of protein dynamics is a key to our understanding the mechanisms underlying protein folding and stability, ligand recognition, allostery, and catalysis. In the last two decades, NMR relaxation measurements have become a powerful tool for characterizing backbone and side chain dynamics in complex biological macromolecules such as proteins and nucleic acids. Accurate analysis of the experimental data in terms of motional parameters is an essential prerequisite for developing physical models of motions to paint an adequate picture of protein dynamics. Here, I describe in detail how to use the software package DYNAMICS that was developed for accurate characterization of the overall tumbling and local dynamics in a protein from nuclear spin-relaxation rates measured by NMR. Step-by-step instructions are provided and illustrated through an analysis of 15N relaxation data for protein G.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Palmer, A. G., 3 rd. (2004) NMR characterization of the dynamics of biomacromolecules. Chem. Rev. 104, 3623–3640.
Sheppard, D., Sprangers, R., and 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.
Godoy-Ruiz, R., Guo, C., and Tugarinov, V. (2010) Alanine methyl groups as NMR probes of molecular structure and dynamics in high-molecular-weight proteins. J. Am. Chem. Soc. 132, 18340–18350.
Cavanagh, J., Fairbrother, W. J., III, A. J. P., and Skelton, N. J. (1996) Protein NMR Spectroscopy, Academic Press, San Diego.
Fushman, D., and Cowburn, D. (1999) The effect of noncollinearity of 15 N-1 H dipolar and 15 N CSA tensors and rotational anisotropy on 15 N relaxation rates, CSA/DD cross correlation, and TROSY. J. Biomol. NMR 13, 139–147.
Fushman, D., Ohlenschlager, O., and Rüterjans, H. (1994) Determination of the backbone mobility of ribonuclease T1 and its 2’GMP complex using molecular dynamics simulations and NMR relaxation data. J. Biomol. Struct. Dyn. 11, 1377–1402.
Pfeiffer, S., Fushman, D., and Cowburn, D. (2001) Simulated and NMR derived backbone dynamics of a protein with significant flexibility: A comparison of spectral densities for the < beta > ARK PH domain. J. Am. Chem. Soc. 123, 3021–3036.
Maragakis, P., Lindorff-Larsen, K., Eastwood, M. P., Dror, R. O., Klepeis, J. L., Arkin, I. T., Jensen, M. O., Xu, H., Trbovic, N., Friesner, R. A., Palmer, A. G., and Shaw, D. E. (2008) Microsecond molecular dynamics simulation shows effect of slow loop dynamics on backbone amide order parameters of proteins. J. Phys. Chem. B 112, 6155–6158.
Fushman, D., and Cowburn, D. (2001) Nuclear magnetic resonance relaxation in determination of residue-specific 15 N chemical shift tensors in proteins in solution: protein dynamics, structure, and applications of transverse relaxation optimized spectroscopy, in Methods in Enzymology (James, T., Schmitz, U., and Doetsch, V., Eds.), 339, 109–126.
Lipari, G., and Szabo, A. (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 2. J. Am. Chem. Soc. 104, 4559–4570.
Lipari, G., and Szabo, A. (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity, J. Am. Chem. Soc. 104, 4546–4559.
Clore, G. M., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C., and Gronenborn, A. M. (1990) Deviations from the simple two-parameter model-free approach to the interpretation of nitrogen-15 nuclear magnetic relaxation of proteins. J. Am. Chem. Soc 112, 4989–4936.
Woessner, D. (1962) Nuclear spin relaxaion in ellipsoids undergoing rotational brownian motion. J.Chem.Phys. 37, 647–654.
Favro, D. L. (1960) Theory of the Rotational Brownian Motion of a Free Rigid Body. Phys. Rev. 119, 53–62.
Ryabov, Y. E., and Fushman, D. (2007) A Model of Interdomain Mobility in a Multidomain Protein. J. Am. Chem. Soc. 129, 3315–3327.
Tjandra, N., Feller, S. E., Pastor, R. W., and Bax, A. (1995) Rotational diffusion anisotropy of human ubiquitin from 15 N NMR relaxation. J. Am. Chem. Soc. 117, 12562–12566.
Palmer, A. G., 3 rd, Grey, M. J., and Wang, C. (2005) Solution NMR spin relaxation methods for characterizing chemical exchange in high-molecular-weight systems. Methods Enzymol. 394, 430–465.
Fushman, D., Cahill, S., and Cowburn, D. (1997) The main chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: Analysis of 15 N relaxation with monomer/dimer equilibration. J. Mol. Biol. 266, 173–194.
Hall, J. B., and Fushman, D. (2006) Variability of the 15 N Chemical Shielding Tensors in the B3 Domain of Protein G from 15 N Relaxation Measurements at Several Fields. Implications for Backbone Order Parameters. J.Am.Chem.Soc. 128, 7855–7870.
Fushman, D., and Cowburn, D. (1998) Studying protein dynamics with NMR relaxation, in Structure, Motion, Interaction and Expression of Biological Macromolecules (Sarma, R., and Sarma, M., Eds.), pp 63–77, Adenine Press, Albany, NY.
Blackledge, M., Cordier, F., Dosset, P., and Marion, D. (1998) Precision and uncertainty in the characterization of anisotropic rotational diffusion by 15 N relaxation. J.Am.Chem.Soc. 120, 4538–4539.
Fushman, D., Xu, R., and Cowburn, D. (1999) Direct determination of changes of interdomain orientation on ligation: use of the orientational dependence of 15 N NMR relaxation in Abl SH(32). Biochemistry 38, 10225–10230.
Fushman, D., Ghose, R., and Cowburn, D. (2000) The effect of finite sampling on the determination of orientational properties: A theoretical treatment with application to interatomic vectors in proteins. J. Am. Chem. Soc. 122, 10640–10649.
Dosset, P., Hus, J. C., Blackledge, M., and Marion, D. (2000) Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data. J. Biomol. NMR 16, 23–28.
Ghose, R., Fushman, D., and Cowburn, D. (2001) Determination of the Rotational Diffusion Tensor of Macromolecules in Solution from NMR Relaxation Data with a Combination of Exact and Approximate Methods - Application to the Determination of Interdomain Orientation in Multidomain Proteins. J. Magn. Reson. 149, 214–217.
Walker, O., Varadan, R., and Fushman, D. (2004) Efficient and accurate determination of the overall rotational diffusion tensor of a molecule from 15 N relaxation data using computer program ROTDIF. J. Magn. Reson. 168, 336–345.
Fushman, D., Varadan, R., Assfalg, M., and Walker, O. (2004) Determining domain orientation in macromolecules by using spin-relaxation and residual dipolar coupling measurements. Prog. NMR Spectros. 44, 189–214.
Hall, J. B., Walker, O., and Fushman, D. (2004) Characterization of the overall rotational diffusion of a protein from 15 N relaxation measurements and hydrodynamic calculations, in Protein NMR techniques (Methods in Molecular Biology) (A.K.Downing, Ed.), pp 139–160, Humana Press Inc.
Fushman, D., and Cowburn, D. (2002) Characterization of Inter-Domain Orientations in Solution Using the NMR Relaxation Approach, in Protein NMR for the Millenium (Biological Magnetic Resonance Vol 20) (N. R. Krishna, L. B., Ed.), pp 53–78, Kluwer.
Fushman, D. (2002) Determination of protein dynamics using 15 N relaxation measurements, in BioNMR in drug research (O.Zerbe, Ed.), pp 283–308, Wiley-VCH.
Mandel, A. M., Akke, M., and Palmer, A. G. I. (1995) Backbone dynamics of E. coli Ribonuclease HI: correlations with structure and function in an active enzyme. J. Mol. Biol. 246, 144–163.
Press, W. H., Teukolsky, S. A., Vetterling, W. T., and Flannery, B. P. (1992) Numerical Recipes in C, Cambridge University Press, NY.
Fushman, D., Weisemann, R., Thüring, H., and Rüterjans, H. (1994) Backbone dynamics of ribonuclease T1 and its complex with 2’GMP studied by two-dimensional heteronuclear NMR spectroscopy. J. Biomol. NMR 4, 61–78.
Kay, L. E., Torchia, D. A., and Bax, A. (1989) Backbone dynamics of proteins as studies by N15 inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28, 8972–8979.
Hall, J. B., and Fushman, D. (2003) Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: Differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G. J. Biomol. NMR 27, 261–275.
Hall, J. B., and Fushman, D. (2003) Direct measurement of the transverse and longitudinal 15 N chemical shift anisotropy-dipolar cross-correlation rate constants using 1 H-coupled HSQC spectra. Mag. Res. in Chemistry 41, 837–842.
Ryabov, Y. E., Geraghty, C., Varshney, A., and Fushman, D. (2006) An efficient computational method for predicting rotational diffusion tensors of globular proteins using an ellipsoid representation. J. Am. Chem. Soc. 128, 15432–15444.
Acknowledgments
The development of DYNAMICS program was supported by NIH grant GM 065334. My work on this chapter has led to several modifications of the program, which hopefully made it user-friendlier, and I would like to thank the editors, Alex Shekhtman and David Burz, for being so patient with me during this process.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Fushman, D. (2012). Determining Protein Dynamics from 15N Relaxation Data by Using DYNAMICS. In: Shekhtman, A., Burz, D. (eds) Protein NMR Techniques. Methods in Molecular Biology, vol 831. Humana Press. https://doi.org/10.1007/978-1-61779-480-3_24
Download citation
DOI: https://doi.org/10.1007/978-1-61779-480-3_24
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-479-7
Online ISBN: 978-1-61779-480-3
eBook Packages: Springer Protocols