Abstract
In this chapter, we discuss experimental and theoretical methods for characterizing the overall rotational diffusion of molecules in solution. The methods are illustrated for the B3 domain of protein G, a small protein with rotational anisotropy of D par/D perp = 1.4. The rotational diffusion tensor of the protein is determined directly from 15N relaxation measurements. The experimental data are treated assuming various possible models for the overall tumbling: isotropic, axially symmetric, and fully anisotropic, and the results of these analyses are compared to determine an adequate diffusion model for the protein. These experimentally derived characteristics of the protein are compared with the results of theoretical calculations of the diffusion tensor using various hydrodynamic models, to find optimal models and parameter sets for theoretical predictions. We also derive model-free characteristics of internal backbone motions in the protein, to show that different models for the overall motion can result in significantly different pictures of motion. This emphasizes the necessity of accurately characterizing the overall tumbling of a molecule to determine its local dynamics.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.). Adenine Press, Albany, NY, pp. 63–77.
Schurr, J. M., Babcock, H. P., and Fujimoto, B. S. (1994) A test of the model-free formulas: effects of anisotropic rotational diffusion and dimerization. J. Magn. Reson. B105, 211–224.
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.
Tjandra, N., Wingfield, P., Stahl, S., and Bax, A. (1996) Anisotropic rotational diffusion of predeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields. J. Biomol. NMR 8, 273–284.
Luginbuhl, P., Pervushin, K. V., Iwai, H., and Wuthrich, K. (1997) Anisotropic molecular rotational diffusion in 15N spin relaxation studies of protein mobility. Biochemistry 36, 7305–7312.
Eisenmesser, E. Z., Bosco, D. A., Akke, M., and Kern, D. (2002) Enzyme dynamics during catalysis. Science 295, 1520–1523.
Fushman, D., Xu, R., and Cowburn, D. (1999) Direct determination of changes of interdomain orientation on ligation: use of the orientational dependence of 15N NMR relaxation in Abl SH(32) Biochemistry 38, 10,225–10,230.
Varadan, R., Walker, O., Pickart, C., and Fushman, D. (2002) Structural properties of polyubiquitin chains in solution. J. Mol. Biol. 324, 637–647.
Tjandra, N., Garrett, D. S., Gronenborn, A. M., Bax, A., and Clore, G. M. (1997) Defining long range order in NMR structure determination from the dependence of heteronuclear relaxation times on rotational diffusion anisotropy. Nat. Struct. Biol. 4, 443–449.
Akerud, T., Thulin, E., Van Etten, R. L., and Akke, M. (2002) Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding. J. Mol. Biol. 322, 137–152.
Tjandra, N., Feller, S. E., Pastor, R. W., and Bax, A. (1995) Rotational diffusion anisotropy of human ubiquitin from 15N NMR relaxation. J. Am. Chem. Soc. 117, 12,562–12,566.
Lee, L. K., Rance, M., Chazin, W. J., and Palmer, A. G., III. (1997) Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13C alpha nuclear spin relaxation. J. Biomol. NMR 9, 287–298.
Copie, V., Tomita, Y., Akiyama, S. K., et al. (1998) Solution structure and dynamics of linked cell attachment modules of mouse fibronectin containing the RGD and synergy regions: comparison with the human fibronectin crystal structure. J. Mol. Biol. 277, 663–682.
Blackledge, M., Cordier, F., Dosset, P., and Marion, D. (1998) Precision and uncertainty in the characterization of anisotropic rotational diffusion by 15N relaxation. J. Am. Chem. Soc. 120, 4538, 4539.
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.
Fushman, D., Varadan, R., Assfalg, M., et al. (2004). Determining domain orientation in macromolecules by using spin-relaxation and residual dipolar coupling measurements. Prog. NMR Spectr., in press.
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 Spectr., in press.
Gronenborn, A. M., Filpula, D. R., Essig, N. Z., et al. (1991) A novel highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science 253, 657–661.
Achari, A., Hale, S. P., Howard, A. J., et al. (1992) 1.67-A X-ray structure of the B2 immunoglobulin-binding domain of streptococcal protein G and comparison to the NMR structure of the B1 domain. Biochemistry 31, 10,449–10,457.
Derrick, J. P. and Wigley, D. B. (1992) Crystal structure of a streptococcal protein G domain bound to a Fab fragment. Nature 359, 752–754.
Derrick, J. P., Wigley, D. B., Lian, L. Y., et al. (1993) Structure and mechanism of streptococcal protein G. Biochem. Soc. Trans. 21, 333S.
Gallagher, T., Alexander, P., Bryan, P., and Gilliland, G. L. (1994) Two crystal structures of the B1 immunoglobulin-binding domain of streptococcal protein G and comparison with NMR. Biochemistry 33, 4721–4729.
Derrick, J. P. and Wigley, D. B. (1994) The third IgG-binding domain from streptococcal protein G: an analysis by X-ray crystallography of the structure alone and in a complex with Fab. J. Mol. Biol. 243, 906–918.
Lian, L. Y., Derrick, J. P., Sutcliffe, M. J., Yang, J. C., and Roberts, G. C. (1992) Determination of the solution structures of domains II and III of protein G from Streptococcus by 1H nuclear magnetic resonance. J. Mol. Biol. 228, 1219–1234.
Fushman, D., Cahill, S., and Cowburn, D. (1997) The main chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: analysis of 15N relaxation with monomer/dimer equilibration. J. Mol. Biol. 266, 173–194.
Fushman, D., Tjandra, N., and Cowburn, D. (1999) An approach to direct determination of protein dynamics from 15N NMR relaxation at multiple fields, independent of variable 15N chemical shift anisotropy and chemical exchange contributions. J. Am. Chem. Soc. 121, 8577–8582.
Fushman, D. (2002) Determination of protein dynamics using 15N relaxation measurements. In BioNMR in Drug Research (Zerbe, O., ed.). Wiley-VCH, New York, pp. 283–308.
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 (Krishna, N. R., ed.). Kluwer, Dordrecht, The Netherlands, pp. 53–78.
Fushman, D., Cahill, S., and Cowburn, D. (1997) The main-chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: analysis of 15N relaxation with monomer/dimer equilibration. J. Mol. Biol. 266, 173–194.
Fushman, D., Weisemann, R., Thüring, H., and Rüterjans, H. (1994) Backbone dynamics of ribonuclease T1 and its complex with 2′GMP studied by twodimensional heteronuclear NMR spectroscopy. J. Biomol. NMR 4, 61–78.
Draper, N. R. and Smith, H. (1981) Applied Regression Analysis. Wiley, New York.
Goldstein, H. (1980) Classical Mechanics. 2nd ed. Addison-Wesley, Boston.
Koenig, S. (1975) Brownian motion of an ellipsoid. Correction to Perrin’s results. Biopolymers 14, 2421–2423.
Cantor, C. R. and Schimmel, P. R. (1980) Biophysical Chemistry. 3 vols. Freeman, New York.
Tirado, M. M. and de la Torre, J. G. (1980) Rotational dynamics of rigid, symmetric top macromolecules: application to circular cylinders. J. Chem. Phys. 73, 1986–1993.
Fushman, D. (1990) Surface fractality of proteins from theory and NMR data. J. Biomol. Struct. Dyn. 7, 1333–1344.
de la Torre, J. G., Navarro, S., Martinez, M. C. L., Diaz, F. G., and Cascales, J. J. L. (1994) HYDRO: Acomputer program for the prediction of hydrodynamic properties of macromolecules. Biophys. J. 67, 530, 531.
de la Torre, J. G., Huertas, M. L., and Carrasco, B. (2000) HYDRONMR: prediction of NMR relaxation of globular proteins from atomic-level structures and hydrodynamic calculations. J. Magn. Reson. B147, 138–146.
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.
DeLano, W. L. (2002) The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Blake-Hall, J., Walker, O., Fushman, D. (2004). Characterization of the Overall Rotational Diffusion of a Protein From 15N Relaxation Measurements and Hydrodynamic Calculations. In: Downing, A.K. (eds) Protein NMR Techniques. Methods in Molecular Biology™, vol 278. Humana Press. https://doi.org/10.1385/1-59259-809-9:139
Download citation
DOI: https://doi.org/10.1385/1-59259-809-9:139
Publisher Name: Humana Press
Print ISBN: 978-1-58829-246-9
Online ISBN: 978-1-59259-809-0
eBook Packages: Springer Protocols