Analytical Ultracentrifugation: A Valuable Tool to Recognize Crystallization Conditions of Proteins
The use of analytical ultracentrifugation to elucidate crystallization conditions for proteins is discussed. The methods are based on careful enhancement of the protein concentration by sedimentation, leading to a partial formation of associates. The results can be monitored either from the broadening of the moving boundary in a Schlieren pattern or from the considerable concentration increase near the cell base. The conditions under which highly charged proteins are able to undergo a self-association process (nucleation) as prerequisite for crystallization can be determined from the second virial coefficient, to which the excluded volume and the net charge mainly contribute. Crystallization experiments are successful when the repulsive forces of charged proteins are screened by addition of neutral salts and the net charge contribution becomes smaller than that of the excluded volume. Protein crystallization can be considered as an open association event with binding constants in the millimolar range. An exact description of single steps by using solutions of the Lamm differential equation is not possible because of the unknown association kinetics and the large number of parameters that must be estimated. However, based on fitting of simulated concentration profiles we can exclude a statistical binding mode for nucleation.
KeywordsSedimentation Velocity Virial Coefficient Crystallization Condition Analytical Ultracentrifugation Sedimentation Equilibrium
Unable to display preview. Download preview PDF.
- 5.M. Jullien, M.P. Crosio, S. Baudet-Nessler, F. Merola, and J.C. Brochon, Evidence for a dimeric intermediate on the crystallization pathway of ribonuclease-A, Acta Crystallographica D 50:398 (1994).Google Scholar
- 14. F.
- 20.H. Fujita, Mathematical Theory of Sedimentation Analysis. Academic Press, New York (1962).Google Scholar
- 22.J.S. Philo, Measuring sedimentation, diffusion, and molecular weights of small molecules by direct fitting of sedimentation velocity concentration profiles, in: Modern Analytical Ultracentrifugation, T. M. Schuster and T. M. Laue, eds., Birkhäuser, Boston (1994).Google Scholar
- 30.P.R. Wills and D.J. Winzor, in: Analytical Ultracentrifugation in Biochemistry and Polymer Science, S. E. Harding, A. J. Rowe, J. C. Horton, eds., Royal Society, Cambridge, U.K. (1992).Google Scholar
- 31.V.V. Barynin and W.R. Melik-Adamyan, The ultracentrifugation protein crystallization mechanism, Kristallografiya 27:981 (1982).Google Scholar
- 34.J. Wyman and S.J. Gill, Binding and Linkage, University Science Books, Mill Valley, CA (1990).Google Scholar