Abstract
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.
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References
J. Jankaric and S.-H. Kim, Sparse matrix sampling: a screening method for crystallization of proteins, J. Appl Crystal. 24:409 (1991).
E.A. Stura and I.A. Wilson, Applications of the streak seeding technique in protein crystallization, J. Crystal Growth 110:270 (1991).
S.D. Durbin and G. Feher, Studies of crystal-growth mechanisms of proteins by electron microscopy, J. Mol Biol. 212:763(1990).
S.D. Durbin and W.E. Carlson, Lysozyme crystal-growth studied by atomic force microscopy, J. Crystal Growth 122:71 (1992).
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).
M.L. Pusey, Estimation of the initial equilibrium-constants in the formation of tetragonal lysozyme nuclei, J. Crystal Growth 110:60 (1991).
M. Skouri, M. Delsanti, J.P. Munch, B. Lorber, and R. Giegé, Dynamic light scattering-studies of the aggregation of lysozyme under crystallization conditions, Febs- Letters 295:84 (1991).
Y. Georgalis, A. Zouni, and W. Saenger, Dynamics of protein precrystallization cluster formation , J. Crystal Growth 118:360(1992).
Y. Georgalis, A. Zouni, W. Eberstein, and W. Saenger, Formation dynamics of protein precrystallization fractal clusters, J. Crystal Growth 126:245 (1993).
A.J. Malkin, J. Cheung, and A. McPherson, Crystallization of satellite tobacco mosaic-virus. 1. Nucleation phenomena, J. Crystal Growth 126:544 (1993).
A.J. Malkin and A. McPherson, Crystallization of satellite tobacco mosaic virus. 2. Postnucleation events,J Crystal Growth 126:555 (1993).
A. George and W.W. Wilson, Predicting protein crystallization from a dilute solution property, Acta Cryst.D 50:361 (1994).
D.F. Rosenbaum and C.F. Zukowski, Protein interactions and crystallization, J. Cryst. Growth 169:752 (1996).
F. Boue, F. Lefaucheux, M.C. Robert, and I. Rosenman, Small-angle neutron-scattering study of lysozyme solutions, J. Crystal Growth 133:246 (1993).
N. Niimura, Y. Minezaki, M. Ataka, and T. Katsura, Aggregation in supersaturated lysozyme solution studied by time-resolved small angle neutron scattering, J. Crystal Growth 154:136 (1995).
Y. Minezaki, N. Niimura, M. Ataka, and T. Katsura, Small-angle neutron-scattering from lysozyme solutions in unsaturated and supersaturated states, Biophys. Chem. 58:355 (1996).
P. Todd, S.K. Sikdar, C. Walker, and Z.R. Korszun, Application of dewatering to the controlled crystallization of biological macromolecules and organic-compounds, J. Crystal Growth 110:283 (1991).
J. Behlke and A. Knespel, Observation of precrystallization aggregation in protein solutions during centrifugation, J. Crystal Growth 158:388 (1996).
A.M. Lenhoff, P.E. Pjura, J.G. Dilmore, and T.S. Godlewski, Ultracentrifugal crystallization of proteins - transport-kinetic modeling, and experimental behavior of catalase, J. Crystal Growth 180:113 (1997).
H. Fujita, Mathematical Theory of Sedimentation Analysis. Academic Press, New York (1962).
L.A. Holladay, An approximate solution of the Lamm equation, Biophys. Chem. 10:187 (1979).
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).
J.S. Philo, An improved function for fitting sedimentation velocity data for low-molecular weight solutes, Biophys. J. 72:435 (1997).
J. Behlke and O. Ristau, Molecular mass determination by sedimentation velocity experiments and direct fitting of the concentration profiles, Biophys. J. 72:428 (1997).
J. Behlke and O. Ristau, An improved approximate solution of the Lamm equation for the simultaneous estimation of sedimentation and diffusion coefficients from sedimentation velocity experiments, Biophys. Chem. 70:133 (1998).
J.M. Claverie, H. Dreux, and R. Cohen, Sedimentation of generalized systems of interacting particles. I. Solution of systems of complete Lamm equations, Biopolymers 14:1685 (1975).
B. Demeler and H. Saber, Determination of molecular-parameters by fitting sedimentation data to finite element solutions of the Lamm equation, Biophys. J. 74:444 (1998).
P. Schuck, Sedimentation analysis of noninteracting and self-associating solutes using numerical - solutions of the Lamm equation, Biophys. J. 75:1503 (1998).
H. Fujita and V.J. MacCosham, Extension of sedimentation to molecules of intermediates sizes, J. Chem.Phys. 30:291 (1959).
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).
V.V. Barynin and W.R. Melik-Adamyan, The ultracentrifugation protein crystallization mechanism, Kristallografiya 27:981 (1982).
J. Behlke and O. Ristau, Analysis of the thermodynamic non-ideality by sedimentation equilibrium experiments, Biophys. Chem. 76:13 (1999).
V. Mikol, E. Hirsch, and R. Giegé, Diagnostic of precipitant for biomacromolecular crystallization by quasi-elastic light-scattering, J. Mol. Biol. 213:187 (1990).
J. Wyman and S.J. Gill, Binding and Linkage, University Science Books, Mill Valley, CA (1990).
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Behlke, J., Ristau, O. (2001). Analytical Ultracentrifugation: A Valuable Tool to Recognize Crystallization Conditions of Proteins. In: Regel, L.L., Wilcox, W.R. (eds) Processing by Centrifugation. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-0687-4_8
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DOI: https://doi.org/10.1007/978-1-4615-0687-4_8
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