Change of Fractal Dimension during the Nucleation Phase of Lysozyme Crystallization

R. J. Heigl1, J. Stellbrink2, A. Radulescu1, R. Schweins3, T. E. Schrader1, D. Richter2

1Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH, Lichtenbergstr.1, 85748 Garching, Germany

2Jülich Centre for Neutron Science JCNS-1 and Institute for Complex Systems ICS-1, Forschungszentrum Jülich GmbH, 52525 J?ich, Germany

3Institut Laue Langevin (ILL), DS / LSS, 71 avenue des Martyrs, 38000 Grenoble, France

t.schrader@fz-juelich.de

In this study we focused on the question how to grow crystals as large as possible in light of their use as samples for neutron protein crystallography. We concentrated on the early stages of the crystallization process where the directions are set whether many small crystals grow or few large ones. We used lysozyme as a model system since it has been studied well in the past and the phase diagram of its crystal growth is known. We used a combination of three scattering techniques since the involved size ranges require a large q-range. Small angle neutron scattering was used in combination with static light scattering on the same sample in order to obtain structural information on the growing crystal seeds. In situ dynamic light scattering at the neutron scattering sample cell was used to obtain an overview of all sizes present in the crystallization process by measuring their hydrodynamic radii. The small angle neutron scattering technique requires crystallization in heavy water instead of normal water. We found that the crystallization conditions did not differ too much from the ones mentioned in the literature for light water when using a corrected pD value of pD=pH+0.4. The crystallization is initiated by mixing a 60 mg/ml Lysozyme solution with a 6 wt% NaCl acetate buffer solution (both at pD=4.75 and at 298 K) in a 1 :1 ratio. Immediately after mixing, dimers of lysozyme molecules are formed and the structure factor seen in the lysozyme stock solution disappears. Under the chosen conditions we could observe a fractal growth of the cluster of monomers with a change of the fractal dimension from 1.0 to 1.7 in the first 90 minutes. This can be interpreted as clusters being formed first which grow more in a linear manner with little branching. Later, a swelling occurs corresponding to a growth in the dimension perpendicular to the previous linear growth. With these results theoretical models of crystal growth can be improved. Furthermore, the early detection of crystal seeds can be used to rapidly change the crystallization conditions (e. g. temperature) in order to avoid the production of more crystal seeds.