(618a) Use of Crosslinked Hydrogels for the Control of Macromolecular Crystallization
The potential of crosslinked hydrogels in the crystallization of proteins is an area that has not been exploited. The technique of counter-diffusion is a novel method for the crystallization of proteins which entails the minimization of convection effects resulting in high quality crystals. The implementation of this technique in capillary tubes with different diameters has been accepted for the crystallization of proteins, by using a precipitating solution which diffuses through an agarose polymer that controls the diffusion process. The diffusional process using such systems can be controlled but cannot be widely manipulated. The use of crosslinked hydrogels provides better diffusional and environmental control.
This research focuses on the role of crosslinked hydrogels on the nucleation process involved in protein crystallization. The crystallization process was performed using crosslinked Poly(ethyleneglycol) hydrogels and lysozyme as a model protein. The effects of polymer concentration and hydrogel pore size on crystallization were studied. To accomplish this, different dilutions of pre-polymer solutions were prepared using the protein as solvent. These solutions were poured in capillaries of 0.8 millimeters in diameter. Also a membrane or plug of different polymer percentages using water as solvent to control diffusion was employed using in this case, capillaries of 4 millimeters in diameter. Both of the solutions were exposed to a UV light source to induce polymerization. A fluorescent monomer was also employed to determine if the crystal structure is formed around or if it pushes polymer chains apart. Confocal Laser Scanning Microscopy was employed to assess the fluorescence throughout the crystal structure.
Results indicated that the diffusion coefficient calculated from the measurement of the crystallization displacement as a function of time was significantly different for each of the plug conditions including the 80%, 65%, and 50% dilutions. Mesh sizes were also measured and they varied from 10-7nm. Moreover, when the polymer concentration was varied qualitative differences in crystal structures were observed. Studies using a fluorescent crosslinker indicated that the polymer was incorporated into the crystal structure. These results indicated that nucleation and crystal growth can be controlled by the manipulation of polymer composition which cannot be easily obtained with current physically crosslinked hydrogels.