| Popis: |
Dynamic light scattering (DLS) techniques have been used on several systems to distinguish between amorphous and crystalline aggregation of proteins during precipitation. Most of these studies suffer from either of two defects: (i) they have been confined to model protein systems where crystallization conditions are well established, and some cannot be considered as typical proteins in their crystallization behavior, and (ii) the analysis method used to evaluate the DLS results, using a first-cumulant method to give an average hydrodynamic radius, is inappropriate for highly polydisperse systems such as protein solutions containing the free protein together with large aggregates. We present dynamic light scattering (DLS) studies on solutions of aminoacyl-tRNA synthetases as they approach the crystallization point. The systems studied were asparaginyl- (NRSEC), leucyl- (LRSEC), and valyl-tRNA synthetase (VRSEC) fromE. coli. As a control system, we used theE. coli elongation factor Tu (EF-Tu). NRSEC, LRSEC, and VRSEC had not been crystallized before. Apart from the different proteins used here, the methods that we have employed differ from previous studies in that (i) instead of making a series of measurements on individual samples at various concentrations, the protein solutions were titrated with the precipitants, and (ii) the results of the light scattering measurements were analyzed by a new maximum entropy procedure that calculates a particle size distribution in a highly reproducible way. The particle size distributions of protein solutions titrated with precipitants are bimodal in most cases. For both peaks, relative areas and mean diffusion coefficients were determined. From comparing the results on the proteins known to crystallize (EF-Tu) with the amorphously precipitating systems (LRS, NRS) we find two necessary, but not sufficient, conditions for the formation of crystals: (i) the diffusion coefficient of the monomer peak stays constant until very close to the precipitation point and (ii) the percentage of large aggregates stays small ( < 10% of the scattered light intensity) during the titration. For VRS both ammonium sulphate and sodium citrate showed a low percentage of large aggregates and a constant diffusion coefficient of the main (protein monomer) peak below the precipitation point. This indicates that both would be possible precipitants for the crystallization of this enzyme. Although no condition could as yet be found for obtaining crystals with ammonium sulphate solutions, crystals of the enzyme have been obtained with sodium citrate. These grow as multicrystals of fine needles; studies are underway to obtain crystals more suitable for diffraction analysis. |
