PRE-NUCLEATION CRYSTALLIZATION STUDIES ON AMINOACYL-TRANSFER RNA-SYNTHETASES BY DYNAMIC LIGHT-SCATTERING

Dynamic light-scattering (DLS) studies on solutions of proteins approaching their precipitation point were made with asparaginyl- (NRSEC), leucyl- (LRSEC) and valyl- (VRSEC) tRNA synthetases from Escherichia coli. The three aminoacyl-tRNA synthetases have not been crystallized previously. As a control system, we used E. coli polypeptide elongation factor Tu (EF-Tu). Apart from the different proteins used here, the methods we employed differed from previous studies in that (1) instead of making a series of measurements on individual samples at various concentrations, the protein solutions were titrated with the precipitants, and (2) the results of the light-scattering measurements were analysed 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 showed two major peaks in most cases. For both peaks, relative areas and mean diffusion coefficients were determined. The diffusion constants were corrected for the viscosity of the solutions. From comparing the results on the proteins known to crystallize (EF-Tu) with the amorphously precipitating systems (LRSEC, NRSEC) we find two necessary, but not sufficient, conditions for the formation of crystals: the diffusion coefficient of the monomer peak stays constant until very close to the precipitation point; the percentage of large aggregates stays small (<10% of the scattered light intensity) during the titration. For VRSEC, 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. Crystallization trials using both these salts were carried out, and 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. © 1992.