Isolation, characterization, and magnesium‐induced self‐association kinetics of discrete aggregates of RecA protein from Escherichia coli

Abstract Dynamic and static intensity light scattering techniques were employed to identify conditions allowing preparation of homogeneous solutions of distinct oligomeric states of RecA protein. These hydrodynamically distinguishable oligomer populations of RecA protein were obtained in homogeneous pure quantities sufficient for physical studies. Results indicate two fairly narrow distributions of RecA oligomers comprised on average of 42 ± 3 and 18 ± 1 RecA monomers. These structures, denoted RecA 42 and RecA 18 , respectively, could be obtained reproducibly in milligram quantities and were stable for at least one week. This enabled reliable characterizations of their hydrodynamic properties by dynamic and total intensity light scattering. These measurements revealed RecA 42 had an average translational diffusion coefficient, D 20 (L) = 8 ± 2 × 10 −8 cm 2 /s, molecular weight, M r = 1.6 ± 0.1 × 10 6 , and radius of gyration, R G = 465 ± 29 Å. The smaller aggregate, RecA 18 , had D 20 (S) = 20.5 ± 2.5 × 10 −8 cm 2 /s. M r = 7.0 ± 0.4 × 10 5 , and R G = 300 ± 20 Å. Heating RecA 18 at 37°C overnight resulted in conversion to a species with hydrodynamic properties indistinguishable from RecA 42 , called RecA 18/42 . Conversion of RecA 42 to RecA 18 occurred almost instantaneously by 50% dilution at 38°C or very slowly with incubation at 4°C for at least 39 days. Self‐association reactions of the three starting oligomeric states (RecA 18 , RecA 42 , and RecA 18/42 ) induced by MgCl 2 were monitored at several temperatures by dynamic light scattering. Results of these experiments provided evaluations of kinetic activation parameters of the self‐association reactions. The activation parameters found for each starting oligomeric state of the protein were significantly different, revealing the variable influence of MgCl 2 on the activation barriers to RecA self‐association. Highly aggregated equilibrium solutions that ultimately form in solutions of each starting oligomeric species, incubated in MgCl 2 at 38°C for four days, were characterized by total intensity light scattering. Interpretations of these data in terms of characteristic behavior of random polymers suggests the surface morphologies of these highly associated equilibrium states formed from RecA 42 and RecA 18/42 are similar but contrast with that of RecA 18 . Calculated values of the translational diffusion coefficient D 0 were obtained for oligomeric structures modeled as helical arrays of connected monomer spheres. Best agreement with experimentally determined diffusion coefficients required that constituent monomer spheres of RecA 42 have radii 33–40% larger than the monomer spheres of RecA 18 . Results suggest the hydrodynamically distinct oligomeric forms of RecA may reside in conformational states with different surface exposure of hydrophobic residues, which results in substantial differences in local solvation/hydration. © 1996 John Wiley & Sons, Inc.