Kinetic analysis of the pre-equilibrium steps in the self-assembly of RecA protein from Escherichia coli.
Total intensity light scattering is employed to investigate the self-assembly kinetics of RecA protein. Reaction conditions are employed where the kinetics of self-assembly are slow enough to yield reliable scattered intensity measurements over the range of scattering angles from 40 to 130 degrees as a function of time. From these measurements the time-dependent behavior of the weight average molecular weight, Mr, and radius of gyration, RG, of the associating protein species as a function of [MgCl2], [NaCl], [RecA], and pH was determined. The temperature dependence of RecA self-assembly was also investigated and allowed an evaluation of the activation thermodynamic parameters of association. Results reveal RecA self-assembly is bi-phasic under all conditions examined. The first phase, referred to as "filamentation" is second-order in [RecA] and occurs via a quasi linear condensation scheme with an Arrhenius activation energy of 88.6 kcal/mol. Filamentation assembly involves the uptake of one proton, one MgCl2, the release of five to six NaCls, and is driven by the release of approximately 70 water molecules. The evaluated activation parameters of the first kinetic phase are consistent with the proposition that linear self-assembly of RecA protein into ordered filaments is entropically driven. The second kinetic phase, referred to as "bundling" is greater than second-order in both [RecA] and [MgCl2], is considerably slower that filamentation assembly, and is apparently initiated by 2nd order collisions of linear filaments.
Wilson
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DH
Department of Chemistry, University of Illinois, Chicago 60680.
Benight
A S
AS
D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D011938:Rec A Recombinases / Q000096:biosynthesis*; D012542:Scattering, Radiation; D012965:Sodium Chloride / Q000494:pharmacology; D013053:Spectrophotometry; D013816:Thermodynamics; D013997:Time Factors
D046911:Macromolecular Substances; D015636:Magnesium Chloride; D012965:Sodium Chloride; D011938:Rec A Recombinases