(1) Dept. Biochemistry, Weizmann Institure of Science, Rehovot 74024 Israel
(2) Cambridge Centre for Protein Engineering, Medical Research Council Centre,
Hills Road, Cambridge, CB2 2QH, UK
The association rate constant between barnase, an extracellular RNase of Bacillus amyloliquefaciens, and barstar, its intracellular inhibitor, is one of the fastest measured so far. This fast association is facilitated by favourable charge interactions between the proteins. Charge-charge interactions can be masked by increasing the ionic strength of the solution. The association rate constant between barnase and barstar correlates linearly with the electrostatic component of the chemical potential of a solution of NaCl or KCl, over an ionic strength of 5-2000 mM, as calculated from the Debye-Huckel equation. 22 differently-charged mutants of barnase or barstar were analysed to their ionic strength dependence, all of whom, except of one, displayed the linear correlation found for the wild-type complex, but with different slopes and intercepts (which represent the association rate constant at zero ionic strength). Both from the mutant data and from extrapolating the association rate constants to infinite ionic strength the association rate constant for barnase and barstar in the absence of electrostatic forces was calculated as 10**5 M-1s-1. This basal rate of association was found to be some what lower for the association of hirudin and thrombin, and could be decreased further by the addition of glycerol. The linear correlation between the association rate constant and the electrostatic potential of the solution breaks down for multiple charged mutations in barnase or thrombin, in which the association rate constant is slowed down by a factor of 1000 or more compared to the wild-type. Our results imply that the transition state for barnase associating with barstar is an early event in the association, in which only charge-charge interactions are of importance, and even those appear to be week at this stage, with a significant importance to the average potential at the binding sites. The deletion of multiple charges in the binding site shifts the transition state to a closer encounter, in which specific docking of polar and hydrophobic surfaces is of importance. This conclusion was affirmed by the linear correlation found between the average electrostatic potentials at the binding site for different barnase and barstar mutants, as calculated with the DelPhi program, with their experimentally measured association rate constants.