Evaluation of direct and cooperative contributions towards the strength ofburied hydrogen bonds and salt bridges

An experimental approach to evaluate the net binding free energy of buried hydrogen bonds and salt bridges is presented. The approach, which involves a modified multiple-mutant cycle protocol, was applied to selected interact ions between TEM-1-beta-lactamase and its protein inhibitor, BLIP. The sele cted interactions (two salt bridges and two hydrogen bonds) all involving B LIP-D49, define a distinct binding unit. The penta mutant, where all side-c hains constructing the binding unit were mutated to Ala, was used-as a refe rence state to which combinations of sidechains were introduced. At first, pairs of interacting residues were added allowing the determination of inte raction energies in the absence of neighbors, using double mutant cycles. A ddition of neighboring residues allowed the evaluation of their cooperative effects on the interaction. The two isolated salt bridges were either neut ral or repulsive whereas the two hydrogen bonds contribute 0.3 kcal mol(-1) each. Conversely, a double mutant cycle analysis of these interactions in their native environment showed that they all stabilize the complex by 1-1. 5 kcal mol(-1). Examination of the effects of neighboring residues on each of the interactions revealed that the formation of a salt bridge triad, whi ch involves two connected salt bridges, had a strong cooperative effect on stabilizing the complex independent of the presence or absence of additiona l neighbors. These results demonstrate the importance of forming net-works of buried salt bridges. We present theoretical electrostatic calculations w hich predict the observed mode of cooperativity, and suggest that the coope rative networking effect results from the favorable contribution of the pro tein to the interaction. Furthermore, a good correlation between calculated and experimentally determined interaction energies for the hive salt bridg es, and to a lesser extent for the two hydrogen bonds, is shown. The data a nalysis was performed on values of Delta Delta G(Kd)double dagger which ref lect the strength of short range interactions, while Delta Delta G(KD)(o) v alues which include the effects of long range electrostatic forces that alt er specifically Delta Delta G(ka)double dagger were treated separately. (C) 2000 Academic Press.