Experimental assignment of the structure of the transition state for the association of barnase and barstar

Association of a protein complex follows a two step reaction mechanism, wit h the first step being the formation of an encounter complex which evolves into the final complex. Here we present new experimental data for the assoc iation of the bacterial ribonuclease barnase and its polypeptide inhibitor barstar which shed light on the thermodynamics and structure of the transit ion state and preceding encounter complex of association at diminishing ele ctrostatic attraction. We show that the activation entropy at the transitio n state is close to zero, with the activation enthalpy being equal to the f ree energy of binding. This observation was independent of the magnitude of the mutual electrostatic attraction, which were altered by mutagenesis or by addition of salt. The low activation entropy implies that the transition state is mostly solvated at all ionic strengths. The structure of the tran sition state was probed by measuring pairwise interaction energies using do uble-mutant-cycles. While at low ionic strength all proximal charge-pairs f orm contacts, at high salt only a subset of these interactions are maintain ed. More specifically, charge-charge interactions between partially buried residues are lost, while exposed charged residues maintain their ability to form specific interactions even at the highest salt concentration. Uncharg ed residues do not interact at any ionic strength. The results presented he re suggest that the barnase-barstar binding sites are correctly aligned dur ing the transition state even at diminishing electrostatic attraction, alth ough specific short range interactions of uncharged residues are not yet fo rmed. Furthermore, most of the interface desolvation (which contributes to the entropy of the system) has not yet occurred. This picture seems to be v alid at low and high salt. However, at high salt, interactions of the activ ated complex are limited to a more restricted set of residues which are eas ier approached during diffusion, prior to final docking. This suggest that the steering region at high salt is more limited, albeit maintaining its sp ecificity. (C) 2001 Academic Press.