MOLECULAR-ORIGINS OF THE SLOW STREPTAVIDIN-BIOTIN DISSOCIATION KINETICS

The association of streptavidin and avidin with biotin is among the strongest known noncovalent protein-ligand interactions (K-a approximate to 2.5 x 10(13) M(-1)) and is controlled by an exceptionally slow off-rate. We have used this model system to elucidate the role of aromatic tryptophan side-chain binding contacts in the dissociation reaction coordinate and relatedly to the construction of the activation barrier and to the structure of the transition state. The significantly lower dissociation t(1/2) of conservative Trp to Phe site-directed mutants, 35 h (wild-type) > 5.5 h (W79F) > 2 h (W108F) > 0.5 h (W120F), reveals the importance of these Trp contacts in regulating the dissociation rate and also highlights the position dependence of the Trp contributions, most notably the optimized ''capping'' interaction of Trp 120 with biotin. We have also conducted a transition state analysis of the temperature-dependent dissociation kinetics, which along with the independent estimation of the equilibrium biotin-binding free energies and enthalpies has provided thermodynamic profiles defining the enthalpic, entropic, and free energy barriers to dissociation for the mutants relative to wild-type streptavidin. The increased biotin off-rate for W79F, which contacts the valeric acid moiety of biotin, and for W120F, which partially caps the bicyclic ring system, is caused largely by free energy destabilization of the ligand-bound ground state relative to wild-type streptavidin. This free energy destabilization is controlled by a 2.4 kcal mol(-1) entropic destabilization of the ligand-bound W79F ground state relative to wildtype at 298 K, and a 5.1 kcal mol(-1) enthalpic destabilization of the ligand-bound W120F ground state relative to wild-type. W79F displays an increased equilibrium binding enthalpy relative to wild-type, and thus streptavidin sacrifices potential binding enthalpy to minimize the entropic costs of biotin immobilization. This energetic role correlates well with the structural role of Trp 79, where the side chain contacts the valeric acid tail of biotin, the most conformationally flexible portion of the ligand and thus the most entropically costly to immobilize. The 17-fold increase in off-rate for W108F, on the other hand, which contacts the bicyclic ring of biotin from a buried position within the biotin-binding site, is largely due to the stabilization of the transition state, and is driven by a large 6.5 kcal mol(-1) increase in the favorable activation entropy relative to wild-type streptavidin at 298 K. These results are consistent with a snapshot of the transition state where biotin and/or the protein have moved such that Trp 79 and Trp 120 no longer maintain strong contact with biotin, while the contact with Trp 108 remains energetically significant.