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.