Papers Published

  1. Chilkoti, Ashutosh and Stayton, Patrick S., Molecular origins of the slow streptavidin-biotin dissociation kinetics, Journal of the American Chemical Society, vol. 117 no. 43 (1995), pp. 10622 - 10628 .
    (last updated on 2007/04/12)

    The association of streptavidin and avidin with biotin is among the strongest known noncovalent protein-ligand interactions (Ka nearly equals 2.5 × 1013 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. 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. 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. 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.

    Dissociation;Reaction kinetics;Electron energy levels;Activation energy;