Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system

The contribution of the Ser45 hydrogen bond to biotin binding activation an d equilibrium thermodynamics was investigated by biophysical and X-ray crys tallographic: studies. The S45A mutant exhibits a 1,700-fold greater dissoc iation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 degrees C. indicating a crucial role in bindin g energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrog en bonds to biotin retain approximately the same lengths. No additional wat er molecules an observed to replace the missing hydroxyl, in contrast to th e previously studied D128A mutant. The equilibrium Delta G degrees,Delta H degrees, Delta S degrees, Delta C degrees(p), and activation Delta G double dagger of S45A at 37 degrees C Lire -13.7 +/- 0.1 kcal/mol, -21.1 +/- 0.5 kcal/mol, -23.7 +/- 1.8 cal/mol K, -223 +/- 12 cal/mol K, and 20.0 +/- 2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the Delta H double dagger and Delta S double dagger of dissociation. 25.8 +/- 1.2 kcal/mol and 18.7 +/- 4.3 cal/mol K. The large increases of Delta H double dagger and S double dagger in the mut ant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically s tabilizing it, and constraining the transition state entropically. The post ulated existence of a Ser45-mediated hydrogen bond in the wild-type strepta vidin transition state is consistent with potential of mean force simulatio ns of the dissociation pathway and with molecular dynamics simulations of b iotin pullout, where Ser45 is seen to from a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.