Abstract
Although high affinity binding between streptavidin and biotin is widely exploited, the accompanying low rate of dissociation prevents its use in many applications where rapid ligand release is also required. To combine extremely tight and reversible binding, we have introduced disulfide bonds into opposite sides of a flexible loop critical for biotin binding, creating streptavidin muteins (M88 and M112) with novel disulfide-switchable binding properties. Crystal structures reveal how each disulfide exerts opposing effects on structure and function. Whereas the disulfide in M112 disrupts the closed conformation to increase koff, the disulfide in M88 stabilizes the closed conformation, decreasing koff 260-fold relative to streptavidin. The simple and efficient reduction of this disulfide increases koff 19,000-fold, thus creating a reversible redox-dependent switch with 70-fold faster dissociation kinetics than streptavidin. The facile control of disulfide formation in M88 will enable the development of many new applications requiring high affinity and reversible binding.
Highlights
High affinity binding between streptavidin and biotin is widely exploited, the accompanying low rate of dissociation prevents its use in many applications where rapid ligand release is required
To create a more general solution to the central problem of increasing rates of dissociation without compromising the desirability of extremely high binding affinity, we report for the first time streptavidin mutants in which the reversible formation and breakage of a strategically located intramolecular disulfide bond acts as a molecular switch converting between a high affinity/low rate of dissociation state and a low affinity/high rate of dissociation state
It is notable that several streptavidin homologs with naturally occurring disulfide bonds in loop [3,4] have been previously reported[24,25,26,27,28]
Summary
High affinity binding between streptavidin and biotin is widely exploited, the accompanying low rate of dissociation prevents its use in many applications where rapid ligand release is required. In combination with the additional stabilizing effects of Trp-120, the addition of the engineered disulfide bond in M88 explains the substantially decreased rate of biotin dissociation when compared to wild-type streptavidin.
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