Engineering a disulfide-gated switch in streptavidin enables reversible binding without sacrificing binding affinity.
Jesse M Marangoni1, Sau-Ching Wu1, Dawson Fogen1,2
1Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada.
Scientific Reports
|July 29, 2020
Summary
Researchers engineered streptavidin (a protein) variants with disulfide bonds to control binding. This creates a reversible, redox-dependent switch for high-affinity, rapidly releasable biotin binding, enabling new applications.
Area of Science:
- Biochemistry
- Protein Engineering
- Structural Biology
Background:
- The high affinity of streptavidin-biotin interaction is extensively utilized.
- However, the slow dissociation rate limits applications requiring rapid ligand release.
Purpose of the Study:
- To engineer streptavidin variants with tunable, switchable binding properties.
- To combine high affinity with rapid, controllable dissociation kinetics.
Main Methods:
- Introduction of disulfide bonds into a flexible loop of streptavidin.
- Creation of streptavidin muteins (M88 and M112).
- Analysis of crystal structures to understand structural and functional effects.
Main Results:
- Mutein M112 showed increased dissociation (koff) due to disrupted conformation.
- Mutein M88 stabilized the closed conformation, reducing koff 260-fold.
- Reduction of the disulfide in M88 increased koff 19,000-fold, creating a redox switch.
Conclusions:
- Disulfide bonds can be used to engineer switchable streptavidin-biotin binding.
- Mutein M88 offers a reversible, redox-dependent system with significantly faster dissociation than native streptavidin.
- This engineered system facilitates new applications demanding both high affinity and rapid reversibility.
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