Researchers have modified a popular system for protein labelling and modification to reduce the risk of unwanted cross-reactions and so make it more accurate and effective.
With incredible specificity and powerful affinity for each other, the protein streptavidin and its small-molecule target biotin are truly the ‘Dynamic Duo’ of biological research, the researchers explain, and a perennial favourite for use in the design of biochemical experimental techniques. For example, one can easily subject biotin-linked proteins to highly specific labelling with streptavidin-linked fluorophores. Nonetheless, there is an important limitation to the system-streptavidin naturally forms tetramers (assemblies of four protein molecules) that bind up to four molecules of biotin, creating the potential for unexpected cross-linking of biotinylated targets. Efforts to engineer monomeric streptavidin variants have generally resulted in diminished biotin affinity.
Now, Alice Ting and colleagues at Massachusetts Institute of Technology, Cambridge, have developed an alternative approach that involves engineering ‘dead’ streptavidin variants that can bind to each other but not to biotin. By combining the two types of streptavidin monomers in the proper proportions and isolating tetramers that consist of three dead subunits and one active subunit, they obtain streptavidin complexes that are functionally monomeric and bind only one molecule of biotin.
They have demonstrated that the hybrid tetramers retain normal affinity for biotin but induce far less ‘clumping’ of biotinylated targets relative to wild-type streptavidin tetramers. This approach also offers the possibility of building divalent and trivalent tetramers. According to Kai Johnsson of the École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland, “the existing plentitude of applications of the streptavidin-biotin interaction provides an enormous playground for streptavidins with reduced but defined valencies.”
More details can be found in April’s Nature Methods.