Expanding the secondary coordination sphere of streptavidin-based artificial metalloenzymes and their characterization

Over billions of years, enzymes evolved to be nature’s catalyst. Under (aqueous) mild conditions they promote a vast number of chemical reactions, achieving remarkable accelerations and exquisite selectivities. In addition, scientists designed and optimized organometallic catalysts in order to perform new-to-nature chemical transformations. Organometallic catalysts and enzymes have complementary properties that are combined and exploited in artificial metalloenzymes. In this approach, the reactivity of the catalyst can be tuned not only by chemical optimization of the catalyst, but furthermore by genetic optimization of the protein scaffold. These systems can be especially useful in bringing new- to-nature reactions in living systems. Various artificial metalloenzymes are designed, assembled and evolved in the Ward group. Although several host proteins have been exploited, the biotin-streptavidin technology occupies a place of choice in this context. This thesis aims to create a chimeric streptavidin variant and to study its utility as a scaffold for the creation of ArMs. Furthermore, native MS and protein crystallography were used to characterize ArMs