Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold

Abstract

Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.

The Masking Game: Design of Activatable Antibodies and Mimetics for Selective Therapeutics and Cell Control

“…(E) Encoding of the LOV domain enables intracellular control of protein location and immunopurification. 11 , 13 Adapted with permission from refs ( 10 , 12 , 13 , and 22 ). Copyright 2020 Wiley-VCH, and 2019 and 2020 Springer Nature.…”

“…Harnessing the power of a natural protein switch, Avalos and Toettcher groups have jointly created antibody mimetics dubbed “optobinders” ( Figure 2 E). 13 The basis for their design is the insertion of a light-oxygen-voltage domain from Avena sativa phototropin 1 (AsLOV2) into the structure of nanobodies (OptoNBs) 11 and monobodies (OptoMBs). 13 The AsLOV2 domain exerts switching under blue light (447 nm) via a large conformational change triggered by the covalent conjugation of photoexcited flavin chromophore FMN to a conserved cysteine.…”

A light way for nuclear cell biologists

“…Recently, nanobodies have been controlled by light following three strategies: (i) the As LOV2 domain (or, more precisely, an optimized version of it called sLOV) was inserted into a surface-exposed loop of the nanobody following a previously reported method ( 45 ) whereby the blue light-triggered unfolding of the Jα helix brings about a local conformational change, which, in turn, results in either loss or gain of binding capacity by the nanobody towards its antigen ( 72 ). The same method was shown to work also with monobodies ( 73 ), which are synthetic customizable protein binders constructed using a fibronectin type III domain as molecular scaffold; (ii) the localization of the nanobody was controlled by a light-inducible heterodimerizing pair ( 74 ); (iii) the nanobody was split into two fragments, which were brought into physical proximity using a light-inducible heterodimerizer ( 75 ). Importantly, optonanobodies offer the opportunity to control the localization of selected endogenous proteins, as long as nanobodies specific for them can be developed ( Fig.…”

“…1G ). This method was recently used by the Niopek group to create a light-inducible version of the Cas9 inhibitor AcrIIA4 ( 132 ) and by the Avalos and Toettcher groups to obtain optonanobodies and monobodies ( 72 , 73 ). Interestingly, the authors found variants whereby the As LOV2 domain disrupted the binding of the nanobody to its target in the dark rather than in the lit state ( 72 ).…”

Design of Smart Antibody Mimetics with Photosensitive Switches

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either based on split nanobodies (optobody) [7] or hybrid proteins that utilize a photosensitive switch (OptoNB and OptoMB) [8,9] or circularly permuted bacterial dihydrofolate reductase (LAMA). [10] Nonetheless, these engineered intrabodies exhibit a relatively slow activation kinetics or suffer from a relatively low or modest dynamic range of light-induced changes.Bearing these unmet needs in mind, we introduce herein a set of smart monobodies and nanobodies that respond to light within seconds, in the form of ON-switches (sunbody) or OFF-switches (moonbody).

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