NSF/MCB-BSF: De novo design of minimalistic light-switchable protein binding domains

University Of California-San Francisco, San Francisco CA

Investigators

Abstract

Nature frequently uses an external signal, such as light, to switch on/off protein function. Photoswitchable proteins in plants, fungi, and bacteria change their conformation upon absorption of a particular wavelength of light. The conformational change results in activation/deactivation of the downstream pathways that control cell movement, growth, development, and other processes. Photoswitching proved to be an attractive strategy in synthetic biology to achieve control of various cellular processes with precise spatial and temporal resolution. Thus, design and engineering of photoswitchable proteins present great interest for both basic science and many applications in synthetic biology. This project will develop and test new methodology for rational design of minimalistic light switchable single-domain proteins that bind specifically to a particular target protein in only one PSC state. This project will train postdoctoral, graduate, and undergraduate students including members of underrepresented minority groups. This project aims to overcome the limitations of currently used approaches by rationally designing interior binding sites for native and synthetic photoswitchable chromophores (PSCs) into various small protein domains that are well-behaved and are could be engineered to interact with variable targets. In such designs, a binding site for a PSC will be carved into the core of a small protein domain, with a cysteine or another nucleophilic amino acid placed to enable proximity enhanced covalent PSC-protein linkage. With protein core stabilized by ligand-protein interactions, the surface residues of the protein domain will be evolved to bind to a particular target protein. Upon excitation by light and isomerization of the PSC, the binding domain would exhibit local or global unfolding and subsequent disruption of the protein-protein interaction. Switching the chromophore back to the ground state would result in restoration of the original conformation and the binding function, producing a stable and recyclable light-switchable protein binder. Moreover, this project will explore whether light switching function encoded by protein core can be coupled to the binding function within a single protein domain, in contrast to what is observed in the majority of native light-switchable proteins that separate light sensing and output functions. This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.