Supramolecular protein assembly is utilized by nature to control a variety of cellular signaling events, including the response to growth factors as well as cell migration. Thus, the ability to control protein assembly within living cells in response to a small molecule input could provide tunable approach for controlling these diverse cellular pathways. Such approaches could provide fundamental insight into cellular signaling events as well as provide methods for engineering new function into biological systems. Host-guest systems represent a promising avenue towards reaching this goal. However, the identification of genetically encodable host-guest systems capable of functioning in the cellular milieu has proven challenging. To address this difficulty, our laboratory has recently described an approach to identify host-guest systems capable of functioning in living cells. In this approach, libraries of the tenth type-III domain of fibronection (Fn3) are evolved for binding to an environment sensitive fluorophore. Importantly, increases in the fluorescence of the small molecule guest can be used to identity binding as well as bias the system towards supramolecular assembly. Our initial host guest was found to be capable of undergoing supramolecular assembly, at a ratio of 5 proteins to 1 guest, on the surface of living cells. Herein, we investigate the ability to generate orthogonal host-guest systems with different binding stoichiometries by varying the structure of the guest used for evolution of a cognate host. We utilize a battery of biophysical characterization techniques including fluorescence titrations, analytical ultracentrifugation, isothermal titration calorimetry, Cd spectroscopy to characterize these new host-guest systems. The binding stoichiometry of these new host-guest systems as well as their ability to function in living cells is discussed. These new tools provide an expanded repertoire for the controlled assembly of proteins within living systems.

New tools for controlling the supramolecular assembly of proteins in living cells

253rd National Meeting (2017)

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