Photocontrol of Protein Affinity Reagents by Red Light using Azobenzene Switches

Abstract

Remote control of protein function by light is a powerful technique for manipulating biological processes in living organisms. One general method to obtain photoswitchable proteins is to couple the photoisomerization of azobenzene derivatives to conformational changes in the protein of interest. The applicability of these compounds in vivo, however, depends largely on the excitation wavelength that is required for their switching. Short wavelengths of visible light are highly scattered and do not penetrate well in cells and live tissues. In the past few years, novel azo derivatives which operate with red wavelengths and are stable in water were designed in our group. We have now applied these compounds to fynomers: small proteins based on Fyn SH3 scaffold that are developed via phage display selection techniques to target biologically relevant proteins. We chose a fynomer that was optimized for inhibiting the activity of human chymase by binding near its active site. Chymase is a serine protease that is secreted by mast cells and is shown to be involved in cardiovascular diseases as well as pathological inflammatory conditions. Two cysteine residues were introduced by point mutations in the sequence of the fynomer. Subsequent crosslinking of the inhibitor with azobenzenes at those residues allows only the cis isomer of the azo moiety to be compatible with the well-folded inhibitor. The dark-adapted (trans azo) crosslinked fynomer was partially unfolded and showed reduced inhibitory activity. Upon its irradiation with red light (635 nm), the inhibitor was largely folded and better suppressed the activity of chymase. Since the Fyn SH3 scaffold can be broadly utilized to target virtually any protein, these results demonstrate the promise of red light switchable azobenzenes for in vivo functional studies and photopharmacology.