Azo switches in vivo

Together with Vince Tropepe's lab in Cell & Systems Biology, we developed a fluorescent reporter peptide that permits imaging of azobenzene photoisomerization in zebrafish.

This indicates that azobenzene-based photochemical switches may be generally useful for spatiotemporal control in living systems.See: Angew Chem Int Ed Engl. 2011, 50(6):1325-7


Azo switches in bioactive proteins

The energy available from azobenzene isomerization (~10 kcal/mol) is larger than the folding free energies of many proteins. Thus photo-control of protein function via control of folding should be possible.

We have foccused of helical proteins such as the bZIP family as well as well-studied beta sheet domains such as FynSH3.

Photo-control of transcription in living cells together with Katja Arndt's group.

 

Light switching of the activity of a coiled-coil protein, the AP-1 transcription factor (cFos-blue/cJun-green dimer bound to DNA in figure), was achieved via introduction of a designed, azobenzene cross-linked, dominant negative peptide, XAFosW (red in figure). In the dark, XAFosW shows decreased helical content and decreased affinity for target Jun (green) proteins. Irradiation at 365 nm enhances helicity and target affinity, displacing c-fos (blue). Photo-control of XAFosW conformation permits control of AP-1 transcription factor activity in living cells. See: Angew Chem Int Ed Engl. 2010, 49(23):3943-6


Photo-control of FynSH3 together with Scott Prosser, Hue Sun Chan, and Alan Davidson.

 

We chose, as a model, a FynSH3 domain for which the free energy of folding is less than the energy available from photoisomerization of the cross-linker. Taking the experimentally determined structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the trans cross-linker was introduced into this L3C-L29C-T47AFynSH3 mutant, the protein was destabilized so that folded and unfolded forms coexisted. Irradiation of the cross-linker to produce the cis isomer recovered the folded, active state of the protein. This work shows that structure-based introduction of switchable cross-linkers is a feasible approach for photocontrol of folding/unfolding of globular proteins.See: J Am Chem Soc. 2009, 131(6):2283-9.