Photoreactive regulator of protein function and methods of use thereof
The present invention provides a synthetic regulator of protein function, which regulator is a light-sensitive regulator. The present invention further provides a light-regulated polypeptide that includes a subject synthetic regulator. Also provided are cells and membranes comprising a subject light-regulated polypeptide. The present invention further provides methods of modulating protein function, involving use of light. The present invention further provides methods of identifying agents that modulate protein function.
1. A method of restoring light sensitivity to a cell in a retina that has reduced light sensitivity, the method comprising administering to an eye comprising the retina an effective amount of a synthetic regulator, wherein the cell expresses a polypeptide that is regulated by the synthetic regulator, wherein the synthetic regulator has the structure A-B-C, wherein:
a) A comprises a binding moiety that provides for covalent linkage to the polypeptide;
b) B is a photoisomerizable group selected from an azobenzene, an azobenzene derivative, a spiropyran, a spiropyran derivative, triphenyl methane, a triphenyl methane derivative, 4,5-epoxy-2-cyclopentene, a 4,5-epoxy-2-cyclopentene derivative, fulgide, a fulgide derivative, thioindigo, diarylethene, a diarylethene derivative, diallylethene, a diallylethene derivative, an overcrowded alkene, and an anthracene; and
c) C comprises a ligand that binds to a ligand-binding site of the polypeptide,
wherein, upon exposure of the cell to a wavelength of light that induces photoisomerization of B, said photoisomerization of B results in a change in the binding of the ligand to the ligand-binding site, and thereby confers light sensitivity to the cell.
2. The method of claim 1 , wherein the cell is a retinal ganglion cell or a retinal bipolar cell.
3. The method of claim 1 , wherein said administering is by intraocular injection.
4. The method of claim 1 , wherein the polypeptide that is regulated by the synthetic regulator is a potassium channel.
5. The method of claim 1 , wherein the polypeptide that is regulated by the synthetic regulator is a glutamate receptor.
6. The method of claim 1 , wherein the cell is genetically modified to express the polypeptide that is regulated by the synthetic regulator.
7. The method of claim 1 , wherein the synthetic regulator has the structure:
wherein R 1 comprises a binding moiety that provides for covalent linkage with an amino acid residue in the light-regulated polypeptide; and
R 2 has the structure:
wherein n is 0, 1 or 2;
m is 1 or 3; and
R″ is a ligand for the light-regulated polypeptide.