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. An in vitro method of identifying an agent that modulates an activity of a non-light-regulated polypeptide, the method comprising:
a) contacting a cell with a test agent which is a candidate agent for modulating the activity of the non-light-regulated polypeptide which is a calcium channel or a sodium channel, wherein the cell expresses:
i) the non-light-regulated polypeptide; and
ii) a light-regulated polypeptide that regulates the activity of the non-light-regulated polypeptide, wherein the light-regulated polypeptide comprises a photoreactive synthetic regulator of protein function, wherein the photoreactive synthetic regulator comprises:
a-i) a linker domain comprising a binding moiety that provides for stable association with covalent linkage to the light-regulated polypeptide, wherein the binding moiety is other than a bromomethyl moiety;
a-ii) a photoisomerizable group; and
a-iii) a ligand that binds to a ligand binding site of the light-regulated polypeptide;
b) exposing the cell to light of a wavelength such that the ligand binds to a ligand binding site of the light-regulated polypeptide, wherein, in the absence of the test agent, exposure of the cell to light results in activation of the non-light-regulated polypeptide by the light-regulated polypeptide; and
c) determining the effect, if any, of the test agent on the activity of the non-light-regulated polypeptide wherein a test agent that modulates activity of the non-light-regulated polypeptide following said light exposure is a candidate agent for modulating the activity of the non-light-regulated polypeptide.
2. The method of claim 1 , wherein the non-light-regulated polypeptide is a sodium channel.
3. The method of claim 2 , wherein the non-light-regulated polypeptide is a calcium channel.
4. The method of claim 1 , wherein step (c) comprises detecting a change in the intracellular concentration of an ion.
5. The method of claim 1 , wherein step (c) comprises detecting a change in the membrane potential of the cell.
6. The method of claim 1 , wherein the cell is a mammalian cell.
7. The method of claim 1 , wherein the mammalian cell is a cell line.
8. The method of claim 7 , wherein the mammalian cell is selected from a 293 cell, a 3T3 cell, a COS cell, and a PC12 cell.
9. The method of claim 1 , wherein the ligand-binding polypeptide is selected from an ion channel, a cation channel, a ligand-gated ion channel, and a voltage-gated ion channel.
10. The method of claim 1 , wherein the ligand-binding polypeptide is a sodium channel, is a potassium channel, a chloride channel, a GABA receptor, or a serotonin receptor.