Light-activated fusion proteins and uses therefor
The invention, in some aspects relates to synthetic, light-activated fusion proteins and their encoding polynucleotide molecules. In some aspects the invention additionally includes expression of the light-activated fusion proteins in cells and their use in methods such as therapeutic methods and candidate compound screening methods.
1. A fusion protein comprising a peptide ligand domain, a linker polypeptide domain, a photoreceptor polypeptide domain, and a membrane-anchoring polypeptide domain, wherein the peptide ligand is a ligand for an ion channel or receptor, the photoreceptor polypeptide domain is a light-oxygen-voltage (LOV) domain, and the binding specificity of the peptide ligand domain for its cognate channel or receptor is maintained in the presence and absence of a light that alters the conformation of the photoreceptor polypeptide domain.
2. The fusion protein of claim 1 , further comprising a trafficking signal polypeptide domain.
3. The fusion protein of claim 2 wherein the trafficking signal is a secretion signal.
4. The fusion protein of claim 1 , wherein the membrane-anchoring polypeptide domain comprises a glycophosphatidylinositol (GPI) anchoring polypeptide, a one-pass transmembrane polypeptide, a channel complex-anchoring polypeptide, or a channel complex partner anchoring polypeptide.
5. The fusion protein of claim 3 , wherein the secretion signal polypeptide domain comprises a polypeptide derived from a truncated MHC I antigen (ss) polypeptide, a prolactin (prl) polypeptide, an achR beta subunit (acr) polypeptide, or a serine protease I (srl) polypeptide.
6. The fusion protein of claim 1 , wherein the photoreceptor polypeptide domain comprises an amino acid sequence derived from an amino acid sequence of a plant photoreceptor polypeptide.
7. The fusion protein of claim 1 , further comprising a reporter molecule polypeptide domain.
8. A composition comprising the fusion protein of claim 1 and a pharmaceutical carrier.
9. A cell comprising the fusion protein of claim 1 , wherein the cell is an in vitro cell.
10. The cell of claim 9 , wherein the cell is an excitable cell.
11. A pharmaceutical composition comprising the fusion protein molecule of claim 1 .
12. A polynucleotide molecule that encodes the fusion protein of claim 1 .
13. An expression vector comprising the polynucleotide molecule of claim 12 .
14. An in vitro cell comprising the polynucleotide molecule of claim 12 .
15. A method of modulating a functional state of an ion channel in a cell membrane, the method comprising,
expressing in a cell comprising a cell membrane ion channel, a fusion protein comprising a polypeptide ligand domain, a linker polypeptide domain, a membrane-anchoring polypeptide domain, and a photoreceptor polypeptide domain, wherein the ligand is a ligand for the ion channel, the photoreceptor polypeptide domain is a light-oxygen-voltage (LOV) domain, and the binding specificity of the peptide ligand domain for its cognate channel or receptor is maintained in the presence and absence of a light that alters the conformation of the photoreceptor polypeptide domain; and
contacting the expressed fusion protein with an effective dose of a light to modulate a conformation of the fusion protein, wherein the modulation of the conformation of the fusion protein alters a functional state of the ion channel in the membrane.
16. A method of determining the effect of a candidate therapeutic compound on a functional state of an ion channel of a membrane, the method comprising,
expressing in a cell comprising a cell membrane ion channel, a fusion protein comprising a polypeptide ligand domain, a linker polypeptide domain, a membrane-anchoring polypeptide domain, and a photoreceptor polypeptide domain, wherein the polypeptide ligand is a ligand for the ion channel, the photoreceptor polypeptide domain is a light-oxygen-voltage (LOV) domain, and the binding specificity of the peptide ligand domain for its cognate channel or receptor is maintained in the presence and absence of a light that alters the conformation of the photoreceptor polypeptide domain;
contacting the cell with a candidate therapeutic compound;
contacting the expressed fusion protein with a dose of a light effective to modulate a conformation of the fusion protein;
determining the functional state of the ion channel; and
comparing the determined functional state of the ion channel with a control functional state of the ion channel, wherein a difference between the determined functional state and the control functional state indicates an effect of the candidate therapeutic compound on the functional state of the ion channel.
17. The fusion protein of claim 1 , wherein the membrane is a plasma membrane.
18. The fusion protein of claim 1 , further comprising a secretion signal polypeptide domain.
19. The fusion protein of claim 18 , wherein the membrane is a plasma membrane.
20. The fusion protein of claim 1 , wherein the linker polypeptide domain comprises a flexible linker.
21. The fusion protein of claim 7 , wherein the reporter molecule polypeptide domain comprises a fluorescent reporter polypeptide.