Heterotrimeric G-protein
AssigneeThe Johns Hopkins University
InventorsPeter Devreotes, Chris Janetopoulos
Life of this patent
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Abstract
Receptor mediated activation of heterotrimeric G-proteins is visualized in living cells by monitoring fluorescence resonance energy transfer (FRET) between subunits of a G protein fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociates and reassociates upon addition and removal of cognate ligand. Energy transfer pairs of G-proteins enables direct in situ detection and have applications for drug screening and GPCR de-orphaning.
Claims19
1-38. (canceled)
A eukaryotic cell which comprises:
- (a) a nucleic acid encoding a functional α subunit or a heterotrimeric G protein which comprises an amino acid sequence encoding a first fluorescent or luminescent protein;
- and (b) a nucleic acid encoding a functional β of γ subunit of a heterotrimeric G protein which comprises an amino acid sequence encoding a second fluorescent or luminescent protein, wherein the cell expresses a functional heterotrimeric G protein which is capable of FRET or BRET.
The eukaryotic cell of claim 38 wherein the cell is a 3T3 cell.
The eukaryotic cell of claim 38 wherein the cell is a D. discoideum cell.
A method for detecting conformational changes in a protein, comprising:
- detecting changes in fluorescent resonance energy transfer (FRET) of an artificial protein subjected to a change in environmental conditions, wherein the artificial protein comprises at least two subunits, wherein a first subunit comprises a cyan fluorescent protein and a second subunit comprises a yellow fluorescent protein, wherein the cyan fluorescent protein and the yellow fluorescent protein are within 100 angstroms of each other, wherein a change in FRET indicates that the change in environmental conditions caused the artificial protein to change its conformation.
56-76. (canceled)
Dependencies are inferred from the claim text.