Drug discovery and protein-protein interaction assay using fluorescent protein exchange
A novel assay for determining a molecular process using a fluorescent protein exchange assay, and a composition for use thereof, are provided. The assay provides first and second signalling proteins and an exchange protein, wherein the exchange protein interacts with the first signalling protein to form a complex, then introducing the second signalling protein, wherein in response to the molecular process, the exchange protein dissociates from the first protein and associates with the second protein. The change in signal in response to the exchange of the proteins is measured to indicate a molecular process.
1. A method of assaying a molecular process, comprising the steps of:
a) providing an exchange proteins and a first signalling proteins, wherein the exchange proteins interacts with the first signalling proteins to form a first complex,
b) introducing a second signalling protein, wherein in response to the molecular process, the exchange protein dissociates from the first signalling protein in the first complex and associates with the second signalling proteins to form a second complex, and
c) measuring change in signal generated, thereby assaying the molecular process, wherein each of the first signalling protein, the second signaling proteins, and the exchange protein are dimerization dependent fluorescent proteins (ddFPs) and wherein one of the first signalling protein and second signalling protein is a red ddFP, and the other is a green ddFP.
2. The method of claim 1 , wherein the molecular process is a loss of protein-protein interaction or physical connection.
3. The method of claim 1 , wherein the change in signal is a change in fluorescence intensity, a change in fluorescence color, or a change in subcellular localization.
4. The method of claim 1 , wherein the change in signal generated fluorescence is a green-to-red or red-to-green change in fluorescence color.
5. The method of claim 1 , wherein the exchange, first or second proteins are conjugated with one or more further proteins.
6. The method of claim 5 , wherein the one or more further proteins interact with each other, or dissociate from each other, thereby changing the signal.
7. The method of claim 1 , wherein an enzyme catalyzes the interaction or loss of interaction between signalling proteins.
8. The method of claim 1 , wherein the proteins are located in a cell.