Abstract: Cells are genetically modified to express a luminophore, e.g., a modified (F64L, S65T, Y66H) Green Fluorescent Protein (GFP, EGFP) coupled to a component of an intracellular signalling pathway such as a transcription factor, a cGMP- or cAMP-dependent protein kinase, a cyclin-, calmodulin- or phospholipid-dependent or mitogen-activated serine/threonin protein kinase, a tyrosine protein kinase, or a protein phosphatase (e.g. PKA, PKC, Erk, Smad, VASP, actin, p38, Jnk1, PKG, IkappaB, CDK2, Grk5, Zap70, p85, protein-tyrosine phosphatase 1C, Stat5, NFAT, NFkappaB, RhoA, PKB). An influence modulates the intracellular signalling pathway in such a way that the luminophore is being redistributed or translocated with the component in living cells in a manner experimentally determined to be correlated to the degree of the influence.

Abstract: A method for screening a library of compounds to detect a biologically active compound that modulates intracellular translocation of a subunit of a component of an intracellular pathway affecting intracellular processes includes: culturing one or more cells containing a nucleotide sequence coding for a hybrid polypeptide comprising a luminophore linked to the subunit of the component; introducing a compound of the library of compounds into the cell culture; screening the compound to determine whether the compound modulates the intracellular translocation of the subunit of the component; measuring light emitted from the luminophore to determine a first distribution; measuring light emitted from the luminophore to determine a second distribution; computing a variation between the first distribution and the second distribution by processing the measured light, any variation is indicative that the compound is biologically active. The method is also performed with a library of compounds.

Abstract: A GFP with an F64L mutation and E222G mutation is provided. This GFP has a bigger Stokes shift compared to other GFPs making it very suitable for high throughput screening due to a better resolution. This GFP also has an excitation maximum between the yellow GFP and the cyan GFP allowing for clearer band separation when used together with those GFPs. Examples include the sequences in SEQ ID NOs: 3 and 4.

Abstract: The present invention relates to novel variants of the fluorescent protein GFP having improved fluorescence properties.

Abstract: A method is described to assay for protein interactions in living cells, e.g. by the introduction of two heterologous conjugates into the cell. The method uses the measurement of cellular distribution of a detectable component (e.g. a GFP-labeled˜fluorescent probe) to indicate the presence or absence of an interaction between that component and a second component of interest. The method uses the knowledge that certain components can be stimulated to redistribute within the cell to defined locations. Inducible redistribution systems make it possible to determine if specific interactions occur between components. Inducible systems are described where it is demonstrated that the redistribution stimuli are essentially “null”, in that they affect no other system in the cell during the assay period, other than the component whose redistribution can be induced.