Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation
View Patent ↗The invention provides peptide synthons having protected functional groups for attachment of desired moieties (e.g. functional molecules or probes). Also provided are peptide conjugates prepared from such synthons, and synthon and conjugate preparation methods including procedures for identifying the optimum probe attachment site. Biosensors are provided having environmentally sensitive dyes that can locate specific biomolecules within living cells and detect chemical and physiological changes in those biomolecules as the living cell is moving, metabolizing and reacting to its environment. Methods are included for detecting GTP activation of a Rho GTPase protein using polypeptide biosensors. When the biosensor binds GTP-activated Rho GTPase protein, the environmentally sensitive dye emits a signal of a different lifetime, intensity or wavelength than when not bound. New fluorophores whose fluorescence responds to environmental changes are also provided that have improved detection and attachment properties, and that can be used in living cells, or in vitro.
1. A polypeptide biosensor comprising a polypeptide and a fluorescent dye, wherein said polypeptide is linked by a direct bond or a linking group to said fluorescent dye, and
wherein said fluorescent dye on said biosensor comprises a compound of the formula:
each m is separately an integer ranging from 1-3;
n is an integer ranging from 0 to 5;
R 8 , R 11 and R 12 are separately CO, SO 2 , C═C(CN) 2 , S, O or C(CH 3 ) 2 ;
each R 13 is hydrogen, alkyl, branched alkyl or heterocyclic ring derivatized with charged groups to enhance water solubility and enhance photostability;
each R 9 and R 10 is separately hydrogen, an alkyl chain derivatized with charged groups to enhance water solubility or with reactive groups for conjugation to other molecules.
2. The polypeptide biosensor of claim 1 wherein said polypeptide biosensor comprises a protein binding domain of p21 activated kinase 1.
3. The polypeptide biosensor of claim 1 wherein when said polypeptide sensor is bound to a GTP-activated Rho GTPase protein having a fluorophore on said GTP-activated Rho GTPase protein, said fluorescent dye on said polypeptide biosensor undergoes fluorescence resonance energy transfer with said fluorophore, and
wherein said fluorophore on said GTP-activated Rho GTPase protein is Green Fluorescence Protein (GFP), Cyan Fluorescence Protein (CFP), Red Fluorescence Protein (RFP) or enhanced GFP (EGFP).
4. The polypeptide biosensor of claim 3 wherein said Rho GTPase protein is Rae or cdc42.
5. The polypeptide biosensor of claim 1 , wherein the fluorescent dye on the polypeptide biosensor comprises a compound of the formula:
6. The polypeptide biosensor of claim 5 , wherein R10 is an alkyl group with 1 to 6 carbon atoms.
7. A peptide biosensor comprising a peptide and a fluorescent dye, said peptide linked by a direct bond or a linking group to said fluorescent dye, and wherein said fluorescent dye on the peptide biosensor comprises a compound of the formula:
each m is separately an integer ranging from 1-3;
n is an integer ranging from 0 to 5;
R 8 , R 11 and R 12 are separately CO, SO 2 , C═C(CN) 2 , S, O or C(CH 3 ) 2 ;
each R 13 is hydrogen, alkyl, branched alkyl or heterocyclic ring derivatized with charged groups to enhance water solubility and enhance photostability;
each R 9 and R 10 is separately hydrogen, an alkyl chain derivatized with charged groups to enhance water solubility or with reactive groups for conjugation to other molecules.