CRYSTAL-STRUCTURE OF PHOSPHATE-BINDING PROTEIN LABELED WITH A COUMARIN FLUOROPHORE, A PROBE FOR INORGANIC-PHOSPHATE

Crystal structures are presented for the A197C mutant of Escherichia c oli phosphate binding protein (PBP) and the same mutant labeled at Cys 197 with dyl)ethyl]-7-(diethylamino)-coumarin-3-carboxamide (MDCC). Bo th proteins are complexed with inorganic phosphate. The latter molecul e, MDCC-PBP, exhibits a large increase in fluorescence on binding inor ganic phosphate. The resulting high-fluorescence state of the coumarin and the ability of this coumarin to monitor the conformational change s associated with inorganic phosphate binding are interpreted in terms of the specific interactions of MDCC with the protein. The structure helps to explain why this particular label gives a high-fluorescence s tate on binding inorganic phosphate, while several other related label s do not, and hence aids our general understanding of environmentally sensitive fluorescence probes on proteins.