ENGINEERING THE MALTOSE-BINDING PROTEIN FOR REAGENTLESS FLUORESCENCE SENSING

This paper describes a mutant of the maltose binding protein (MBP) in which the serine residue at position 337 is replaced by a cysteine res idue using site-directed mutagenesis. The mutant MBP has an approximat ely 2-fold lower affinity for maltose, and the cysteine residue can be modified with 4-[N-(2-(iodoacetoxy) thyl)-N-methylamino]-7-nitrobenz- 2-oxa-1,3-diazole (IANBD) and 6-acryloyl-2-(dimethylamino)-naphthalene (acrylodan). This combined genetic and chemical modification places t he fluorophores close to the maltose binding site such that when the l igand is added the fluorescence intensity of the labels increases by 6 0-180% over that of the ligand-free form. This change is consistent wi th the fluorophores being buried when the conformation of the protein changes with maltose binding. Titration of the labeled mutant proteins yields dissociation constants for maltose of 62 +/- 0.2 and 0.8 +/- 0 .01 mu M respectively for the IANBD and acrylodan modifications. The a pplication of this strategy of combined genetic and chemical modificat ion to the development of reagentless fluorescence sensing is discusse d.