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 residue using site-directed mutagenesis. The mutant MBP has an approximately 2-fold lower affinity for maltose, and the cysteine residue can be modified with 4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD) and 6-acryloyl-2-(dimethylamino)-naphthalene (acrylodan). This combined genetic and chemical modification places the fluorophores close to the maltose binding site such that when the ligand is added the fluorescence intensity of the labels increases by 60-180% over that of the ligand-free form. This change is consistent with 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 microM respectively for the IANBD and acrylodan modifications. The application of this strategy of combined genetic and chemical modification to the development of reagentless fluorescence sensing is discussed.
Gilardi
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Biochemistry Department, Imperial College of Science, Technology and Medicine, London, U.K.
Zhou
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Hibbert
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Cass
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D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes / Q000737:chemistry*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008024:Ligands; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010069:Oxadiazoles / Q000737:chemistry*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010069:Oxadiazoles; C461599:maltose transport system, E coli; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; D008320:Maltose; C038518:acrylodan; D015081:2-Naphthylamine; D003545:Cysteine