Spectroscopic properties of an engineered maltose binding protein.
The maltose binding protein (MBP) has been site specifically labelled with a nitrobenzoxadiazole (NBD) group following mutation of a serine to a cysteine residue at position 337. The resulting protein shows a large ligand (maltose or beta-cyclodextrin) dependent increase in its steady-state fluorescence intensity. Analysis of the static (intensity and anisotropy) and dynamic (lifetime distributions) fluorescence of the NBD label as well as the tryptophan residues in both ligand-bound and ligand-free states of this molecule reveals complex multi-component decays that are interpreted in terms of a ligand-induced solvent shielding mechanism. In the context of the known crystal structures of the various forms of the maltose-binding protein (MBP), ligand-dependent changes in both the fluorescence parameters as well as the circular dichroism spectra of the NBD group are interpreted by a twisted intramolecular charge-transfer (TICT) mechanism, wherein ligand binding locks the NBD group into a conformation that prevents efficient relaxation of the excited state.
Gilardi
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Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
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D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005818:Genetic Engineering; D007202:Indicators and Reagents / Q000378:metabolism; D008320:Maltose / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007202:Indicators and Reagents; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D008320:Maltose; D009327:4-Chloro-7-nitrobenzofurazan