title: Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors.
abstract: A new general method, post-photoaffinity-labeling modification (PPALM), for constructing fluorescent saccharide biosensors based on naturally occurring saccharide-binding proteins, lectins, is described in detail. An active-site-directed incorporation of a masked reactive site into a lectin was conducted by using a photoaffinity labeling technique followed by demasking and then chemical modification to yield a fluorescent lectin. Two photoaffinity labeling reagents were designed and synthesized in this study. The labeling reagent with a photoreactive site appended through a disulfide link to a mannoside unit was bound to the saccharide-binding pocket of the lectin concanavalin A (Con A). After light irradiation, the mannoside unit was cleaved by reduction. The unique thiol group thus produced was site-specifically modified with various fluorescent groups (dansyl, coumarin, or dimethylaminobenzoate derivatives) to afford fluorescent Con As. The labeling site was characterized by protease-catalyzed digestion followed by HPLC, MALDI-TOF MS, and tandem mass-mass spectrometry; these methods indicated that the photolabeling step is remarkably site specific. Strong fluorescence was observed in the engineered Con A with a fluorophore, and the emission changed sensitively upon saccharide complexation. The binding constants for various saccharides were determined by fluorescence titration and demonstrated that the binding selectivity and affinity of the engineered Con As are comparable to those of native Con A. The red shift of the emission maximum, the decrease in the fluorescence anisotropy of the dansyl unit, and the increase in the twisted intramolecular charge transfer emission caused by sugar binding to the engineered Con A explicitly indicate that the microenvironment of the appended fluorophores changes from a restricted and relatively hydrophobic environment into a rather freely mobile and hydrophilic environment.
authors.affiliation: Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka, 812-8581, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Nagase
authors.forename: Eiji
authors.initials: E
authors.lastname: Nakata
authors.forename: Seiji
authors.initials: S
authors.lastname: Shinkai
authors.forename: Itaru
authors.initials: I
authors.lastname: Hamachi
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D032449:Canavalia / Q000737:chemistry; D003208:Concanavalin A / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D008358:Mannose / Q000737:chemistry; D008958:Models, Molecular; D019920:Photoaffinity Labels / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D004220:Disulfides; D005456:Fluorescent Dyes; D019920:Photoaffinity Labels; D003208:Concanavalin A; D008358:Mannose