title: Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins.
abstract: Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. Here we compare the effects of two direct immobilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respectively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand binding and signal transduction but with adversely affected affinities and signal amplitudes for the thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically oriented layer to maleimide-derivatized plates using a His(2)Cys(2) zinc finger domain fused at either their N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with the immobilized proteins are on the order of seconds.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: de Lorimier
authors.forename: Yaji
authors.initials: Y
authors.lastname: Tian
authors.forename: Homme W
authors.initials: HW
authors.lastname: Hellinga
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D001710:Biotin / Q000737:chemistry; D005453:Fluorescence; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008301:Maleimides / Q000737:chemistry; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D033902:Periplasmic Binding Proteins / Q000737:chemistry*; D012266:Ribose / Q000378:metabolism; D015398:Signal Transduction; D019809:Streptavidin / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties; D046968:Thermoanaerobacter / Q000737:chemistry; D020124:Thermotoga maritima / Q000737:chemistry; D016335:Zinc Fingers
chemical_list: D008024:Ligands; D008301:Maleimides; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D013438:Sulfhydryl Compounds; C043592:maleimide; D012266:Ribose; D001710:Biotin; D019809:Streptavidin; D005947:Glucose