Binding and signaling of surface‐immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins

Abstract

Development of biosensor devices typically requires incorporation of themolecular recognition element into as olid surface for interfacing with as ignal detector.O ne approach is to immobilize the signal transducingp rotein directlyo nasolids urface. Herew ec ompare the effects of two directi mmobilization methods on ligand binding, kinetics, ands ignal transduction of reagentless fluorescent biosensors based on engineeredperiplasmic binding proteins. We used thermostable ribose andglucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima,r espectively.T otest thebehavior of these proteins in semispecifically oriented layers, we covalently modified lysiner esidues with biotin or sulfhydrylf unctions,a nd attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively.T he immobilized proteins retained ligand bindinga nd signal transduction butw ith adverselya ffected affinities and signala mplitudes for the thiolated, butn ot the biotinylated, proteins. We also immobilized these proteins in am ore specifically oriented layertomaleimide-derivatized platesusing aHis 2 Cys 2 zinc finger domain fusedateither their No rCtermini.P roteins immobilized this way either retained,o rd isplayed enhanced, ligand affinity and signal amplitude. In allc ases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loadinga nd elution. The kinetics of ligand exchange with the immobilized proteins areo nt he order of seconds.Keywords: biosensor; surface immobilization; periplasmicb inding protein; fluorescence;z inc finger; biotin Soluteb inding members of thep eriplasmic bindingp rotein( PBP) superfamily have been intensively studied as receptors fors ensora pplications (Hellinga andM arvin 1998). Theseproteins exhibit high specificity and affinity for their natural cognate ligandsa nd canb ed esigned to bind nonnatural ligands ( Marvin andH ellinga 2001; Looger et al. 2003; Allert et al. 2004). Ligand binding is accompanied by conformational changes in theprotein, which canbelinked to changes affecting site-specificallyattached fluorophores, therebyt ransducing binding into af luorescents ignal (Gilardie ta l. 1994; Marvin et al. 1997;de Lorimier et al. 2002). This engineered reagentless sensing mechanism is potentially well suited forrealtime sensing applications.Development of sensor devicesr equires incorporation of sensing proteins into ad etector element by encapsulation or surface immobilization on asuitable material for interfacing with detectors. Here we describe studiesf or the immobilization of engineeredfluorescent signal transducing PBPs. Reagentless sensings ystemsm ay have the signal transducing proteins eparated from thef luid sampleb yadiffusion barrier.F or example, the protein may be entrapped in ap orousm aterial through which small molecules diffuse to reache quilibrium with the immobilized receptor (Topoglidis et al. 1998; Alarcon et al. Article published onlinea head of pri...

Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins

“…Subsequently, the binding of glucose to GBP induces a conformational change which may apparently bring closer both these two fluorescent proteins and therefore there is an increase in FRET efficiency. Several research workers have reported that FRET is apparently a very complex process when free and immobilized whole-cells or cell-free extracts are involved [19][20][21][22]. Regarding the differences in FRET efficiencies between the free and immobilized nanosensors, there are a number of factors affecting immobilized and soluble proteins such as restricted mobility on immobilization, chemical modification due to the immobilization method used, nature of the microenvironment and diffusion limitation [20].…”

“…Apparently, this difference may be due to the reduced flexibility of the linker domain between the fluorescent proteins because of the attachment to solid surface. This issue emphasizes the importance to retain flexibility of both the fusion proteins in devising FRET pairs for immobilization to solid surfaces [19,21]. In general, FRET theory assumes that in a FRET couple only a single donor and a single acceptor are present with very weak coupling.…”

Fluorescent Biosensors for Protein Interactions and Drug Discovery

“…Enzymes are very specific, however in some cases the catalysis is not desirable, thus some enzymes have to be modified to impair the activity and conserve only the ligand binding property, or the ideal case is to use a protein that only bind the analyte to monitor. Accordingly, a family of proteins in the periplasmic space of bacteria fulfill the last requirement (Looger, Dwyer et al 2003;de Lorimier, Tian et al 2006). These proteins named periplasmic binding proteins (PBPs), present a conformational change upon ligand binding, as a first step to interact with a membrane transporters (ABC proteins), previous of the translocation of ligand to the interior to the cell (de Lorimier, Tian et al 2006;Medintz and Deschamps 2006;Tsukiji, Miyagawa et al 2009).…”

“…Accordingly, a family of proteins in the periplasmic space of bacteria fulfill the last requirement (Looger, Dwyer et al 2003;de Lorimier, Tian et al 2006). These proteins named periplasmic binding proteins (PBPs), present a conformational change upon ligand binding, as a first step to interact with a membrane transporters (ABC proteins), previous of the translocation of ligand to the interior to the cell (de Lorimier, Tian et al 2006;Medintz and Deschamps 2006;Tsukiji, Miyagawa et al 2009). The different members of these proteins are able to bind a large number of analytes, such a as: carbohydrates, amino acids, ions, hormones, heme-groups, etc.…”

Construction of a novel glucose‐sensing molecule based on a substrate‐binding protein for intracellular sensing

“…As such, it has been expected to be a suitable sensing element for blood or intracellular glucose monitoring. Indeed, there have been several reports about fluorescentbased glucose-sensing systems using GBP conjugated with fluorophores that change in fluorescent intensity with a GBP conformational change (de Lorimier et al, 2002;Der and Dattelbaum, 2008;Salins et al, 2001;Thomas et al, 2006;Tolosa et al, 1999), and also about the design of FRET-based methods using GBP (de Lorimier et al, 2006;Deuschle et al, 2005;Fehr et al, 2003Fehr et al, , 2005Ge et al, 2004;Khan et al, 2008), which allow glucose sensing in vitro and also in mammalian cells. We have been engaged in the development of glucose-sensing molecules with GBP such a glucosespecific SBP with a K d value suitable for the measurement of physiological glucose concentrations and a novel luminescence-based glucose-sensing molecule combining with a luciferase (Taneoka et al, 2009).…”