Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.

The cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp PCC 7002) was grown turbidostatically in white light at three levels of irradiance: 20, 200, and 1260 microeinsteins per square meter per second. Phycobilisomes were isolated from each culture and analyzed by absorbance, gel electrophoresis, and electron microscopy. The ratio of phycocyanin to allophycocyanin decreased 1.8-fold from the lowest to highest irradiance. This change was due entirely to an approximately 2.5-fold decrease in one structural unit of rod domains, the complex of phycocyanin, and a 33-kilodalton linker polypeptide (LR33). For a given irradiance, phycobilisomes from cells grown on ammonium as the nitrogen source had 10 to 20% more phycocyanin than those from nitrate cultures. Total RNA was isolated from all cultures and probed with gene fragments specific to phycocyanin and allophycocyanin subunits and LR33. The relative level of RNAs encoding phycocyanin and allophycocyanin was found to vary with light intensity in parallel with the phycobiliprotein ratio. Hence, the light-harvesting capacity of phycobilisomes is directly regulated by relative levels of phycobiliprotein mRNA. The LR33 transcript occurs as a 3' extension on about 10% of phycocyanin transcripts. The ratio of RNA encoding LR33 to that encoding phycocyanin did not vary with irradiance, although the protein ratio changed 1.7-to twofold between extremes. Based on these and other observations, we propose that the LR33 protein is constitutively synthesized at a rate higher than that required to complex with available phycocyanin.The cyanobacterium Agmenellum quadruplicatum (Synechococcus sp PCC 7002) was chosen for this study. The phycobilisomes of this organism are well described in terms ofoverall structure and the function ofindividual polypeptide components (reviewed in refs. 2 and 6). The major components of this complex are the pigmented proteins, PC' and AP. The former is localized in rod substructures, where it is bound to linker polypeptides called LRC29, LR33, and LR9. Rods are linked to a central core domain by LRC29. The core contains AP and its associated linker polypeptides.The genes encoding the a and 13 subunits of PC (cpcA and cpcB, respectively) are linked in tandem with those encoding LR33 (cpcC) and LR9 (cpcD) (7-9, 20) (Fig. 4). Genes encoding AP a and ,B subunits (apcA and apcB, respectively) and one linker polypeptide (LC8.5) are similarly clustered (4). Transcripts encoding these cpc and apc loci have been mapped (12). The major transcript of each locus encodes only the a and ,B subunits of the respective phycobiliproteins. Minor transcripts extend further in the 3' direction to encode linker polypeptides. Having this knowledge of phycobilisome structure and gene organization in A. quadruplicatum, we could readily investigate responses to changes in light intensity.In addition to irradiance, we simultaneously examined the effect of nitrogen source on phycobilisome structure. This is of interest because phycobiliproteins are strongly...

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...