FRET-based nanosensors for monitoring and quantification of alcohols in living cells

Abstract

Upon binding of alcohols, ObpIIa undergoes conformational changes resulting in transfer of energy in the form of FRET from donor to the acceptor fluorophore.

Engineering genetically encoded FRET-based nanosensors for real time display of arsenic (As3+) dynamics in living cells

“…The K d of SenALiB was measured after mixing the sensor protein (diluted in 20 mM MOPS buffer, pH 8.0) with different concentrations of As 3+ . To determine the affinity constant, ligand saturation curve was fitted in the binding isotherm equation 50 : S = (r − r apo )/(r sat − r apo ) = [As 3+ ]/( K d + [As 3+ ]), where S is saturation of the binding site; r is ratio; r apo is ratio in the absence of arsenite; r sat is ratio at saturation with arsenite and [As 3+ ] is arsenite concentration. All the readings were taken in triplicates in a 96-well microtitre plate.…”

Biotechnological applications of mammalian odorant-binding proteins

“…More recently, it was described the potential of OBP-based sensors to screen volatile organic chemicals (VOCs) emitted by organisms, as components of noninvasive medical procedures, to monitor a patient's metabolic state or diagnose pathological conditions [30]. For example, a genetically encoded fluorescence resonance energy transfer (FRET)based nanosensor was described to monitor and quantify ethanol and other alcohols in living cells [93]. The nanosensor was designed using a human OBP (hOBP IIa ) flanked by two fluorescent proteins, enhanced cyan fluorescent protein (ECFP) and Venus at C and N-terminus of OBP, respectively.…”

“…The nanosensor was designed using a human OBP (hOBP IIa ) flanked by two fluorescent proteins, enhanced cyan fluorescent protein (ECFP) and Venus at C and N-terminus of OBP, respectively. The sensor revealed a dissociation constant (Kd) of 4.16 lM for ethanol and due to its characteristics is a noninvasive sensing device with the potential to be used in bacteria, yeast, and mammalian cells [93].…”

A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer

“…Previous reports 19 have used Cyt b 562 and a GFP mutant to construct a chimera via a conventional recombinant protein fusion method leading to bio-inspired tools. 20 Nagamune and co-workers utilized a Gly–Ser linker to prepare a fusion protein displaying 65% of energy transfer from the GFP mutant towards heme, while Jones and co-workers inserted Cyt b 562 into the GFP sequence to prepare a di-domain protein scaffold demonstrating the importance of inter-domain interactions in the energy transfer process. Although these previous efforts were successful in expression of the fusion protein, conjugation sites of the components were limited, and optimization of an appropriate linker is necessary.…”