Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor

Ahmad, Mohammad; Ameen, Seema; Siddiqi, Tariq Omar; Khan, Parvez; Ahmad, Altaf

doi:10.1016/j.bios.2016.06.049

Cited by 25 publications

(16 citation statements)

References 33 publications

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“…Although, LivJ binds to all the three branched chain amino acids, it binds with the highest affinity to isoleucine (K d with Ile = 0.9 µM, K d with Leu = 2.3 µM, K d with Val = 4 µM). The ECFP and EYFP (Enhanced Yellow Fluorescent Protein) have been commonly used as FRET pair in a number of studies [19,20]. The performance of this FRET pair was further improved by substituting EYFP with Venus; a variant of the EYFP.…”

Section: Resultsmentioning

confidence: 99%

“…Since FRET efficiency depends on the relative orientation and is inversely proportional to the sixth power of the donor-acceptor distance. This basic principle has been used to construct nanosensors for a number of metabolites like sugars, amino acids, and metal ions [20][21][22]. A periplasmic binding protein (PBP), from a bacterial source usually, serves as the scaffold for such a sensor, to which FRET pair of fluorescent proteins are attached.…”

Section: Introductionmentioning

confidence: 99%

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Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor

Singh

Sharma

Alqarawi

et al. 2019

Sensors

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Isoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flux of this amino acid in live cells. This study presents a novel genetically-encoded nanosensor for real-time monitoring of isoleucine in living cells. This nanosensor was developed by sandwiching a periplasmic binding protein (LivJ) of E. coli between a fluorescent protein pair, ECFP (Enhanced Cyan Fluorescent Protein), and Venus. The sensor, named GEII (Genetically Encoded Isoleucine Indicator), was pH stable, isoleucine-specific, and had a binding affinity (Kd) of 63 ± 6 μM. The GEII successfully performed real-time monitoring of isoleucine in bacterial and yeast cells, thereby, establishing its bio-compatibility in monitoring isoleucine in living cells. As a further enhancement, in silico random mutagenesis was carried out to identify a set of viable mutations, which were subsequently experimentally verified to create a library of affinity mutants with a significantly expanded operating range (96 nM–1493 μM). In addition to its applicability in understanding the underlying functions of receptors of isoleucine in metabolic regulation, the GEII can also be used for metabolic engineering of bacteria for enhanced production of isoleucine in animal feed industries.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor

Singh

Sharma

Alqarawi

et al. 2019

Sensors

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“…We expect that this study will help to develop many more FRET-based genetically encoded nanosensors based on steady-state fluorescence anisotropy. Up to now, a diverse range of FRET-based genetically encoded nanosensors have been designed and constructed by exploring different sets of fluorescent proteins and different ligand binding periplasmic proteins to acquire live-cell imaging of bacterial cells for the ideal representation of the nanosensors [30][31][32]. The FLIP-SP will be useful for studying sulfate uptake, translocation, and regulatory mechanisms controlling compartmental SO 4 2− homeostasis.…”

Section: Discussionmentioning

confidence: 99%

A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells

Fatima

Okla

Mohsin

et al. 2020

IJMS

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Sulfur (S) is an essential element for all forms of life. It is involved in numerous essential processes because S is considered as the primary source of one of the essential amino acids, methionine, which plays an important role in biological events. For the control and regulation of sulfate in a metabolic network through fluxomics, a non-invasive tool is highly desirable that opens the door to monitor the level of the sulfate in real time and space in living cells without fractionation of the cells or tissue. Here, we engineered a FRET (fluorescence resonance energy transfer) based sensor for sulfate, which is genetically-encoded and named as FLIP-SP (Fluorescent indicator protein for sulfate). The FLIP-SP can measure the level of the sulfate in live cells. This sensor was constructed by the fusion of fluorescent proteins at the N-and C-terminus of sulfate binding protein (sbp). The FLIP-SP is highly specific to sulfate, and showed pH stability. Real-time monitoring of the level of sulfate in prokaryotic and eukaryotic cells showed sensor bio-compatibility with living cells. We expect that this sulfate sensor offers a valuable strategy in the understanding of the regulation of the flux of sulfate in the metabolic network.

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“…Recently, well optimized FRET based nanosensors have been developed for measuring Ras and Rap1 activity [41] and imaging glutamate levels in brain [42]. We have successfully constructed special FRET based nanosensors for quantification of leucine [43] and methionine [44], for in vivo monitoring of zinc concentrations [45], lysine flux [46] and glycine betaine levels [47] and vitamin B12 levels [48].…”

Section: Labelling Through Genetic Tagsmentioning

confidence: 99%

Nanoscale gizmos – the novel fluorescent probes for monitoring protein activity

Manzoor

Soleja

Mohsin

2018

Biochemical Engineering Journal

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a b s t r a c t Nanobiotechnology has emerged inherently as an interdisciplinary field, with collaborations from researchers belonging to diverse backgrounds like molecular biology, materials science and organic chemistry. Till the current times, researchers have been able to design numerous types of nanoscale fluorescent tool kits for monitoring protein-protein interactions through real time cellular imagery in a fluorescence microscope. It is apparent that supplementing any protein of interest with a fluorescence habit traces its function and regulation within a cell. Our review therefore highlights the application of several fluorescent probes such as molecular organic dyes, quantum dots (QD) and fluorescent proteins (FPs) to determine activity state, expression and localization of proteins in live and fixed cells. The focus is on Fluorescence Resonance Energy Transfer (FRET) based nanosensors that have been developed by researchers to visualize and monitor protein dynamics and quantify metabolites of diverse nature. FRET based toolkits permit the resolution of ambiguities that arise due to the rotation of sensor molecules and flexibility of the probe. Achievements of live cell imaging and efficient spatiotemporal resolution however have been possible only with the advent of fluorescence microscopic technology, equipped with precisely sensitive automated softwares.

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Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor

Cited by 25 publications

References 33 publications

Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor

Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor

A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells

Nanoscale gizmos – the novel fluorescent probes for monitoring protein activity

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