Arsenic poisoning has been a major concern that causes severe toxicological damages. Therefore, intricate and inclusive understanding of arsenic flux rates is required to ascertain the cellular concentration and establish the carcinogenetic mechanism of this toxicant at real time. The lack of sufficiently sensitive sensing systems has hampered research in this area. In this study, we constructed a fluorescent resonance energy transfer (FRET)-based nanosensor, named SenALiB (Sensor for Arsenic Linked Blackfoot disease) which contains a metalloregulatory arsenic-binding protein (ArsR) as the As
3+
sensing element inserted between the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus. SenALiB takes advantage of the ratiometic FRET readout which measures arsenic with high specificity and selectivity. SenALiB offers rapid detection response, is stable to pH changes and provides highly accurate, real-time optical readout in cell-based assays. SenALiB-676n with a binding constant (
K
d
) of 0.676 × 10
−6
M is the most efficient affinity mutant and can be a versatile tool for dynamic measurement of arsenic concentration in both prokaryotes and eukaryotes
in vivo
in a non-invasive manner.
Silver is commonly
used in wound dressing, photography, health
care products, laboratories, pharmacy, biomedical devices, and several
industrial purposes. Silver (Ag+) ions are more toxic pollutants
widely scattered in the open environment by natural processes and
dispersed in soil, air, and water bodies. Ag+ binds with
metallothionein, macroglobulins, and albumins, which may lead to the
alteration of various enzymatic metabolic pathways. To analyze the
uptake and metabolism of silver ions in vitro as well as in cells,
a range of high-affinity fluorescence-based nanosensors has been constructed
using a periplasmic protein CusF, a part of the CusCFBA
efflux complex, which is involved in providing resistance against
copper and silver ions in Escherichia coli. This nanosensor was constructed by combining of two fluorescent
proteins (donor and acceptor) at the N- and C-terminus of the silver-binding
protein (CusF), respectively. SenSil (WT) with a
binding constant (K
d) of 5.171 μM
was more efficient than its mutant variants (H36D and F71W). This
nanosensor allows monitoring the level of silver ions in real time
in prokaryotes and eukaryotes without any disruption of cells or tissues.
Nitrate (NO3
–) is a critical source
of nitrogen (N) available to microorganisms and plants. Nitrate sensing
activates signaling pathways in the plant system that impinges upon,
developmental, molecular, metabolic, and physiological responses locally,
and globally. To sustain, the high crop productivity and high nutritional
value along with the sustainable environment, the study of rate-controlling
steps of a metabolic network of N assimilation through fluxomics becomes
an attractive strategy. To monitor the flux of nitrate, we developed
a non-invasive genetically encoded fluorescence resonance energy transfer
(FRET)-based tool named “FLIP-NT” that monitors the
real-time uptake of nitrate in the living cells. The developed nanosensor
is suitable for real-time monitoring of nitrate flux in living cells
at subcellular compartments with high spatio-temporal resolution.
The developed FLIP-NT nanosensor was not affected by the pH change
and have specificity for nitrate with an affinity constant (K
d) of ∼5 μM. A series of affinity
mutants have also been generated to expand the physiological detection
range of the sensor protein with varying K
d values. It has been found that this sensor successfully detects
the dynamics of nitrate fluctuations in bacteria and yeast, without
the disruption of cellular organization. This FLIP-NT nanosensor could
be a very important tool that will help us to advance the understanding
of nitrate signaling.
Since the last decade, a lot of advancement has been made to understand biological processes involving complex intracellular pathways. The major challenge faced was monitoring and trafficking of metabolites in real time. Although a range of quantitative and imaging techniques have been developed so far, the discovery of green fluorescent proteins (GFPs) has revolutionized the advancement in the field of metabolomics. GFPs and their variants have enabled researchers to 'paint' a wide range of biological molecules. Fluorescence resonance energy transfer (FRET)-based genetically encoded sensors is a promising technology to decipher the real-time monitoring of the cellular events inside living cells. GFPs and their variants, due to their intrinsic fluorescence properties, are extensively being used nowadays in cell-based assays. This review focuses on structure and function of GFP and its derivatives, mechanism emission and their use in the development of FRET-based sensors for metabolites.
Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (K d) of 5.09 × 10−7 M, thus providing a detection range for Hg2+ quantification between 0.210 µM and 1.196 µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.
Upon binding of alcohols, ObpIIa undergoes conformational changes resulting in transfer of energy in the form of FRET from donor to the acceptor fluorophore.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.