Engineering an FMN-based iLOV protein for the detection of arsenic ions

Ravikumar, Yuvaraj; Nadarajan, Sivakumar; Lee, Chong‐Soon; Yun, Hyungdon

doi:10.1016/j.ab.2017.02.012

Cited by 21 publications

(16 citation statements)

References 22 publications

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“…Few studies have examined how mutations around the FMN‐binding sites in FbFPs impacts the affinity for metals. Reported examples have focused on how mutations affect As III binding in iLOV and Hg II binding in a Presudomonas putida ‐derived FbFP, but have not focused on essential metals such as copper. Our work sets the foundation for future mutagenesis studies to explore the copper‐binding pocket and interactions with the flavin chromophore.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, Ravikumar and co‐workers reported that the flavin‐based fluorescent protein iLOV binds Cu II ( K d =4.72 μ m at pH 7.4) . The same group also reported binding of FbFP variants to arsenic(III) and mercury(II) . Metal interactions with other flavin‐based fluorescent proteins such as CreiLOV have not been reported and these proteins have not been used to develop high affinity single protein sensors or FRET‐based sensors.…”

Section: Introductionmentioning

confidence: 95%

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Live‐Cell Copper‐Induced Fluorescence Quenching of the Flavin‐Binding Fluorescent Protein CreiLOV

2020

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CreiLOV is a flavin‐binding fluorescent protein derived from the blue‐light photoreceptor protein family that contains light‐oxygen‐voltage (LOV) sensing domains. Flavin‐binding fluorescent proteins represent a promising foundation for new fluorescent reporters and biosensors that can address limitations of the well‐established green fluorescent protein (GFP) family. Flavin‐binding fluorescent proteins are smaller than GFPs, are stable over a wider pH range, offer rapid chromophore incorporation, and are oxygen‐independent so can be applied to live anaerobic organisms. Among the flavin‐binding fluorescent proteins, CreiLOV has a high quantum yield and excellent photophysical properties, making it promising for cellular applications. Here, we investigated the suitability of CreiLOV as an intensity‐ and fluorescence‐lifetime‐based metal sensor. CreiLOV selectively binds copper(II) over other biologically relevant metals with low‐micromolar affinity, resulting in fluorescence quenching and a decrease in the fluorescence lifetime that can be observed in cuvettes and live bacterial cells.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

Live‐Cell Copper‐Induced Fluorescence Quenching of the Flavin‐Binding Fluorescent Protein CreiLOV

2020

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“…A significant number of whole-cell arsenic and/or cadmium biosensors has been already described in literature and is based on the realization of reporter systems containing regulatory cis-acting sequences interacting with a transcriptional repressor belonging to the ArsR/SmtB family [ 15 , 16 ]. Biosensors are not intended to fully replace chemical methods but have the advantage of lower fabrication cost and higher stability and can offer on-site monitoring of even trace levels of targeted compounds in comparison with non-portable analytical methodologies [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system

et al. 2018

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BackgroundThe characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differently from other microbes, are not organized into a single operon. They encode the proteins: arsenate reductase, TtArsC, arsenic efflux membrane transporter, TtArsX, and transcriptional repressor, TtSmtB.ResultsIn this work we show that the arsenic efflux protein TtArsX and the arsenic responsive transcriptional repressor TtSmtB are required to provide resistance to cadmium. We analyzed the sensitivity to Cd(II) of mutants lacking TtArsX, finding that they are more sensitive to this metal than the wild type strain. In addition, using promoter probe reporter plasmids, we show that the transcription of TtarsX is also stimulated by the presence of Cd(II) in a TtSmtB-dependent way. Actually, a regulatory circuit composed of TtSmtB and a reporter gene expressed from the TtarsX promoter responds to variation in Cd(II), As(III) and As(V) concentrations.ConclusionsOur results demonstrate that the system composed by TtSmtB and TtArsX is responsible for both the arsenic and cadmium resistance in T. thermophilus. The data also support the use of T. thermophilus as a suitable chassis for the design and development of As-Cd biosensors.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0918-7) contains supplementary material, which is available to authorized users.

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“…Also, the iLOV protein has been successfully modified either to improve its photostability or to shift its absorption and emission optical bands to longer or shorter wavelengths (Christie et al, 2012; Davari et al, 2016; Khrenova et al, 2017; Khrenova et al, 2015). The iLOV protein has also been modified into a highly sensitive sensor for intracellular arsenic ions (Ravikumar et al, 2017). These modifications make iLOV a versatile tool for enhancing future studies to characterize SHFV infections in vitro and in vivo .…”

Section: Discussionmentioning

confidence: 99%

Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV)

et al. 2018

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Recombinant SHFV infectious cDNA clones expressing a foreign gene from an additional sg mRNA were constructed. Two 3′ genomic region sites, between ORF4′ and ORF2b and between ORF4 and ORF5, were utilized for insertion of the myxoma M013 gene with a C-terminal V5 tag followed by one of the three inserted transcription regulatory sequences (TRS), TRS2′, TRS4′ or TRS7. M013 insertion at the ORF4′/ORF2b site but not the ORF4/ORF5 site generated progeny virus but only recombinant viruses with an inserted TRS2′ retained the entire M013 gene through passage four. Insertion of an auto-fluorescent protein gene, iLOV, with an inserted TRS2′ at the ORF4′/ORF2b site generated viable progeny virus and iLOV expression was maintained through passage eight. Although regulation of SHFV subgenomic RNA synthesis is complex, the ORF4′/ORF2b site, which is located between the two sets of minor structural proteins, is able to tolerate foreign gene insertion.

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Engineering an FMN-based iLOV protein for the detection of arsenic ions

Cited by 21 publications

References 22 publications

Live‐Cell Copper‐Induced Fluorescence Quenching of the Flavin‐Binding Fluorescent Protein CreiLOV

Live‐Cell Copper‐Induced Fluorescence Quenching of the Flavin‐Binding Fluorescent Protein CreiLOV

Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system

Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV)

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