A Single Point Mutation Converts a Proton-pumping Rhodopsin into a Turn-on Fluorescent Sensor for Chloride

The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a turn-on fluorescent sensor for chloride in detergent micelles and in live Escherichia coli. This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The addition of chloride tunes the pKa of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single cell fluorescence microscopy experiments with E. coli, expressing a GR1 fusion with cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of chloride. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin fluorescent chloride sensors for cellular applications and learn how proteins can evolve and adapt to bind anions in water.

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A single point mutation converts a proton-pumping rhodopsin into a red-shifted, turn-on fluorescent sensor for chloride

Chemical Science ◽

10.1039/d0sc06061e ◽

2021 ◽

Author(s):

Jasmine N. Tutol ◽

Jessica Lee ◽

Hsichuan Chi ◽

Farah N. Faizuddin ◽

Sameera S. Abeyrathna ◽

...

Keyword(s):

Point Mutation ◽

Fluorescent Sensor ◽

Single Point ◽

Proton Pumping ◽

Single Point Mutation ◽

Turn On ◽

Gloeobacter Violaceus

By utilizing laboratory-guided evolution, we have converted the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride.

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Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v20i3.1825 ◽

2021 ◽

Vol 20 (3) ◽

Author(s):

Hairul Aini Hamzah ◽

Rahmatullah Sirat ◽

Mohammed Imad A. Mustafa Mahmud ◽

Roesnita Baharudin

Keyword(s):

Mutational Analysis ◽

Single Point ◽

Point Mutations ◽

Multidrug Resistant ◽

Quinolone Resistance ◽

Resistant Bacteria ◽

Single Point Mutation ◽

Phenotypic Resistance ◽

Drug Effectiveness ◽

E Coli

Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

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Enhancement of coenzyme binding by a single point mutation at the coenzyme binding domain of E. coli lactaldehyde dehydrogenase

Protein Science ◽

10.1110/ps.073277108 ◽

2008 ◽

Vol 17 (3) ◽

pp. 563-570 ◽

Cited By ~ 11

Author(s):

José Salud Rodríguez-Zavala

Keyword(s):

Point Mutation ◽

Single Point ◽

Binding Domain ◽

Single Point Mutation ◽

E Coli ◽

Coenzyme Binding

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Identification of plasmid- and integron-borne bla IMP-1 and bla IMP-10 in clinical isolates of Serratia marcescens

Journal of Medical Microbiology ◽

10.1099/jmm.0.006874-0 ◽

2009 ◽

Vol 58 (2) ◽

pp. 217-221 ◽

Cited By ~ 16

Author(s):

Zhuting Hu ◽

Wei-Hua Zhao

Keyword(s):

Serratia Marcescens ◽

Single Point ◽

Gene Cassette ◽

Clinical Isolates ◽

Single Point Mutation ◽

Class 1 Integron ◽

Gene Cassettes ◽

E Coli ◽

Class 1 ◽

P2 Promoter

The emergence of carbapenem-hydrolysing metallo-β-lactamases (MBLs) is a serious threat to the clinical utility of carbapenems. This study identified plasmid- and integron-borne bla IMP-1 and bla IMP-10 in clinical isolates of Serratia marcescens. The bla IMP-1 and bla IMP-10 gene cassettes were carried by a class 1 integron and followed by the aac(6′)-IIc gene cassette. The bla IMP-1 and bla IMP-10 gene cassettes were preceded by a weak Pant promoter, TGGACA(N)17TAAGCT, and an inactive P2 promoter, TTGTTA(N)14TACAGT. These genes were easily transferred to Escherichia coli by conjugation and transformation, indicating that they are located on transferable plasmids. Due to the acquisition of bla IMP-1, the susceptibility of E. coli transconjugants to imipenem, meropenem, panipenem and biapenem decreased by 32-, 256-, 64- and 128-fold, respectively. In comparison, after gaining bla IMP-10, the susceptibility of E. coli transconjugants to the four carbapenems decreased by 64-, 2048-, 256- and 64-fold, respectively. Strains harbouring bla IMP-10 showed higher-level resistance to imipenem, meropenem and panipenem than the strains harbouring bla IMP-1, although the nucleotide sequences of the class 1 integrons carrying bla IMP-10 and bla IMP-1 were identical except for a single point mutation.

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1.8 Å bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants

Biochemical Journal ◽

10.1042/bj20061401 ◽

2007 ◽

Vol 402 (1) ◽

pp. 35-42 ◽

Cited By ~ 174

Author(s):

Andre C. Stiel ◽

Simon Trowitzsch ◽

Gert Weber ◽

Martin Andresen ◽

Christian Eggeling ◽

...

Keyword(s):

Blue Light ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Uv Light ◽

Structural Features ◽

Trans Isomerization ◽

Fluorescent State ◽

Green Fluorescent ◽

New Applications ◽

Key Events

RSFPs (reversibly switchable fluorescent proteins) may be repeatedly converted between a fluorescent and a non-fluorescent state by irradiation and have attracted widespread interest for many new applications. The RSFP Dronpa may be switched with blue light from a fluorescent state into a non-fluorescent state, and back again with UV light. To obtain insight into the underlying molecular mechanism of this switching, we have determined the crystal structure of the fluorescent equilibrium state of Dronpa. Its bicyclic chromophore is formed spontaneously from the Cys62–Tyr63–Gly64 tripeptide. In the fluorescent state, it adopts a slightly non-coplanar cis conformation within the interior of a typical GFP (green fluorescent protein) β-can fold. Dronpa shares some structural features with asFP595, another RSFP whose chromophore has previously been demonstrated to undergo a cis–trans isomerization upon photoswitching. Based on the structural comparison with asFP595, we have generated new Dronpa variants with an up to more than 1000-fold accelerated switching behaviour. The mutations which were introduced at position Val157 or Met159 apparently reduce the steric hindrance for a cis–trans isomerization of the chromophore, thus lowering the energy barrier for the blue light-driven on-to-off transition. The findings reported in the present study support the view that a cis–trans isomerization is one of the key events common to the switching mechanism in RSFPs.

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Emergence and molecular basis of azithromycin resistance in typhoidal Salmonella in Dhaka, Bangladesh

10.1101/594531 ◽

2019 ◽

Author(s):

Yogesh Hooda ◽

Senjuti Saha ◽

Mohammad S I Sajib ◽

Hafizur Rahman ◽

Stephen P Luby ◽

...

Keyword(s):

Efflux Pump ◽

Single Point ◽

Salmonella Typhi ◽

Oral Drug ◽

Single Point Mutation ◽

E Coli ◽

Vaccine Introduction ◽

Rnd Efflux Pump ◽

Azithromycin Resistance

With rising fluoroquinolone and ceftriaxone-resistant Salmonella Typhi, azithromycin, a macrolide, has become the last oral drug available against typhoid. Between 2009-2016, we isolated 1,082 Salmonella Typhi and Paratyphi A strains in Bangladesh, 13 (12 Typhi and 1 Paratyphi A) of which were azithromycin-resistant. When compared to 462 previously sequenced Typhi strains, the genomes of the 12 azithromycin-resistant Typhi strains (4.3.1 sub-clade, H58) harbored an exclusive non-synonymous single-point mutation R717Q in AcrB, an RND-efflux pump. Expression of AcrB-R717Q in E. coli and Typhi strains increased its minimum inhibitory concentration (MIC) for azithromycin by 11- and 3-fold respectively. The azithromycin-resistant Paratyphi A strain also contained a mutation at R717 (R717L), whose introduction in E. coli and Paratyphi A strains increased MIC by 7- and 3-fold respectively, confirming the role of R717 mutations in conferring azithromycin resistance. With increasing azithromycin use, strains with R717 mutations may spread leading to treatment failures, making antibiotic stewardship and vaccine introduction imperative.

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Efficient switching of mCherry fluorescence using chemical caging

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617280114 ◽

2017 ◽

Vol 114 (27) ◽

pp. 7013-7018 ◽

Cited By ~ 11

Author(s):

Bas M. C. Cloin ◽

Elke De Zitter ◽

Desiree Salas ◽

Vincent Gielen ◽

Gert E. Folkers ◽

...

Keyword(s):

Single Molecule ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Functional Characterization ◽

Fluorescence Emission ◽

Total Recovery ◽

X Ray Crystallography ◽

Chemically Induced ◽

Wide Range ◽

Fluorescent State

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores.

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A photoactivatable green-fluorescent protein from the phylum Ctenophora

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2009.1774 ◽

2009 ◽

Vol 277 (1685) ◽

pp. 1155-1160 ◽

Cited By ~ 25

Author(s):

Steven H. D. Haddock ◽

Nadia Mastroianni ◽

Lynne M. Christianson

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Green Fluorescent Proteins ◽

Optical Probes ◽

Fluorescent Emission ◽

The Family ◽

Fluorescent State ◽

Green Fluorescent

Genes for the family of green-fluorescent proteins (GFPs) have been found in more than 100 species of animals, with some species containing six or more copies producing a variety of colours. Thus far, however, these species have all been within three phyla: Cnidaria, Arthropoda and Chordata. We have discovered GFP-type fluorescent proteins in the phylum Ctenophora, the comb jellies. The ctenophore proteins share the x YG chromophore motif of all other characterized GFP-type proteins. These proteins exhibit the uncommon property of reversible photoactivation, in which fluorescent emission becomes brighter upon exposure to light, then gradually decays to a non-fluorescent state. In addition to providing potentially useful optical probes with novel properties, finding a fluorescent protein in one of the earliest diverging metazoans adds further support to the possibility that these genes are likely to occur throughout animals.

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Fatal amyloid formation in a patient’s antibody light chain is caused by a single point mutation

eLife ◽

10.7554/elife.52300 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 3

Author(s):

Pamina Kazman ◽

Marie-Theres Vielberg ◽

María Daniela Pulido Cendales ◽

Lioba Hunziger ◽

Benedikt Weber ◽

...

Keyword(s):

Conformational Changes ◽

Light Chain ◽

Single Point ◽

Proteolytic Cleavage ◽

Terminal Segment ◽

Fibril Formation ◽

Single Point Mutation ◽

Amyloid Formation ◽

E Coli ◽

Depth Analysis

In systemic light chain amyloidosis, an overexpressed antibody light chain (LC) forms fibrils which deposit in organs and cause their failure. While it is well-established that mutations in the LC’s VL domain are important prerequisites, the mechanisms which render a patient LC amyloidogenic are ill-defined. In this study, we performed an in-depth analysis of the factors and mutations responsible for the pathogenic transformation of a patient-derived λ LC, by recombinantly expressing variants in E. coli. We show that proteolytic cleavage of the patient LC resulting in an isolated VL domain is essential for fibril formation. Out of 11 mutations in the patient VL, only one, a leucine to valine mutation, is responsible for fibril formation. It disrupts a hydrophobic network rendering the C-terminal segment of VL more dynamic and decreasing domain stability. Thus, the combination of proteolytic cleavage and the destabilizing mutation trigger conformational changes that turn the LC pathogenic.

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