A single point mutation converts a proton-pumping rhodopsin into a red-shifted, 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 Turn-on Fluorescent Sensor for Chloride

10.26434/chemrxiv.13028192 ◽

2020 ◽

Author(s):

Jasmine Tutol ◽

Jessica Lee ◽

Hsichuan Chi ◽

Farah Faizuddin ◽

Sameera Abeyrathna ◽

...

Keyword(s):

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Sensor ◽

Single Point ◽

Proton Pumping ◽

Single Point Mutation ◽

E Coli ◽

Turn On ◽

Natural Function ◽

Fluorescent State

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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Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Applied and Environmental Microbiology ◽

10.1128/aem.03116-14 ◽

2015 ◽

Vol 81 (6) ◽

pp. 1996-2005 ◽

Cited By ~ 21

Author(s):

Gajendar Komati Reddy ◽

Steffen N. Lindner ◽

Volker F. Wendisch

Keyword(s):

Corynebacterium Glutamicum ◽

Point Mutation ◽

Single Point ◽

Proton Pumping ◽

Nadh:Ubiquinone Oxidoreductase ◽

Single Point Mutation ◽

Content Type ◽

Suppressor Mutations ◽

Substrate Level ◽

Suppressor Mutants

ABSTRACTCorynebacterium glutamicumuses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) fromClostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154–7160, 2010,http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth ofCorynebacterium glutamicumwith glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression ofudhAencoding soluble transhydrogenase fromEscherichia colipartly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants ofC. glutamicumNDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-IID213Gand thus by coupling to electron transport phosphorylation (ETP).

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Faculty Opinions recommendation of Unraveling adaptive evolution: how a single point mutation affects the protein coregulation network.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1041005.489903 ◽

2006 ◽

Author(s):

Mark D Adams

Keyword(s):

Point Mutation ◽

Adaptive Evolution ◽

Single Point ◽

Single Point Mutation

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Faculty Opinions recommendation of One small step for a yeast--microevolution within macrophages renders Candida glabrata hypervirulent due to a single point mutation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.724428301.793504585 ◽

2015 ◽

Author(s):

Joseph Heitman ◽

Blake Billmyre

Keyword(s):

Point Mutation ◽

Candida Glabrata ◽

Single Point ◽

Single Point Mutation ◽

Small Step

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Dominant-negative inhibition of pheromone receptor signaling by a single point mutation in the G protein α subunit. Vol. 279 (2004) 35287-35297

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)56430-2 ◽

2005 ◽

Vol 280 (33) ◽

pp. 29988

Author(s):

Yuh-Lin Wu ◽

Shelley B. Hooks ◽

T. Kendall Harden ◽

Henrik G. Dohlman

Keyword(s):

G Protein ◽

Point Mutation ◽

Single Point ◽

Dominant Negative ◽

Single Point Mutation ◽

Receptor Signaling ◽

Α Subunit ◽

Pheromone Receptor ◽

G Protein Α Subunit

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Can a Micronutrient Mixture Delay the Onset and Progression of Symptoms of Single-Point Mutation Diseases?

Journal of the American College of Nutrition ◽

10.1080/07315724.2021.1910592 ◽

2021 ◽

pp. 1-10

Author(s):

Kedar N. Prasad ◽

Stephen C. Bondy

Keyword(s):

Point Mutation ◽

Single Point ◽

Single Point Mutation

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Structural basis for a complex I mutation that blocks pathological ROS production

Nature Communications ◽

10.1038/s41467-021-20942-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhan Yin ◽

Nils Burger ◽

Duvaraka Kula-Alwar ◽

Dunja Aksentijević ◽

Hannah R. Bridges ◽

...

Keyword(s):

Point Mutation ◽

Complex I ◽

Structural Changes ◽

Ischemia Reperfusion ◽

Single Point ◽

Structural Basis ◽

Single Point Mutation ◽

Ros Production

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

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