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Mechanism of ArcLight derived GEVIs involves electrostatic interactions that can affect proton wires.

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Genetically encoded voltage indicators were improved by adding negative charges to fluorescent proteins (FPs), enhancing voltage-dependent signals. Mutants showed wavelength-specific responses, revealing new ways to map cellular electrical activity.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Neuroscience

Background:

  • Genetically encoded voltage indicators (GEVIs) like ArcLight utilize fluorescent proteins (FPs) for voltage sensing.
  • Intermolecular electrostatic interactions between FPs mediate voltage-dependent optical signals.

Purpose of the Study:

  • To improve the voltage sensitivity of ArcLight and its derivatives.
  • To investigate the mechanisms underlying voltage-dependent fluorescence changes.
  • To explore wavelength-specific responses and potential applications in cellular electrophysiology.

Main Methods:

  • Random mutagenesis of FPs to introduce external charges.
  • Voltage-clamp fluorometry with alternating excitation wavelengths (390 nm and 470 nm).
  • Analysis of mutant responses to varying membrane potentials.

Main Results:

  • A negative charge mutation increased signal >10-fold.
  • Repositioning the charge reversed signal polarity, indicating "hot spots" of interaction.
  • Mutants exhibited wavelength-dependent, ratiometric signals with opposing polarities.
  • Specific mutation (E222H) showed voltage-dependent fluorescence increase at 390 nm, affecting the proton wire.

Conclusions:

  • External electrostatic pathways in FPs influence fluorescence in a wavelength-specific manner.
  • Mutated FPs offer novel ways to probe chromophore fluorescence and conformational changes.
  • These engineered sensors provide new tools for mapping cellular electrical activity.