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Related Concept Videos

Reporter Genes02:11

Reporter Genes

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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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Engineering a Fluorescent Protein Color Switch Using Entropy-Driven β-Strand Exchange.

Anna Miriam John1, Harsimranjit Sekhon1, Jeung-Hoi Ha1

  • 1Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, United States.

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|January 10, 2022
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Researchers developed a novel biosensor using protein conformational switches. Ligand binding triggers a color change in a fluorescent protein, enabling sensitive detection in vitro and in cells.

Keywords:
alternate frame foldingfluorescent protein biosensorloop entropyprotein conformational switchprotein engineering

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Protein conformational switches are crucial for biosensing, linking ligand binding to optical signals.
  • A key challenge is effectively coupling input (ligand binding) and output (optical readout) domains via conformational changes.

Purpose of the Study:

  • To engineer a biosensor where input domain folding drives output domain conformational change for signal transduction.
  • To demonstrate a novel mechanism for coupling ligand binding to a fluorescent protein color shift.

Main Methods:

  • Engineered a biosensor using circularly permuted FK506 binding protein (cpFKBP) as the input domain and a GFP variant as the output domain.
  • Utilized the loop-closure entropy principle for coupling ligand-induced cpFKBP folding to GFP β-strand exchange.
  • Investigated the mechanism in vitro and in cultured cells.

Main Results:

  • Achieved a sixfold green-to-yellow ratiometric fluorescence change in vitro upon FK506 or rapamycin binding.
  • Observed a 35-fold intensiometric fluorescence increase in cultured cells.
  • Identified high-energy barriers in GFP folding influencing the mechanism in vitro versus in cells.

Conclusions:

  • The designed biosensor demonstrates a proof-of-concept for genetically encoded, modular protein switches.
  • The mechanism highlights the potential of loop-closure entropy for signal coupling in biosensors.
  • The study suggests that GFP folding dynamics influence the observed signal transduction mechanism.