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Related Experiment Videos

A monomeric red fluorescent protein.

Robert E Campbell1, Oded Tour, Amy E Palmer

  • 1Department of Pharmacology. University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

Proceedings of the National Academy of Sciences of the United States of America
|June 13, 2002
PubMed
Summary
This summary is machine-generated.

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Researchers engineered a monomeric red fluorescent protein (mRFP1) from Discosoma red fluorescent protein (DsRed) to overcome limitations of tetrameric structure for fusion tagging. This improved red fluorescent protein enables functional fusion proteins, enhancing cell biology research.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Coelenterate fluorescent proteins often exhibit quaternary structures, like the obligate tetramerization of Discosoma red fluorescent protein (DsRed).
  • The tetrameric nature of DsRed significantly restricts its utility as a genetically encoded fusion tag in biological research.
  • Aequorea green fluorescent protein (GFP) dimerizes weakly, but its quaternary structure has not impeded its widespread use.

Purpose of the Study:

  • To engineer a monomeric red fluorescent protein (mRFP1) from DsRed.
  • To overcome the limitations imposed by the tetrameric structure of DsRed for fusion protein applications.
  • To create a red fluorescent protein suitable for use as a genetically encoded fusion tag.

Main Methods:

  • Stepwise evolution of DsRed by disrupting subunit interfaces and employing random and directed mutagenesis.

Related Experiment Videos

  • Introduction of arginine residues to disrupt quaternary structure, followed by 17 (dimer) and 33 (mRFP1) amino acid substitutions to restore fluorescence.
  • Construction and functional assessment of fusion proteins using connexin43 and the engineered red fluorescent proteins.

    • Main Results:

    • Successful engineering of DsRed into a tandem dimer and subsequently a monomeric red fluorescent protein (mRFP1).
    • mRFP1 fusions with connexin43 formed fully functional gap junctions, unlike fusions with tetrameric DsRed or its dimer.
    • mRFP1 exhibits faster maturation (>10x) and red-shifted spectra (excitation 584 nm, emission 607 nm) compared to DsRed, offering similar brightness in living cells.

    Conclusions:

    • Monomeric red fluorescent protein (mRFP1) provides a viable alternative to tetrameric DsRed for fusion tagging applications.
    • The red-shifted spectrum and improved functionality of mRFP1 enhance its utility in live-cell imaging and tissue penetration.
    • Engineered fluorescent proteins like mRFP1 expand the toolkit for advanced biological research and imaging techniques.