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Related Experiment Videos

Circular permutation and receptor insertion within green fluorescent proteins.

G S Baird1, D A Zacharias, R Y Tsien

  • 1Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0647, USA.

Proceedings of the National Academy of Sciences of the United States of America
|September 29, 1999
PubMed
Summary

Genetically engineered green fluorescent proteins (GFPs) can now undergo major rearrangements and insertions while retaining fluorescence. This robustness enables the creation of novel genetically encoded protein sensors for biological research.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Biochemistry

Background:

  • Green fluorescent protein (GFP) fusions are vital tools in biology and biotechnology for protein labeling.
  • Traditionally, GFP is used as an intact unit, fused to the termini or inserted within a host protein.
  • Major structural alterations were presumed to abolish GFP fluorescence due to its complex folding and chromophore formation.

Purpose of the Study:

  • To investigate the impact of significant structural modifications on GFP fluorescence.
  • To explore the potential of creating novel fluorescent protein-based biosensors through GFP engineering.
  • To assess the robustness of the GFP folding process.

Main Methods:

  • Constructing circularly permuted variants of GFP by interchanging amino and carboxyl portions.

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  • Inserting foreign protein domains into specific sites within the GFP structure, such as Tyr-145 in enhanced yellow fluorescent protein (EYFP).
  • Evaluating the fluorescence properties, pKa values, and chromophore orientation of modified GFPs.
  • Testing the functionality of engineered GFPs as biosensors, e.g., calmodulin insertion for Ca(2+) detection.

    • Main Results:

    • Circularly permuted GFPs retain fluorescence, albeit with altered pKa and chromophore orientation.
    • Specific internal sites within GFP tolerate the insertion of entire protein domains.
    • Insertion of calmodulin or a zinc finger domain into EYFP resulted in proteins whose fluorescence is modulated by metal binding.
    • A calmodulin-grafted EYFP successfully monitored cytosolic Ca(2+) in single mammalian cells.

    Conclusions:

    • GFP folding is remarkably robust, tolerating extensive structural rearrangements and insertions.
    • Engineered GFPs offer a versatile platform for developing genetically encodable, physiological indicators.
    • This work expands the toolkit for creating novel fluorescent protein-based biosensors for diverse biological applications.