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Related Experiment Videos

What causes hyperfluorescence: folding intermediates or conformationally flexible native states?

John Ervin1, Edgar Larios, Szabolcs Osváth

  • 1Department of Chemistry, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Illinois 61801, USA.

Biophysical Journal
|June 25, 2002
PubMed
Summary

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Hyperfluorescence during protein unfolding may indicate a loosened native state, not necessarily a folding intermediate. This study suggests caution in interpreting hyperfluorescence solely as proof of thermodynamic intermediates.

Area of Science:

  • Biophysics
  • Protein Folding
  • Structural Biology

Background:

  • Hyperfluorescence during protein unfolding titrations is often interpreted as evidence of thermodynamic folding intermediates.
  • An alternative explanation suggests hyperfluorescence might arise from a "pretransition" state of the native protein.

Purpose of the Study:

  • To investigate whether hyperfluorescence indicates a "pretransition" loosened native state rather than a thermodynamic folding intermediate.
  • To model such native states using mutants of a fluorescent ubiquitin variant.

Main Methods:

  • Studied mutants of a fluorescent ubiquitin variant with cavities near the tryptophan fluorophore.
  • Utilized circular dichroism, fluorescence intensity unfolding titrations, fluorescence anisotropy, and molecular dynamics simulations.

Related Experiment Videos

  • Correlated protein flexibility with enhanced fluorescence intensity.

    • Main Results:

    • Developed a model for hyperfluorescence based on static and dynamic conformational properties of the native state.
    • Observed hyperfluorescence potentially linked to conformational loosening of the native state.
    • Analyzed examples including apomyoglobin denaturant unfolding and phosphoglycerate kinase cold denaturation.

    Conclusions:

    • Hyperfluorescence during unfolding titrations is not conclusive proof of thermodynamic folding intermediates.
    • A loosened native state ("pretransition" state) can also cause hyperfluorescence.
    • Caution is advised when interpreting hyperfluorescence as solely indicative of thermodynamic intermediates.