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Related Experiment Videos

Protein stability parameters measured by hydrogen exchange

Y Bai1, J S Milne, L Mayne

  • 1Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104-6059.

Proteins
|September 1, 1994
PubMed
Summary

Hydrogen exchange (HX) rates in cytochrome c reveal protein unfolding thermodynamics. This method offers insights into protein stability below melting transitions, accounting for proline isomerization.

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Area of Science:

  • Biochemistry
  • Protein dynamics
  • Thermodynamics

Background:

  • Cytochrome c is a crucial protein in cellular respiration.
  • Understanding protein unfolding is vital for comprehending protein function and stability.
  • Standard denaturation methods may not capture all aspects of protein stability.

Purpose of the Study:

  • To investigate the thermodynamic parameters of global unfolding in oxidized cytochrome c using hydrogen exchange (HX).
  • To compare HX-derived stability data with traditional denaturation methods.
  • To explore the influence of proline isomerization on protein stability measurements.

Main Methods:

  • Utilizing one-dimensional nuclear magnetic resonance (1D NMR) to measure hydrogen exchange rates.
  • Analyzing HX rates of peptide group NH hydrogens in equine cytochrome c.

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  • Applying thermodynamic principles to interpret unfolding equilibrium and denaturant binding.

    • Main Results:

    • HX rates are governed by transient global unfolding equilibrium in cytochrome c.
    • Thermodynamic parameters of unfolding can be determined under mild conditions below the melting transition.
    • Discrepancies between HX and denaturation methods arise from slow proline cis-trans isomerization.
    • HX experiments at low denaturant concentrations show increased protein stability, aligning with the denaturant binding model.

    Conclusions:

    • Hydrogen exchange is a powerful tool for characterizing protein stability and unfolding thermodynamics.
    • The study highlights the importance of considering proline isomerization in stability assessments.
    • Results support the denaturant binding model for explaining protein stability deviations.