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Related Experiment Videos

Salt-stabilized globular protein structure in 7 M aqueous urea solution

V Dötsch1, G Wider, G Siegal

  • 1Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.

FEBS Letters
|September 25, 1995
PubMed
Summary

This study investigates how salts and compounds affect protein folding equilibrium in urea solutions. Researchers identified conditions to stabilize folded protein for structural analysis in urea.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Chemical Physics

Background:

  • High concentrations of urea are commonly used to unfold proteins.
  • The 434-repressor N-terminal domain exists as a mixture of folded and unfolded states in 7M urea.
  • Conformational interconversion is slow, allowing NMR to monitor equilibrium.

Purpose of the Study:

  • To investigate the influence of various salts and non-ionic compounds on the conformational equilibrium of the 434-repressor N-terminal domain in urea solutions.
  • To identify solution conditions that favor the folded state of the protein in the presence of high urea concentrations.
  • To establish a basis for NMR structure determination of the folded protein in concentrated urea and study its solvation.

Main Methods:

  • Nuclear Magnetic Resonance (NMR) spectroscopy to monitor protein conformation.

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  • Preparation of a 63-residue N-terminal domain of the 434-repressor.
  • Systematic variation of solution conditions (urea concentration, pH, temperature, presence of salts and non-ionic compounds).

    • Main Results:

    • A 7 M aqueous urea solution at pH 7.5 and 18°C contains approximately 10% folded and 90% unfolded 434-repressor N-terminal domain.
    • Separate NMR resonances indicate slow interconversion between folded and unfolded states.
    • Specific salts or non-ionic compounds were found to shift the equilibrium towards the folded state.

    Conclusions:

    • The conformational equilibrium of the 434-repressor N-terminal domain in urea is sensitive to solution additives.
    • Conditions were found that stabilize the folded protein in 6-7 M urea.
    • These findings provide a foundation for structural studies of proteins in denaturing conditions and for understanding protein solvation in mixed aqueous-organic environments.