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Measuring the stability of partly folded proteins using TMAO
Cecilia C Mello1, Doug Barrick
1Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Protein Science : a Publication of the Protein Society
|June 26, 2003
Summary
This study introduces a novel method combining urea denaturation and trimethylamine N-oxide (TMAO) stabilization to accurately measure protein unfolding free energy. This approach successfully quanties thermodynamic parameters for partly folded proteins, overcoming limitations of standard techniques.
Area of Science:
- Biochemistry and Molecular Biophysics
- Protein Thermodynamics
- Chemical Denaturation Studies
Background:
- Standard methods for protein unfolding free energy measurement require observable native baselines, which are often unattainable for partly folded proteins.
- Neither chemical denaturation nor osmolyte-induced refolding alone provides both native and denatured baselines for accurate thermodynamic analysis of partly folded proteins.
- Partly folded proteins present a challenge for traditional thermodynamic studies due to incomplete baseline resolution.
Purpose of the Study:
- To develop and validate a combined method using urea denaturation and trimethylamine N-oxide (TMAO) stabilization for measuring protein unfolding free energy.
- To accurately determine thermodynamic unfolding parameters for partly folded proteins.
- To assess the independent contributions of urea and TMAO to protein stability.
Main Methods:
- Combined urea denaturation with trimethylamine N-oxide (TMAO) stabilization to induce baseline-resolved structural transitions.
- Studied two-state equilibrium unfolding of Barnase and the Notch ankyrin domain.
- Utilized a global fit analysis assuming additive, linear dependence of unfolding free energy on cosolvent concentrations.
Main Results:
- The free energy of unfolding (DeltaG degrees) was found to depend linearly on TMAO concentration for both proteins.
- The sensitivity of DeltaG degrees to urea (m-value) was independent of TMAO concentration, indicating independent cosolvent effects.
- Thermodynamic parameters derived from the combined method closely matched those from standard urea-induced unfolding.
Conclusions:
- The combination of urea denaturation and TMAO stabilization provides a robust method for measuring the free energy of unfolding in partly folded proteins.
- Urea and TMAO exert independent effects on protein stability, allowing for accurate thermodynamic parameter determination.
- This approach enhances the ability to study the thermodynamics of proteins with complex folding behaviors.