Measuring the stability of partly folded proteins using TMAO
Open accessAbstract
Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N‐oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte‐induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline‐resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two‐state equilibrium unfolding, we found that Δ G° for unfolding depends linearly on TMAO concentration, and that the sensitivity of Δ G° to urea (the m‐value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline‐resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of Δ G° on each cosolvent are similar to those obtained by standard urea‐induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full‐length Notch ankyrin domain.
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TMAO is reportedly capable of directly inducing conformational changes in proteins and of stabilizing protein folding and acting as a small-molecule protein chaperone.
“TMAO, a small quaternary amine with an aliphatic character, is reportedly capable of directly inducing conformational changes in proteins and of stabilizing protein folding and acting as a small-molecule protein chaperone (48, 49).”
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“TMAO, a small quaternary amine with an aliphatic character, is reportedly capable of directly inducing conformational changes in proteins and of stabilizing protein folding and acting as a small-molecule protein chaperone (48, 49).”
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- Authors
- Mello, C. C., & Barrick, D.
- Affiliations
- Johns Hopkins University (US)
- Corresponding author
- Barrick, Doug
- ORCID
- Barrick, Doug
- Venue
- Protein Science
- Publisher
- Wiley
- Published
- 2003-07
- Volume
- 12
- Issue
- 7
- Pages
- 1522-1529
- ISSN
- 0961-8368 (ISSN-L)
- 1469-896X
- PMC
- PMC2323936
- OpenAlex
- W2047168931
- Cited by
- 151
- References
- 37
- Citations by year
- 2026: 3 · 2025: 4 · 2024: 3 · 2023: 3 · 2022: 5 · 2021: 1 · 2020: 6 · 2019: 3 · 2018: 5 · 2017: 9 · 2016: 9 · 2015: 6 · 2014: 9 · 2013: 7 · 2012: 13
- FWCI
- 1.84
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Protein Science (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- National Institute of General Medical Sciences (R01GM060001)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Physical Sciences › Chemistry › Physical and Theoretical Chemistry › thermodynamics and calorimetric analyses
- Keywords
- trimethylamine N-oxide, urea denaturation, two-state unfolding, m-value, barnase
- MeSH terms
- Methylamines, Osmolar Concentration, Protein Denaturation, Proteins, Solvents, Thermodynamics, Urea, Oxidants, Protein Folding
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