Denaturation midpoint of a protein is defined as the
temperature
Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measured with a thermometer.
Thermometers are calibrated in various temperature scales that historically have relied o ...
(T
m) or
concentration
In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', ''molar concentration'', ''number concentration'', an ...
of
denaturant (C
m) at which both the
folded and
unfolded states are equally populated at equilibrium (assuming two-state
protein folding
Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduci ...
). T
m is often determined using a
thermal shift assay
A thermal shift assay (TSA) measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mut ...
.
If the widths of the folded and unfolded wells are assumed to be equal both these states will have identical free energies at the midpoint. However, for natural
proteins
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
this is not the case. There is an inherent asymmetry as evidenced by the difference in
heat capacities between them - the folded ensemble has a lower heat capacity (in other words, lower
fluctuations thus indicating a narrower well) than the unfolded ensemble. This would mean that the free energy of the folded state is lower at the denaturation midpoint than the unfolded state. In such a scenario, the temperature at which both the wells have identical free energies is termed the characteristic temperature (T
o).
[{{cite journal, last1=Munoz, first1=V., last2=Sanchez-Ruiz, first2=J.M., date=2004, title=Exploring protein-folding ensembles: A variable-barrier model for the analysis of equilibrium unfolding experiments, journal= Proceedings of the National Academy of Sciences, volume=101, issue=51, pages=17646–17651, pmid=15591110, doi=10.1073/pnas.0405829101, pmc=539728, bibcode=2004PNAS..10117646M, doi-access=free]
References
Protein structure