Thermal protein unfolding by differential scanning calorimetry and circular dichroism spectroscopy Two-state model versus sequential unfolding

Joachim Seelig, Hans-Joachim Schönfeld
2016 Quarterly Reviews of Biophysics (print)  
AbstractThermally-induced protein unfolding is commonly described with the two-state model. This model assumes only two types of protein molecules in solution, the native (N) and the denatured, unfolded (U) protein. In reality, protein unfolding is a multistep process, even if intermediate states are only sparsely populated. As an alternative approach we explore the Zimm–Bragg theory, originally developed for theα-helix-to-random coil transition of synthetic polypeptides. The theory includes
more » ... ermediate structures with concentrations determined by the cooperativity of the unfolding reaction. We illustrate the differences between the two-state model and the Zimm–Bragg theory with measurements of apolipoprotein A-1 and lysozyme by differential scanning calorimetry (DSC) and CD spectroscopy. Nine further protein examples are taken from the literature. The Zimm–Bragg theory provides a perfect fit of the calorimetric unfolding transitions for all proteins investigated. In contrast, the transition curves and enthalpies predicted by the two-state model differ considerably from the experimental results. Apolipoprotein A-1 is ~50%α-helical at ambient temperature and its unfolding follows the classicalα-helix-to-random coil equilibrium. The unfolding of proteins with littleα-helix content, such as lysozyme, can also be analyzed with the Zimm–Bragg theory by introducing the concept of 'folded' and 'unfolded' peptide units assuming an average unfolding enthalpy per peptide unit. DSC is the method of choice to measure the unfolding enthalpy,$\Delta H_{\rm exp} ^0 $, but CD spectroscopy in combination with the two-state model is often used to deduce the unfolding enthalpy. This can lead to erroneous result. Not only are different enthalpies required to describe the CD and DSC transition curves but these values deviate distinctly from the experimental result. In contrast, the Zimm–Bragg theory predicts the DSC and CD unfolding transitions with the same set of parameters.
doi:10.1017/s0033583516000044 pmid:27658613 fatcat:5vjlnbdzl5h4xfdkkjrorzdage