Global analysis of thermal and chemical denaturation using CheMelt: Thermodynamic dissection of highly thermostable de novo designed proteins

ABSTRACT De novo protein design often produces thermostable proteins that denature above 100 °C, which complicates the analysis of their stability. Thermostable proteins can be unfolded by combined chemical and thermal denaturation followed by global analysis of multiple melting curves. Here, we have developed CheMelt, a new online tool for global analysis of unfolding data via an intuitive graphical user interface. We use nanoscale differential scanning fluorimetry followed by CheMelt data analysis to dissect the combined thermal and chemical denaturation of thirty-five de novo designed protein binders. Fifteen present sufficient fluorescence changes to extract thermodynamic parameters of unfolding. These de novo designed proteins have systematically lower ΔCp and m-values than comparable natural proteins, which implies that they expose fewer hydrophobic residues upon unfolding. We show that a high thermostability of a designed protein does not necessarily imply a high equilibrium stability; and demonstrate the potential of CheMelt in dissecting thermodynamic properties for protein design and engineering. - protein stability - de novo protein design - protein unfolding - CheMelt - eSPC Competing Interest Statement The authors have declared no competing interest. Footnotes Some equations and greek letters were not rendered correctly in the first version.