Beyond thermal unfolding: urea-gradient nanoDSF approach for thermostability analysis of kinetically stable hyperthermophilic proteins

Abstract In this study, we demonstrate that urea enables reliable melting temperature (T m ) determination of hyperthermostable proteins by nano differential scanning fluorimetry (nanoDSF) Under native conditions, Pfu DNA polymerase and its Sso7d-fusion variant showed no detectable unfolding transitions, despite their T m values falling within the instrument’s operational range, reflecting their extreme kinetic stability. In the presence of up to 7 M urea, intrinsic tyrosine and tryptophan fluorescence revealed clear unfolding transitions, yielding extrapolated T m values of 104.8 ± 0.09 °C for Pfu and 106.8 ± 0.33 °C for its Sso7d-fusion variant. These results demonstrate that urea-gradient nanoDSF overcomes both instrumental and kinetic limitations, providing a simple and robust method for assessing the thermal stability of (hyper)thermostable proteins. Graphical abstract