Rusinek, W.; Dorawa, S.

In this study, we demonstrate that urea enables reliable melting temperature (Tm) 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 Tm values falling within the instruments 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 Tm values of 104.8 {+/-} 0.09 {degrees}C for Pfu and 106.8 {+/-} 0.33 {degrees}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.