Beyond thermal unfolding: urea-gradient nanoDSF approach for thermostability analysis of kinetically stable hyperthermophilic proteins
Rusinek, W.; Dorawa, S.
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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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/717478v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1b8c5e3org.highwire.dtl.DTLVardef@1c7d395org.highwire.dtl.DTLVardef@14093forg.highwire.dtl.DTLVardef@16b2f25_HPS_FORMAT_FIGEXP M_FIG C_FIG
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