Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

We show that using sample volumes 10-50-fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodynamic and kinetic stability, as well as folding/unfolding kinetics

Gopinath Chattopadhyay; Raghavan Varadarajan

2019

Scholarcy highlights

  • Isothermal, unfolding studies for two proteins, homodimeric Escherichia coli CcdB and monomeric hen egg white lysozyme using both a conventional fluorimeter and nanoDSF
  • Changes in the structure of the protein have an effect on both the intensity and the emission wavelength, especially of tryptophan fluorescence
  • Kinetic stability on the other hand is related to the activation free energy barrier separating the folded and nonnative states
  • Isothermal denaturation of CcdB in 10 or 200 mM HEPES, pH 8.4 was carried out at a fixed protein concentration of 47 μg/mL and HEWL in PBS, pH 7.5 was used at 15 μg/mL
  • Refolding kinetic traces of fluorescence intensity in 1.5 M GdnCl as a function of time for CcdB in 10 mM or 200 mM HEPES, pH 8.4 were normalized from 0 to 1 between native and denatured baseline at 1.5 M GdnCl. Unfolding kinetic traces of fluorescence intensity in 3 M GdnCl as a function of time for CcdB in 10 mM or 200 mM HEPES, pH 8.4 were normalized from 0 to 1 between native and denatured baseline at 3 M GdnCl. The data was analyzed using SigmaPlotTM version 12.5 for WindowsTM scientific graphing software, from Systat Software, Inc., San Jose, CA, USA,, and plots were fitted to a 5 parameter equation for exponential decay for refolding + c*exp(−dx)), yielding slow and fast phase rate constants and a 3 parameter exponential rise for unfolding as described previously, where x is the time of refolding/unfolding. where yf and yu are the folded and unfolded parameters at zero denaturant concentration

Need more features? Save interactive summary cards to your Scholarcy Library.