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Protein folding assay

MB Maria Babu
FF Filippo Favretto
AO Alain Ibáñez de Opakua
MR Marija Rankovic
SB Stefan Becker
MZ Markus Zweckstetter
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RNaseT1 (purchased from Thermo Fischer) was unfolded by incubating in 6.9 M urea at 10 °C for 2 h in 100 mM Tris-HCl buffer at pH 8. Refolding of RNaseT1 was initiated by diluting it 35 times with same buffer such that the final concentration of RNaseT1 was 2.27 µM and urea 0.197 M. Tryptophan fluorescence emission was measured at 320 nm (excitation wavelength = 280 nm) during refolding for 1 h on a Cary Eclipse Fluorescence Spectrophotometer at 10 °C. In experiments with PR20, PPIA, or both, they were added to the dilution buffer and incubated at 10 °C for 2 h prior to mixing. Data were normalized and averaged for graphical representation. The exponential constants, k, for the increasing fluorescence intensities were obtained from fitting a mono-exponential function to the experimental data in Graph Pad Prism.

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