Stopped-flow fluorescence

Aliquots of guanidinium hydrochloride (GdmHCl) were prepared by dispensing the appropriate volume of stock solution of GdmHCl in sodium phosphate buffer (50 mM sodium phosphate buffer (pH 6.8), 150 mM NaCl) using a Hamilton Microlab ML510B dispenser (Hamilton). For each protein, two aliquots (3 mL) were prepared to a final concentration of 10 μM of protein. One aliquot was fully folded in sodium phosphate buffer (or low concentrations of GdmHCl), and the other denatured in 6 M GdmHCl. Samples were equilibrated at 10 or 25°C for 2 h. The proteins and the GdmHCl solutions were mixed at a 1:5 ratio. An excitation wavelength of 280 nm was used, and the emission was measured using a 330-nm cutoff filter. Unfolded protein was refolded by rapid mixing with increasing concentrations of GdmHCl up to the denaturation midpoint as defined by equilibrium denaturation. Folded protein was unfolded by rapid mixing with increasing concentrations of GdmHCl above the equilibrium denaturation midpoint. Multiple traces were acquired at each GdmHCl concentration, averaged and then fitted to a single exponential or a double exponential in GraphPad Prism.

Chevron plots that showed nonlinear folding and/or unfolding arms were fitted using a broad transition state barrier model originally described by Oliveberg and coworkers (40). Nevertheless, the fit was simply qualitative, as the refolding rates of these CTPR proteins are faster than the limit of detection of our instrument: