Figure 3.
Single-jump ensemble kinetics. A, ensemble refolding (white) and unfolding (black) rates of MSG shown as kobs. Both phases of refolding, i.e. fast (circles) and slow (triangles), exhibit a rollover where denaturant-assisted refolding is observed under strongly native conditions, a classic sign of misfolded intermediate formation. The refolding rates at 0 m urea (white square and diamond) were obtained by pH jump (pH 11 to pH 7.9) refolding of MSG in the native buffer. Two unfolding arms of the chevron (black circles and triangles) intersect near 5.5 m urea. The inset represents origin of new fastest phase rates (diamonds) of unfolding at >7.5 m urea. B, comparison of refolding rates from Trp fluorescence (white), ANS fluorescence (red), and ellipticity (black cross) is shown. Inset represents a good correlation of averaged refolding rates from Trp fluorescence probe with the CD probe. C and D, fractional amplitudes of refolding and unfolding phases, respectively. The signal lost within the dead time of mixing (red) in both the cases is found to be independent of urea concentration. E, fractional amplitudes of two refolding phases as probed by ANS fluorescence. Error bars wherever shown represent standard deviation from three individual experiments.