Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin
Figure 4
Unfolding profiles of wt-ub and (4R)-FPro-ub.
(A) GdmCl-dependent equilibrium unfolding profiles of wt-ub and (4R)-FPro-ub at 25°C and pH 2.0, monitored by changes in Tyr fluorescence at 310 nm upon excitation at 278 nm. (B) Chevron plot analyses of wt-ub and (4R)-FPro-ub. Refolding and unfolding reactions were monitored by changes in the fluorescence above 300 nm of the single Tyr 59 at pH 2.0 and 25°C. Equilibrium and kinetic data were globally fitted according to a linear three-state model with a high energy intermediate. Black line (wt-ub) and red line ((4R)-FPro-ub) represent best fit to the model.