Figure 1.
Tertiary structure of human ubiquitin according to the 1.8 Å crystal structure (1UBQ.pdb).
All three proline residues (shown in red) display the Cγ-exo conformation and were simultaneously substituted with (2S,4R)-4-fluoroproline ((4R)-FPro-ub).
Figure 2.
ESI-MS analyses of wt-ub (A), (4R)-FPro-ub (B). The minor peak corresponds to the double substituted protein. (C) wt-ub and (4R)-FPro-ub after purification on 15% SDS-PAGE.
Figure 3.
Profiles of melting temperature against pH and unfolding enthalpy against melting temperature.
(A) Dependence of denaturation temperature Tm on pH for wt-ub (black circles) and (4R)-FPro-ub (red circles). (B) Unfolding enthalpy versus denaturation temperature for wt-ub (black circles) and (4R)-FPro-ub (red circles).
Table 1.
Thermal unfolding parameters of wt-ub and (4R)-FPro-ub.
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.
Table 2.
Kinetic data for the refolding/unfolding of wt-ub and (4R)-FPro-ub monitored by changes in Tyr fluorescence using GdmCl denaturant.
Figure 5.
Autoubiquitination assay in the presence of E6 associating protein.
After one hour of incubation, the band corresponding to the E6 associating protein (100 kDa) disappeared and was substituted by a band of higher molecular weight corresponding to the polyubiquitinated protein. M: protein marker, 1: wt-ub at t = 0 h; 2: wt-ub at t = 1 h; 3: wt-ub at t = 2 h; 4: (4R)-FPro-ub at t = 0 h; 5: (4R)-FPro-ub at t = 1 h; 6: (4R)-FPro-ub at t = 2 h.