Unfolding/refolding transition

Mitraki, A., J.M. Betton, M. Desmadril, and J.M. Yon. 1987. Quasi-irreversibility in the unfolding-refolding transition of phosphoglycerate kinase induced by guanidine hydrochloride. Eur J Biochem 163 29-34. 

The study of temperature effects on the reactivation of an enzyme that has been completely unfolded allows one to distinguish between reactivation (referring to kinetic analysis exclusively) and renaturation, the latter of which would reflect both the refolding transition and the formation of misfolded or aggregated byproducts. 

Urea-induced unfolding of bovine serum albumin and one of its fragments containing domain II+III has been studied by difference spectral and fluorescence emission measurements. The unfolding-refolding curves of both the proteins showed the presence of at least one stable intermediate when the transition was monitored at 288 nm. The presence... 

Under conditions of the equilibrium for unfolding-refolding and dissociation-association processes, side reactions become preponderant. For this reason, conditions for kinetic studies are generally chosen far from this equilibrium so that the reactions are practically irreversible (i.e., the rate constants for reverse reactions are negligible). In the critical range of the monomer-oligomer transition, a kind of hysteresis, i.e., a noncoincidence of, on the one hand, deactivation-denaturation, on the other hand. 

Prolyl isomerases are enzymes. In protein folding they catalyze cis trans isomerizations in both directions (Miicke and Schmid, 1992) and show equal efficiencies in unfolding and refolding experiments under identical conditions near the midpoint of the unfolding transition. They carry no information about the isomeric states of the prolyl peptide bonds in the protein substrates. The native isomer is selected by the refolding protein itself simply because the molecules... 

Fig. 4. Urea-induced equilibrium unfolding transitions of murine PrP (121-231) at 22°C. (A) Dependence of reversible, urea-induced unfolding on pH. Open symbols represent refolding experiments, and closed symbols represent unfolding experiments. (B) Dependence on ionic strength of the formation of the acid-induced unfolding intermediate of PrP(121-231) at pH 4.0 and a protein concentration of 29 pM. Unfolding experiments were performed at 22°C in 50 mM formic acid/NaOH, pH 4.0 (ionic strength 32 mM), containing 0 M (O), 50 mM ( ), 100 mM (A), or 150 mM ( ) sodium chloride.

Fig. 5. Kinetics of folding of murine PrP( 121-231) (variant F175W) at pH 7.0 and 4°C, measured by stopped-flow fluorescence. PrP(l21-231 )-F175W was unfolded with 8 M urea and diluted with refolding buffer to a final concentration of 3.7 M urea. The extrapolated value at t = 0 s corresponds to the expected value from the urea-induced equilibrium transition (excitation 280 nm emission > 320 nm).

Fig. 7. Conservation of the unfolding/folding mechanism of cold-shock proteins (Csp) from B. subtilis (Bs), B. caldolyticus (fid), and Thermotoga maritima (Tm). (a) Equilibrium unfolding transitions of Csp from Bs (A), Be ( ), and Tm ( ) induced by GdmCI at 25° and monitored by intrinsic fluorescence. Least-squares fit analyses based on the two-state model yield stabilization energies AGstab = 11.3,20.1, and 26.2 kJ/mol for Csp from Bs, Be, and Tm, respectively, (b) Kinetics of unfolding (open symbols) and refolding (closed symbols) of Bs (A, A), Be ( , ) and T Csp (O, ), respectively. The apparent rate constants, X, are plotted against the GdmCI concentration. The fits are on the basis of the linear two-state model. ...

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