Unfolding rates

F NMR was used to monitor the folding kinetics of TmCsp labelled with 5-fluorotryptophan. It was found that the increased thermostability of TmCsp (rui = 87°C) compared to CspB (ri =52°C) was due to lower unfolding rate constants over a wide temperature range. This suggested that entropic factors play an important role in the thermostabilization of TmCsp. 

Fig. 7 Left Arrhenius plot of the approximate folding and unfolding rates of 15. Right At low temperatures, the relaxation is better fitted by a stretched exponential or biexponential the stretching exponent is shown here as a convenient measure of the deviation from single exponentiality...

As the pH decreases, the unfolding rates are measured at progressively lower values of temperature. At each value of pH, the deviation, from linearity is imperceptible. But the value of Adecreases with the decreasing average temperature of each set. 

Fraction folded Fraction folded Fraction folded Fold and unfold rates Fold and unfold rates... 

H. Sugiura, Y. Nigorikawa, Y. Saiki, K. Nakamura, M. Yamaguchi, Marked Effect of Aromatic Solvent on Unfolding Rate of Helical Ethynylhelicene Oligomer, J. Am. Chem. Soc. 2004, 126, 14858-14864. 

The molecular mechanisms by which the extension of the N-terminus by the extra methionine residue destabilized recombinant a-lactalbumin remain unclear. Additional conformational entropy of the extra methionine residue in the unfolded state could account for the destabilization and unfolding-rate acceleration of the recombinant protein [22]. Ishikawa and coworkers reported the destabilization of recombinant bovine a-lactalbumin, similarly induced by the extra N-terminal methionine residue, and showed that the enthalpy change of thermal unfolding was the same for the authentic and recombinant proteins, indicating that the destabilization was caused by an entropic effect [42]. However, the destabilization by the extra methionine residue in the lysozyme homologous to a-lactalbumin was rather enthalpic and accompanied by a disruption of hydrogen-bond networks in the N-terminal region [43,44]. 

The unfolding behaviors of the authentic and recombinant forms of goat a-lactalbumin are remarkably different, although both forms have an identical three-dimensional structure. The recombinant form was found to be l.lkcalmol-1 less stable than the authentic form, and the recombinant form unfolded at a ninefold faster rate than the authentic form. The destabilization and unfolding-rate acceleration were due to the presence of an extra methionine residue at the N-terminus in the recombinant protein. 

The model simply proposes that hydrogen exchange may occur (with different rates) in both the folded and unfolded forms of the protein, and that the folding/unfolding rates are not affected by replacement of amide hydrogens by deuteriums. (Note that experiments in D2O, since there are no... 

Similar results (not shown) were obtained for numerous other C22A-FKBP segments. Observation of a common unfolding rate for each of many segments of the protein backbone further establishes a two-state (folded/unfolded) equilibrium for C22A-FKBP in 3.5 and 4.5 M urea. The unfolding rate constant, kunfold = 1 -8 +... 

Fig. 17.5c, solid line) to determine the values of AG and Ax. These force-clamp experiments show that the distance to the transition state of unfolding of the 127 protein, Ax = 2.4 A, which is the size of a water molecule [24]. In addition, the value of the unfolding rate extrapolated to zero force measures the size of the activation energy barrier AG. However, AG obtained from the Arrhenius fits depends on the value used for the attempt frequency A (Fig. 17.4), which needs to be measured independently for each reaction [24]. By contrast, the value of Ax is measured from the slope of the Arrhenius plot, and thus is independent of the value of the attempt frequency A. 

Fig. 17.12. Unfolding data obtained from ubiquitin, Ubig, polyproteins at a constant pulling force of 110 pN. (a) The length versus time traces show 20 nm stepwise increases in length each time a single protein domain unfolds. The time of occurrence of the unfolding events (At) is probabilistic, (b) Histogram of unfolding dwell times is measured at 110 pN. The dashed line is a single exponential fit with an unfolding rate, fc, of 0.6 s [10,18]. The solid line is a stretched exponential fit with p(t) = exp[—(fct) ] with b = 0.7...

Slow motions in phase space are characterized by the local Lyapunov exponent XN, which defines the unfolding rate of nearby orbits,... 

In the presence of SDS, the unfolding rate accelerates and can be measured at lower temperature. SDS inhibits the refolding of the 7 species back to the native. There is no detectable aggregation in SDS and the transformation from N to 7 and then to M is quantitative. The unfolding mechanism of tailspike protein... 

Here, and k2 are the unfolding rate constants for the unfolding transition from N to I and from I to respectively. The unfolding kinetics can be quantitatively analyzed from the scanned intensities of the Coomassie blue stained tailspike bands on SDS gels. Figure 1 depicts results from a typical thermal unfolding experiment for wild type tailspike protein, which was performed in Tris buffer 8) and 2% SDS at 65 C. Kinetic analysis yields two rate constants 1.1 x 10 s and 4.0 x 10 s for the conversion from N to I and from I to A/, respectively. 

Figure 2. Dependence of unfolding rate constants on pH. Wild type tailspike protein was prepared in 50 mM Tris, 1.7 mM 2-mercaptoethanol and 2% SDS and adjust to different pH values by 1 N HCl. Thermal unfolding was done at 65°C and followed by SDS-PAGE at about 20°C. Sample pH values shown here have been corrected to 65°C. kj (a) and k2 ( ) shown in log are the thermal unfolding rate constants for the conversions from N to I and from I to M, respectively. The linear lines through the data points are the results of least-square fit to each individual pH phase for both kj and k2 data. The calculated slopes of the fitting lines for kj are -0.46 and 0.35 for the low and high pH phases, respectively and for k2 are -1.9 and 1.1 for the low and high pH phases, respectively.

During unfolding, E4M displayed the higher unfolding rate when it is present at 10 %. This would suggest the ease at which E4M is able to penetrate and unfold at the interface. 

Concentration Diffusion Rate Unfolding Rate, Rearrangement Rate,... 

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