Unfolding transition, effect

Fig. 8. Dependence of (A) corrected diffusion coefficient (D), (B) steady-state fluorescence intensity, and (C) corrected number of particles in the observation volume (N) of Alexa488-coupled IFABP with urea concentration. The diffusion coefficient and number of particles data shown here are corrected for the effect of viscosity and refractive indices of the urea solutions as described in text. For steady-state fluorescence data the protein was excited at 488 nm using a PTI Alphascan fluorometer (Photon Technology International, South Brunswick, New Jersey). Emission spectra at different urea concentrations were recorded between 500 and 600 nm. A baseline control containing only buffer was subtracted from each spectrum. The area of the corrected spectrum was then plotted against denaturant concentrations to obtain the unfolding transition of the protein. Urea data monitored by steady-state fluorescence were fitted to a simple two-state model. Other experimental conditions are the same as in Figure 6.

The structures of the intermediate and transition state can be perturbed by radical mutations that cause significant decreases in stability.61 Interestingly, there is a mixture of the Hammond effect, as found for CI2 in Figure 19.7, and the very rare anti-Hammond effect (discussed in detail in referance 61). Molecular dynamics simulations are consistent with the experimental data and show that radical mutations in the major helix cause it to be more unfolded in the transition state for unfolding (the anti-Hammond effect) but the j8 sheet is less unfolded (Hammond Effect).62... 

Where Kunf and Kfiuct are the equilibrium constants for the unfolding transitions and fluctuations respectively. For simplicity, both the unfolded and the fluctuated conformations are assumed to exchange with the rate determined from unstructured dipeptides (Bai et al., 1993 Connelly et al., 1993). This may be a poor assumption, especially for the fluctuated form, but it allows the rates to be normalized for temperature, pH and amino acid sequence effects. Kpp, the equilibrium constant determined from kobs / is then given by... 

Renaturation. The effects of i) reduction of the disulfide bond of p-lactamase, ii) renaturation buffer pH, iii) concentration of protein, iv) GuHCl in the denaturing solution and finally v) sucrose concentration on the reversibility of the unfolding transition were investigated. 

To facilitate conformational transitions in the before-mentioned adenylate kinase, Elamrani and co-workers scaled all atomic masses by a large factor thus allowing the use of a high effective simulation temperature of 2000K ([Elamrani et al. 1996]). To prevent protein unfolding, elements of secondary structure had to be constrained. 

The novel element in these models is the introduction of a third phase in the Hashin-Rosen model, which lies between the two main phases (inclusions and matrix) and contributes to the progressive unfolding of the properties of the inclusions to those of the matrix, without discontinuities. Then, these models incoporate all transition properties of a thin boundary-layer of the matrix near the inclusions. Thus, this pseudo-phase characterizes the effectiveness of the bonding between phases and defines a adhesion factor of the composite. 

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. 

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