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Peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy are used to study the equilibrium and kinetics of canine milk lysozyme folding/unfolding. The results have shown that the folding initiation site is different between the two proteins, thereby providing an example of the phenomenon.

Rapid mixing methods remain the method of choice for studies of protein folding reactions far from equilibrium transition region where intermediate states are most likely to accumulate. Rapid mixing experiments involve large changes in denaturant concentration, which is the case of protein folding or unfolding, pose additional challenges related to the increase in solvent viscosity, which slows down the mixing process, convective flow due to density gradients across the mixer and the heat of mixing which can give rise to thermal artifacts.

A study based on stopped-flow measurements proposed that the prion protein folds via a transient intermediate formed on the submillisecond time scale, and mutations linked to familial diseases result in a pronounced increase in the population of this intermediate. Findings demonstrate that the populations of the intermediate is significantly increased at mildly acidic pH and in the presence of urea the partially structured intermediate might be a direct monomeric precursor of the misfolded Pr[P.sup.Sc] oligomer.

An investigation of the kinetic refolding of recombinant canine milk lysozyme is presented by means of circular dichroism (CD) at 10 different wavelengths with a stopped-flow technique, and obtained the CD spectra of the burst-phase and second kinetic intermediates observed during the refolding process. A schematic model of the refolding of canine milk lysozyme that is consistent with the hierarchical mechanism of protein folding is proposed.

The monitoring the refolding of Cycle3 from the acid-denatured state by chromophore fluorescence, tryptophan fluorescence, and far-UV CD are investigated using stopped-flow apparatus. Thus, it is reported that the refolding kinetics are found to involve five kinetics phases.

Equilibrium unfolding and kinetic refolding of staphylococcal nuclease were examined in relation to the response of staphylococcal nuclease to the effects of proline isomerizations. Fluorescence spectroscopy of a tryptophan residue and circular dichroism in the peptide region were also conducted to explore the folding and unfolding mechanisms. Four single mutants and four triple mutants were constructed to permit a better understanding of the mechanics of protein folding. Results show that the effects of individual prolines were remarkably different.

Abstract: Despite close structural similarity, the ferric and ferrous forms of cytochrome c differ greatly in terms of their ligand binding properties, stability, folding, and dynamics. The reduced heme iron binds diatomic ligands such as CO only under destabilizing conditions that promote weakening or disruption of native methionine–iron linkage. This makes CO a useful conformational probe for detecting partially structured states that cannot be observed in the absence of endogenous ligands. Heme absorbance, circular dichroism, and NMR were used to characterize the denaturant-induced unfolding equilibrium of ferrocytochrome c in the presence and in the absence of CO. In addition to the native state (N), which does not bind CO, and the unfolded CO complex (U-CO), a structurally distinct CO-bound form (M-CO) accumulates to high levels (∼75% of the population) at intermediate guanidine HCl concentrations. Comparison of the unfolding transitions for different conformational probes reveals that M-CO is a compact state containing a native-like helical core and regions of local disorder in the segment containing the native Met80 ligand and adjacent loops. Kinetic measurements of CO binding and dissociation under native, partially denaturing, and fully unfolded conditions indicate that a state M that is structurally analogous to M-CO is populated even in the absence of CO. The binding energy of the CO ligand lowers the free energy of this high-energy state to such an extent that it accumulates even under mildly denaturing equilibrium conditions. The thermodynamic and kinetic parameters obtained in this study provide a fully self-consistent description of the linked unfolding/CO binding equilibria of reduced cytochrome c. [Copyright &y& Elsevier]