Equilibrium and kinetics of the folding of equine lysozyme studied by circular dichroism spectroscopy.

The research explores the folding dynamics of equine lysozyme, a calcium-binding protein, using circular dichroism spectroscopy. The findings suggest that folding/unfolding follows a three-stage mechanism, involving an intermediate, molten globule state that is more stable and closer to the native state than previously studied proteins.

Study Approach

• The research investigated the unfolding and folding of equine lysozyme using circular dichroism spectra in the far and near-ultraviolet regions. This spectroscopy technique measures differences in the absorption of left-handed and right-handed polarized light, which helps to study secondary and tertiary structural changes in proteins.
• The study also noted the transitions in the unfolding process of the protein, catalysed by guanidine hydrochloride, a known protein denaturant. Using guanidine hydrochloride stimulates the unfolding of the proteins, which enables the researchers to analyze the transition stages.

Findings

• The study demonstrated that equine lysozyme unfolding happens through a three-state mechanism. These states include the native protein state, unfolded state, and an intermediate state known as the molten globule state. The molten globule state, which has a more significant extent of the protein’s structure intact than the fully unfolded state, acts as a stable intermediate during unfolding and refolding.
• Compared to alpha-lactalbumin, another protein studied, the molten globule state of equine lysozyme was found to be more stable and more closely similar to its native form. This indicates that the folding process of proteins might be more complex than previously assumed.
• Through stopped-flow circular dichroism, the kinetic unfolding and refolding of the protein was measured. In the presence of low concentrations of guanidine hydrochloride, the kinetic refolding displayed a burst-phase change.
• Under strong native conditions, almost all equine lysozyme molecules rapidly transition from the unfolded state into the molten globule, suggesting a highly efficient folding process, which then proceeds to refold from the molten state.

Conclusions

• The findings of this study propose a sequential model of protein folding, arguing that the molten globule state acts as a necessary intermediate step in the folding process of proteins.
• Transition state characterization by guanidine hydrochloride concentration-dependence of the rate constants of refolding and unfolding indicated that this state is more native-like, with more than 80% of the hydrophobic surface of the protein buried in the interior of the molecule.
• These results provide novel insights into the process of protein folding/unfolding, offering a more nuanced understanding of these critical biological processes.