Partially folded states of equine lysozyme. Structural characterization and significance for protein folding.

This research investigates the folding behavior of a protein called equine lysozyme, primarily focusing on how the protein’s structure changes when heated or exposed to specific chemical conditions, and how the binding of calcium affects these processes.

Understanding the Folding Behavior of Equine Lysozyme

• The study focuses on the protein known as equine lysozyme, which is a calcium-binding protein found in equine milk. It particularly investigates how the structure of this protein unfolds or changes when subjected to various conditions, inclusive of heat, pH changes and in presence of guanidine hydrochloride, a potent denaturant.
• The unfolding behavior of this protein has significant implications for understanding protein folding in general, as it provides an evolutionary link between two protein families – c-type lysozymes and alpha-lactalbumins – that exhibit notably different unfolding behaviors.

Characterizing the Unfolding Process

• The research used techniques such as Differential Scanning Calorimetry, fluorescence emission, UV difference absorption spectroscopy, and 1H NMR to carefully track and characterize the unfolding process of equine lysozyme under varying conditions.
• Two distinct stages of unfolding were observed. In the absence of calcium, the initial stage occurs at 44 degrees Celsius at pH 4.5 or in 0.8 M guanidine hydrochloride (GdnHCl) at a pH 7.5, 25 degrees Celsius, and the second stage occurs near 70 degrees Celsius at pH 4.5 or in 3.7 M GdnHCl at a pH of 7.5, 25 degrees Celsius.

Role of Calcium Binding

• The presence of calcium was found to influence the unfolding behavior of equine lysozyme, shifting the parameters for the first transition point significantly, but not affecting the second transition point.
• In the presence of calcium, the first transition takes place with a midpoint of 55 degrees Celsius or in excess of 2.5 M GdnHCl, indicating the potential for calcium to stabilize the protein in some sense and resist unfolding to an extent.

Details of the Unfolding States

• The study found that the first transition generates an intermediate state with some aromatic side chains being hidden from the solvent, indicating an inner movement and reshuffling of the protein’s structure.
• The intermediate state was found to possess extensive secondary and tertiary structure, despite having significant disorder, suggesting that this state may be quite flexible or adaptable.
• The second transition was interpreted as denaturation, a process where the protein loses its structure entirely, leading to a highly unfolded state.