Protein micelles from lipoxygenase 3

Area of Science:

• Biochemistry
• Protein Chemistry
• Structural Biology

Background:

• Lipoxygenase 3 (LOX3) is an enzyme involved in various biological processes.
• Understanding protein conformational changes under thermal stress is crucial for enzyme stability and function.

Purpose of the Study:

• To characterize heat-induced conformational changes in lipoxygenase 3.
• To investigate the structural basis of aggregate formation in lipoxygenase 3.
• To elucidate the mechanism of soluble aggregate formation in lipoxygenase 3.

Main Methods:

• Differential scanning calorimetry (DSC) to detect thermal transitions.
• Variable-temperature circular dichroism (VT-CD) to monitor secondary structure changes.
• Size exclusion chromatography (SEC), native polyacrylamide gel electrophoresis (native-PAGE), dynamic light scattering (DLS), and electron microscopy (EM) to analyze aggregate structure.

Main Results:

• Thermal transitions of lipoxygenase 3 were buffer-composition dependent.
• Heating lipoxygenase 3 in carbonate buffer (pH 8.0) induced formation of large soluble aggregates.
• Aggregate formation was not associated with unfolding of the alpha-helical C-terminal domain.
• Aggregates were spherical particles (average hydrodynamic radius of 26 nm, molecular weight ~10,000,000 Da).

Conclusions:

• Heat-induced aggregation of lipoxygenase 3 involves unfolding of the N-terminal beta-barrel domain.
• Exposed hydrophobic residues drive micelle formation, with hydrophilic C-terminal domains forming the surface.
• These findings provide insight into the structural dynamics and aggregation propensity of lipoxygenase 3.