The lipoxygenase enzyme 15-LOX-2 oxidizes arachidonoyl substrates in a membrane, and 15-LOX-2 activity has been linked to elevated low-density lipoprotein levels and development of atherosclerotic plaques. Liquid surface X-ray scattering techniques such as X-ray reflectivity (XR) and grazing incidence X-ray diffraction (GIXD) are powerful tools for elucidating structure and interactions of peripheral membrane proteins. XR results are analyzed to determine the electron density profile of a phospholipid monolayer and associated proteins at an air/water interface. In GIXD, diffraction is measured from semi-crystalline species present at the air/water interface, such as condensed phospholipid tails. Preliminary evidence suggests that 15-LOX-2 may undergo a conformational change when binding the membrane in order to facilitate passage of the lipid substrate into the active site. The goal of this work is to determine the orientation of 15-LOX-2 binding the membrane, and also to investigate the impacts of 15-LOX-2 enzyme activity on membrane structure. Experiments with lipid monolayers show that 15-LOX-2 has limited binding with membranes made of pure DSPC and pure SAPC, but exhibited stable interactions with membranes composed of 80:20 DSPC:SAPC. Lipid oxidation was also detectable by XR, and 15-LOX-2 activity caused a rearrangement in membrane structure. Furthermore, 15-LOX-2 binding resulted in a decrease in area per lipid, effectively compressing the lipids that were packed in a condensed phase, while simultaneously reducing the bulk amount of condensed-phase lipids. Overall, this work is beginning to deconvolute the structural transitions that occur from 15-LOX-2 binding membranes and oxidizing arachidonoyl substrates.

Liquid surface x-ray scattering analysis of lipoxygenase (15-LOX-2) binding to DSPC/SAPC lipid monolayers

ACS Spring Meeting 2021

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