Endothelial Nitric Oxide Synthase Oxygenase on Lipid Nanodiscs: A Nano-assembly Reflecting Native-like Function of eNOS

Ghaith AlTawallbeh, Cleveland State University
Mohammad M. Haque, Cleveland Clinic
Kiril A. Streletzky, Cleveland State University
Dennis J. Stuehr, Cleveland Clinic
Mekki Bayachou, Cleveland State University

This work was supported by National Institutes of Health Grant EB019739 to M.B. and by GM51491 to D.J.S. M.B and G.A recognize a DRA award from the Office of Sponsored programs of Cleveland State University.

Abstract

Endothelial nitric oxide synthase (eNOS) is a membrane-anchored enzyme. To highlight the potential role and effect of membrane phospholipids on the structure and activity of eNOS, we have incorporated the recombinant oxygenase subunit of eNOS into lipid nanodiscs. Two different size distribution modes were detected by multi-angle dynamic light scattering both for empty nanodiscs, and nanodiscs-bound eNOSoxy. The calculated hydrodynamic diameter for mode 1 species was 9.0 nm for empty nanodiscs and 9.8 nm for nanodisc bound eNOSoxy. Spectroscopic Griess assay was used to measure the enzymatic activity. Remarkably, the specific activity of nanodisc-bound eNOSoxy is ~65% lower than the activity of free enzyme. The data shows that the nano-membrane environment affects the catalytic properties of eNOS heme domain.