[WS2] Frontiers in Bio-metal science: the roles of metals in life

[WS2-02] Probing functional and structural differences of quinol dependent Nitric Oxide Reductases’ (qNOR) within a lipidic nanodisc environment

^○Chai Gopalasingam¹, Hiroaki Yamaoka¹, Akitoshi Shibata¹, Kouki Fukumoto¹, Hideki Shigematsu², Takehiko Tosha², Kazumasa Muramoto¹, Yoshitsugu Shiro¹ (1.Graduate School of Life Sciences, University of Hyogo, 2.RIKEN SPring-8 Center)

The structure and function of membrane proteins is highly influenced by the surrounding hydrophobic lipid bilayer, yet, many studies utilise detergent to probe the functional and structural properties. Detergent offers a non-native method to study membrane proteins, with possibilities of altered activity and structural artifacts. Lipid nanodisc technology attempts to overcome this by tailoring of lipid(s) combined with an amphipathic helix (membrane scaffold protein) to form a discoidal particle, in which the target protein can be incorporated with complete detergent removal.
The membrane integrated, heme containing quinol-dependent Nitric Oxide Reductase (qNOR) from the pathogenic bacterium Neisseria meningitidis exhibits the highest rate of NO reduction of any NOR studied and is implemented in pathogen survival within the host.
Here we present efforts to reconstitute qNOR dimer and monomer fractions into several ‘flavours’ of lipid nanodiscs, to probe possible functional differences. We also attempt to reveal their structure by cryogenic Electron Microscopy (cryoEM), with emphasis on the 90 kDa monomer fraction, where no catalytically active structure has been solved. Functional data reveals losses of activity (~30-40%) compared to detergent qNOR samples, with cryo electron microscopy analysis underway to reveal the native structure of qNOR.