Reconstitution of Membrane-Bound Enzymes for Neutron Scattering Studies: A Case Study of Human Dihydroorotate Dehydrogenase

Abstract: Membrane proteins are challenging to study due the necessity to maintain a lipid-like environment to preserve their structure and function. Particularly difficult to study are membrane-bound enzymes that interact with lipophilic substrates. The aim of this doctoral thesis was to investigate the mechanism of action and inhibition of membrane-bound enzymes that interact with lipids and lipophilic substrates under non-crystalline, physiologically relevant conditions using neutron scattering methods. Neutron scattering techniques are suitable for the study of biological systems. Neutrons have no net charge and can therefore penetrate deeply into matter. Using neutrons, it is also possible to distinguish between isotopes of the same element, as is the case with the isotopes of hydrogen (protium and deuterium). Selective deuteration can be used to determine the structure and location of biological molecules in complex systems. Neutron reflectometry (NR) is a surface scattering technique capable of determining the structure and composition of thin films in the direction perpendicular to the surface. NR is therefore highly suitable for the study of membrane proteins and lipid bilayers. A relevant example of this type of membrane proteins is human dihydroorotate dehydrogenase (HsDHODH). HsDHODH in an integral protein found in the inner mitochondrial membrane (IMM), where it catalyzes the oxidation of dihydroorotate to orotate with the concomitant reduction of ubiquinone Q10 (coenzyme Q10), thus linking pyrimidine biosynthesis and the respiratory chain. HsDHODH is the target of anti-inflammatory drugs and anti-proliferative compounds, and mutations in the HsDHODH gene have been identified as the cause of Miller syndrome, a rare genetic disorder characterized by malformations of the head, face, and limbs. Firstly, the production of the protein reagents needed for this thesis was established. A full biochemical characterization of HsDHODH and truncated HsDHODH as well as three variants associated with Miller syndrome (G19E, E52G, R135C) was performed. A particular focus was placed on their interaction with lipids including quartz crystal microbalance with dissipation (QCM-D) measurements of their binding to supported lipid bilayers (SLB). Miller syndrome mutants displayed lower activities compared to the wild-type enzymes, but showed also decreased stability, a probably impaired mitochondrial import, as well as differences in interactions with lipids (Papers I – III). As part of this thesis, the interactions between SLBs consisting of either synthetic lipids or a complex lipid mixture extracted from yeast with or without Q10 and a soluble truncated form of HsDHODH, as well as the soluble bacterial analog from E. coli (EcDHODH), were investigated by NR. Q10 was found to be located at the center of all the bilayers studied, between the lipid leaflets. Both enzymes were found to penetrate into the outer lipid leaflet of the bilayer upon interaction. The bacterial enzyme displayed a stronger binding and better retention than the human enzyme. Binding was also found to depend on the lipid composition of the bilayer as both enzymes displayed a stronger binding to complex bilayers consisting of several lipid species as opposed to those consisting of a few synthetic lipids. The interaction between both enzymes and ubiquinone was found to be mediated by protein penetration, as opposed to Q10 migration (Paper IV). The reconstitution of HsDHODH into supported lipid bilayers was attempted using three different methodologies: adsorption of lipid-detergent micelles prepared with dodecyl D-maltoside (DDM), fusion of proteoliposomes, and hybrid approaches combining the adsorption of DDM-protein micelles and lipid vesicle fusion and characterized by NR. Micelle adsorption resulted in membranes with low lipid and protein coverage and with residual detergent. Proteoliposomes yielded a good lipid bilayer coverage but with a low protein content. The hybrid approaches resulted in good protein incorporation but also in the formation of an additional floating layer. The information obtained from these approaches can be used to guide and inform the reconstitution of proteins structurally similar to HsDHODH (Paper V).