Preparation of human dihydroorotate dehydrogenase for interaction studies with lipid bilayers

• Human dihydroorotate dehydrogenase (DHODH) is an integral protein of the inner mitochondrial membrane (IMM) that catalyzes the fourth step of the de novo pyrimidine biosynthesis and is functionally connected to the respiratory chain via its lipophilic co-substrate, ubiquinone Q10. DHODH is the target for drugs approved for the treatment of rheumatoid arthritis and multiple sclerosis, and mutations in its sequence have been identified as the cause of Miller syndrome, a rare genetic disorder. The N-terminus of DHODH consists of a signal peptide for mitochondrial import (MS), a transmembrane domain (TM), followed by a microdomain which interacts with the lipids of the IMM and has been proposed to form the binding site for ubiquinone Q10. However, the mechanism by which DHODH interacts with the membrane-embedded Q10 and the lipids of the IMM remains unknown. We present the preparation and characterization of proteins necessary for investigating the structural interactions of DHODH with the lipids of the IMM, including expression and purification of full-length and N-terminally truncated (without MS and TM) DHODH. We characterized the interaction of truncated DHODH with lipid bilayers containing some key lipids of the IMM using Quartz Crystal Microbalance with Dissipation monitoring and compared it to the DHODH from E. coli, a DHODH that naturally lacks a TM. Our results suggest that although cardiolipin enhances the interaction of truncated DHODH with lipid bilayers, the presence of the TM in human DHODH is necessary for stable binding to and securing its location at the outer surface of the IMM.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

protein membrane interactions

protein production

Pyrimidine biosynthesis

QCM-D

ubiquinone

Publikations- och innehållstyp

art (ämneskategori)

ref (ämneskategori)