title: Structure determination of functional membrane proteins using small-angle neutron scattering (sans) with small, mixed-lipid liposomes: native beef heart mitochondrial cytochrome c oxidase forms dimers.
abstract: The low-resolution three-dimensional structure of purified native beef heart mitochondrial cytochrome c oxidase (COX) in asolectin unilamellar liposomes has been measured by small-angle neutron scattering under the conditions where the protein remains fully functional. From a neutron scattering perspective, the use of mixed-lipid liposomes provided for a more homogeneous matrix than can be achieved using a single lipid. As a result, the measurements were able to be performed under conditions where the liposome scattering was essentially eliminated (contrast-matched conditions). The protein structure in the membrane was modeled as a simple parallelepiped with side lengths of (59 × 70 × 120) Å with uncertainties, respectively, (11, 12, 20 Å). The molecular mass calculated for a typical protein with this volume is estimated to be (410 ± 124) kDa, which indicates the mass of a COX dimer. The longest dimension has some uncertainty due to intermolecular scattering contributing to the data. Nevertheless, that length was estimated using an average protein density and the known dimer molecular mass. Using the same cross sectional dimensions for the structure, the length is estimated to be 120 Å. However, the measured scattering curve of the dimer in the liposome differs significantly from that calculated from the X-ray structure of the dimer in a crystal of mixed micelles (PDB 3AG1). The calculated SANS scattering from the crystal structure was fit with a parallelepiped, measuring (59 × 101 × 129) Å with fitting uncertainties, respectively, (2, 3, 3 Å). Our results suggest that COX is a functional dimer when reconstituted into mixed-lipid liposomes.
authors.affiliation: NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. rubinson@wright.edu
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Rubinson
authors.forename: Christine
authors.initials: C
authors.lastname: Pokalsky
authors.forename: Susan
authors.initials: S
authors.lastname: Krueger
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Prochaska
mesh_terms: D000818:Animals; D002417:Cattle; D019281:Dimerization; D003576:Electron Transport Complex IV / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology*; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology*; D053838:Scattering, Small Angle
chemical_list: D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins; D003576:Electron Transport Complex IV