X-ray diffraction data is the main source of 3-dimensional structure information at atomic resolution for proteins. While a great deal of information is potentially available from this technique, its application requires the ability to prepare crystals of size and order suitable for X-ray analysis. This has proved especially difficult for membrane proteins. Complex II (succinate: ubiquinol oxidoreductase) is a membrane protein complex, which funnels electrons from succinate into the mitochondrial respiratory chain. The respiratory chain is responsible for biological oxidation and for conservation of the energy released in the form of a proton electrochemical potential gradient across the mitochondrial inner membrane. Energy from this gradient is then used to synthesize ATP or to do work by transporting substances across the membrane. A number of mitochondrial myopathies and CNS disorders have been shown to be due to defects in the mitochondrial electron transport chain and in some cases in complex II. In the course of our work on the mitochondrial bc1 complex we have prepared and solubilized mitochondria from a number of vertebrate sources. We have learned to prepare complex II as a by-product of the cytochrome bc1 preparation. In the case of the chicken enzyme, we have been able to crystallize the enzyme. The crystals can be flash-frozen for cryogenic data collection with diffraction up to 3.5 Angstrom units. The data can be indexed on an orthorhombic lattice with unit cell edges 69.036 x 83.735 x 290.214 Angstrom units, and systematic absences suggest the spacegroup P212121. It seems reasonable to expect that with improvement in size and mosaicity the crystals will diffract beyond 3.0 Angstrom units, which would be sufficient to build an atomic model of the protein once the data is phased. It is proposed to (a) Improve the quality of the crystals with respect to size and mosaicity, by using recrystallization and seeding techniques and more carefully controlling the crystallization conditions. (B) Collect diffraction data and phase it, either by molecular replacement using the coordinates from fumarate reductase of E. coli or W. succinogenes, or (in case neither of those structures is available) by isomorphous derivative and multiwavelength anomalous dispersion techniques using heavy atom derivatives and the intrinsic irons of the complex. (C) Build an atomic model of the protein into the electron density map and refine it against the diffraction data, for submission to the protein data bank.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062563-02
Application #
6387296
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Preusch, Peter C
Project Start
2000-05-01
Project End
2003-04-30
Budget Start
2001-05-01
Budget End
2002-04-30
Support Year
2
Fiscal Year
2001
Total Cost
$158,980
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Other Basic Sciences
Type
Organized Research Units
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Berry, Edward A; Walker, F Ann (2008) Bis-histidine-coordinated hemes in four-helix bundles: how the geometry of the bundle controls the axial imidazole plane orientations in transmembrane cytochromes of mitochondrial complexes II and III and related proteins. J Biol Inorg Chem 13:481-98
Huang, Li-shar; Sun, Gang; Cobessi, David et al. (2006) 3-nitropropionic acid is a suicide inhibitor of mitochondrial respiration that, upon oxidation by complex II, forms a covalent adduct with a catalytic base arginine in the active site of the enzyme. J Biol Chem 281:5965-72
Huang, Li-Shar; Shen, John T; Wang, Andy C et al. (2006) Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II, and the identity of the ligand in the ""oxaloacetate-inhibited"" state. Biochim Biophys Acta 1757:1073-83
Huang, Li Shar; Borders, Toni M; Shen, John T et al. (2005) Crystallization of mitochondrial respiratory complex II from chicken heart: a membrane-protein complex diffracting to 2.0 A. Acta Crystallogr D Biol Crystallogr 61:380-7
Huang, Li-Shar; Cobessi, David; Tung, Eric Y et al. (2005) Binding of the respiratory chain inhibitor antimycin to the mitochondrial bc1 complex: a new crystal structure reveals an altered intramolecular hydrogen-bonding pattern. J Mol Biol 351:573-97
Cobessi, D; Huang, L S; Ban, M et al. (2002) The 2.6 A resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic 'special position'. Acta Crystallogr D Biol Crystallogr 58:29-38