The aim of our research is to determine the structure of enzymes and supercomplexes that make up the mitochondrial and bacterial respiratory chain by cryo-electron microscopy and state of the art image analysis techniques. The importance of understanding the function and relationships between enzymes in the respiratory chain is highlighted by the fact that many mitochondrial diseases can be traced back to defects in the genes encoding complex I. Examples are LHON-disease (Leber's hereditary optic neuropathy), Ligh's disease and Kearns-Sayre syndrome. We will determine the native structures of complex I from the strictly aerobic yeast Yarrowia lipolytica and from the bacterium Aquifex aeolicus. The arrangement of the subunits in complex I will be determined. Knowledge of their architecture within the complex will provide the information needed to test existing functional models and identify the subunits involved. Three-dimensional immuno cryo-electron microscopy with monoclonal and polyclonal antibodies against different subunits will be used to localize subunits and determine their architecture in the complex. We will calculate structures of the complex with and without antibody labeling. The location of the binding epitopes will be determined from the difference between each two structures in three dimensions. We will further analyze supercomplexes of the complete electron transfer chain from the membrane of P. denitrificans to investigate direct coupling of the enzymes. We will determine the structure of this supercomplex by three-dimensional electron microscopy using either tomographic techniques of individual particles or the Random Conical Reconstruction Technique applied to large data sets with extensive classification. Antibody labeled supercomplexes will be analyzed to identify the relative arrangement of the individual electron transfer chain complexes within the supercomplex. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068650-02
Application #
6781736
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Chin, Jean
Project Start
2003-08-01
Project End
2007-07-31
Budget Start
2004-08-01
Budget End
2005-07-31
Support Year
2
Fiscal Year
2004
Total Cost
$303,000
Indirect Cost
Name
University of Vermont & St Agric College
Department
Physiology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Angerer, Heike; Radermacher, Michael; Ma?kowska, Michalina et al. (2014) The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity. Proc Natl Acad Sci U S A 111:5207-12
Hatle, Ketki M; Gummadidala, Phani; Navasa, Nicolás et al. (2013) MCJ/DnaJC15, an endogenous mitochondrial repressor of the respiratory chain that controls metabolic alterations. Mol Cell Biol 33:2302-14
Xie, Xiaocong; Li, Yi; Fox, Joseph M (2013) Selective syntheses of ?(?,?) and ?(?,?) butenolides from allylic cyclopropenecarboxylates via tandem ring expansion/[3,3]-sigmatropic rearrangements. Org Lett 15:1500-3
Panish, Robert; Chintala, Srinivasa R; Boruta, David T et al. (2013) Enantioselective synthesis of cyclobutanes via sequential Rh-catalyzed bicyclobutanation/Cu-catalyzed homoconjugate addition. J Am Chem Soc 135:9283-6
Fisher, Laural A; Smith, Natalee J; Fox, Joseph M (2013) Chiral cyclopropenyl ketones: reactive and selective Diels-Alder dienophiles. J Org Chem 78:3342-8
Yu, Lingbo; Snapp, Robert R; Ruiz, Teresa et al. (2013) Projection-based volume alignment. J Struct Biol 182:93-105
Tarwade, Vinod; Selvaraj, Ramajeyam; Fox, Joseph M (2012) Facially selective Cu-catalyzed carbozincation of cyclopropenes using arylzinc reagents formed by sequential I/Mg/Zn exchange. J Org Chem 77:9900-4
DeAngelis, Andrew; Dmitrenko, Olga; Fox, Joseph M (2012) Rh-catalyzed intermolecular reactions of cyclic ýý-diazocarbonyl compounds with selectivity over tertiary C-H bond migration. J Am Chem Soc 134:11035-43
Dröse, Stefan; Krack, Stephanie; Sokolova, Lucie et al. (2011) Functional dissection of the proton pumping modules of mitochondrial complex I. PLoS Biol 9:e1001128
Hassink, Matthew; Liu, Xiaozhong; Fox, Joseph M (2011) Copper-catalyzed synthesis of 2,4-disubstituted allenoates from ?-diazoesters. Org Lett 13:2388-91

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