Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans
| Autor: | Michael J.O. Wakelam, Ian M. Fearnley, John E. Walker, Martin G. Montgomery, Ian N. Watt, Qifeng Zhang, Edgar Morales-Ríos, Shujing Ding |
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| Přispěvatelé: | Montgomery, Martin [0000-0001-6142-9423], Wakelam, Michael [0000-0003-4059-9276], Walker, John [0000-0001-7929-2162], Apollo - University of Cambridge Repository |
| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
crystallization
Protein subunit Immunology subunits General Biochemistry Genetics and Molecular Biology Mass Spectrometry chemistry.chemical_compound Adenosine Triphosphate Bacterial Proteins F-ATPase Cardiolipin α-proteobacteria lcsh:QH301-705.5 Research Articles chemistry.chemical_classification paracoccus denitrificans biology Research General Neuroscience Inhibitor protein biology.organism_classification Transmembrane protein Protein Subunits Proton-Translocating ATPases Enzyme chemistry Biochemistry lcsh:Biology (General) f-atpase Electrophoresis Polyacrylamide Gel Paracoccus denitrificans cardiolipin Adenosine triphosphate Protein Binding |
| Zdroj: | Open Biology, Vol 5, Iss 9 (2015) 'Open Biology ', vol: 5, pages: 150119-1-150119-9 (2015) Open Biology |
| ISSN: | 2046-2441 |
| Popis: | The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex. his work was funded by the intramural programme of the Medical Research Council via MRC programme U105663150 to J.E.W., and by support from the Biotechnology and Biological Sciences Research Council to M.J.O.W. |
| Databáze: | OpenAIRE |
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