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  Mutations in a Helix-1 Motif of the ATP Synthase c-Subunit of Bacillus pseudofirmus OF4 Cause Functional Deficits and Changes in the c-Ring Stability and Mobility on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Liu, J., Fackelmayer, O. J., Hicks, D. B., Preiß, L., Meier, T., Sobie, E. A., et al. (2011). Mutations in a Helix-1 Motif of the ATP Synthase c-Subunit of Bacillus pseudofirmus OF4 Cause Functional Deficits and Changes in the c-Ring Stability and Mobility on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis. Biochemistry, 50(24), 5497-5506. doi:10.1021/bi2005009.

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 Creators:
Liu, Jun1, Author
Fackelmayer, Oliver J.1, Author
Hicks, David B.1, Author
Preiß, Laura2, Author           
Meier, Thomas2, Author           
Sobie, Eric A.1, Author
Krulwich, Terry A.1, Author
Affiliations:
1Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, New York 10029, USA, ou_persistent22              
2Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068291              

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Free keywords: Genetics; Membranes; Peptides and proteins; Stability; Monomers
 Abstract: The ATP synthase of the alkaliphile Bacillus pseudofirmus OF4 has a tridecameric c-subunit rotor ring. Each c-subunit has an AxAxAxA motif near the center of the inner helix, where neutralophilic bacteria generally have a GxGxGxG motif. Here, we studied the impact of four single and six multiple Ala-to-Gly chromosomal mutations in the A16xAxAxA22 motif on the capacity for nonfermentative growth and, for most of the mutants, on ATP synthesis by ADP- and Pi-loaded membrane vesicles at pH 7.5 and 10.5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analyses of the holo-ATP synthases were used to probe stability of the mutant c-rotors and mobility properties of the c-rotors as well as the monomeric c-subunits that are released from them by trichloroacetic acid treatment. Mutants containing an Ala16-to-Gly mutation exhibited the most severe functional defects. Via SDS–PAGE, most of the mutant c-monomers exhibited increased mobility relative to the wild-type (WT) c-subunit, but among the intact c-rings, only Ala16-to-Gly mutants exhibited significantly increased mobility relative to that of the WT c-ring. The hypothesis that these c-rings have a decreased c-subunit stoichiometry is still untested, but the functional impact of an Ala16-to-Gly mutation clearly depended upon additional Ala-to-Gly mutation(s) and their positions. The A16/20G double mutant exhibited a larger functional deficit than both the A16G and A16/18G mutants. Most of the mutant c-rings showed in vitro instability relative to that of the WT c-ring. However, the functional deficits of mutants did not correlate well with the extent of c-ring stability loss, so this property is unlikely to be a major factor in vivo.

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Language(s): eng - English
 Dates: 2010-05-102010-11-052011-05-232011-06-21
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/bi2005009
PMID: 21568349
PMC: PMC3115392
 Degree: -

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Title: Biochemistry
Source Genre: Journal
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Publ. Info: Columbus, Ohio : American Chemical Society
Pages: - Volume / Issue: 50 (24) Sequence Number: - Start / End Page: 5497 - 5506 Identifier: ISSN: 0006-2960
CoNE: https://pure.mpg.de/cone/journals/resource/954925384103