Document Detail

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.
MedLine Citation:
PMID:  21568349     Owner:  NLM     Status:  MEDLINE    
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 P(i)-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.
Jun Liu; Oliver J Fackelmayer; David B Hicks; Laura Preiss; Thomas Meier; Eric A Sobie; Terry A Krulwich
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2011-05-23
Journal Detail:
Title:  Biochemistry     Volume:  50     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2011 Jun 
Date Detail:
Created Date:  2011-06-14     Completed Date:  2011-08-24     Revised Date:  2014-09-16    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5497-506     Citation Subset:  IM    
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MeSH Terms
Amino Acid Motifs
Amino Acid Sequence
Amino Acid Substitution
Bacillus / enzymology*,  genetics*
Bacterial Proton-Translocating ATPases / chemistry*,  genetics*,  metabolism
Electrophoresis, Polyacrylamide Gel
Enzyme Stability
Glucosides / pharmacology
Models, Molecular
Molecular Sequence Data
Molecular Weight
Mutagenesis, Site-Directed
Protein Structure, Secondary
Protein Subunits
Recombinant Proteins / chemistry,  genetics,  metabolism
Sequence Homology, Amino Acid
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Grant Support
Reg. No./Substance:
0/Glucosides; 0/Protein Subunits; 0/Recombinant Proteins; 29836-26-8/octyl-beta-D-glucoside; EC 3.6.1.-/Bacterial Proton-Translocating ATPases

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