Document Detail


Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies.
MedLine Citation:
PMID:  23193105     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The bacterial flagellar motor is an intricate nanomachine powered by a transmembrane electrochemical gradient. Rotation is driven by the cumulative action of several peptidoglycan-anchored stator complexes on the rotor. In proton-motive force-driven motors, the stator complex is composed of a motility protein B (MotB) dimer surrounded by four copies of MotA, where both MotA and MotB are integral membrane proteins. The lack of full-length MotA and MotB structures hinders understanding of the mechanism of torque generation. Given the low levels of expression and low stability of detergent-solubilized MotB, a soluble chimaeric variant was engineered, where the two transmembrane helices of the MotB dimer were replaced by a leucine zipper. The biochemical and biophysical analysis of the resultant protein showed that it was properly folded, stable, behaved as a monodisperse dimer at low pH, had molecular dimensions close to those expected for native MotB and yielded reproducible crystals. The chimaeric protein is, therefore, a good candidate for structural studies. This 'solubilization by design' approach may be generally applicable to the production of soluble forms of other dimeric, trimeric and tetrameric single-span membrane proteins for functional and structural studies.
Authors:
Daniel A Andrews; Meng Xie; Victoria Hughes; Matthew C Wilce; Anna Roujeinikova
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of the Royal Society, Interface / the Royal Society     Volume:  10     ISSN:  1742-5662     ISO Abbreviation:  J R Soc Interface     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2012-11-29     Completed Date:  2013-05-01     Revised Date:  2014-02-14    
Medline Journal Info:
Nlm Unique ID:  101217269     Medline TA:  J R Soc Interface     Country:  England    
Other Details:
Languages:  eng     Pagination:  20120717     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Bacterial Proteins / chemistry*,  isolation & purification
Chromatography, Gel
Circular Dichroism
Crystallization
DNA Primers / genetics
Models, Molecular*
Molecular Motor Proteins / chemistry*,  isolation & purification
Molecular Sequence Data
Protein Conformation*
Protein Engineering / methods*
Recombinant Proteins / chemistry*,  isolation & purification
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/DNA Primers; 0/Molecular Motor Proteins; 0/MotB protein, Bacteria; 0/Recombinant Proteins
Comments/Corrections

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