Document Detail

Cross-linking of dimeric CitS and GltS transport proteins.
MedLine Citation:
PMID:  21599460     Owner:  NLM     Status:  Publisher    
Abstract CitS of Klebsiella pneumoniae and GltS of Escherichia coli are Na(+)-dependent secondary transporters from different families that are believed to share the same fold and quaternary structure. A 10 kDa protein tag (Biotin Acceptor Domain [BAD]) was fused to the N-terminus of both proteins (CitS-BAD1 and GltS-BAD1, respectively) and inserted in the central cytoplasmic loop that connects the two halves of the proteins (CitS-BAD260 and GltS-BAD206). Both CitS constructs and GltS-BAD206 were produced and shown to be active transporters, but GltS-BAD1 could not be detected in the membrane. Distance relationships in the complexes were studied by cross-linking studies. Both CitS constructs were shown to be in the dimeric state after purification in detergent by cross-linking with glutaraldehyde. The concentration of glutaraldehyde resulting in 50% cross-linking was significantly higher for CitS-BAD1 than for CitS and CitS-BAD260. Remarkably, GltS and GltS-BAD260 were not cross-linked by glutaraldehyde because of the lack of productive reactive sites. Cross-linking of GltS was observed when the N-terminal 46 residues of CitS with or without BAD at the N-terminus were added to the N-terminus of GltS. The stretch of 46 residues contains the first transmembrane segment of CitS that is missing in the GltS structure. The data support an orientation of the monomers in the dimer with the N-termini close to the dimer interface and the central cytoplasmic loops far away at the ends of the long axis of the dimer structure in a view perpendicular to the membrane.
Tomasz Krupnik; Adam Dobrowolski; Juke S Lolkema
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-5-20
Journal Detail:
Title:  Molecular membrane biology     Volume:  -     ISSN:  1464-5203     ISO Abbreviation:  -     Publication Date:  2011 May 
Date Detail:
Created Date:  2011-5-23     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9430797     Medline TA:  Mol Membr Biol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute , University of Groningen , The Netherlands.
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