PMID:21599460
| Citation |
Krupnik, T, Dobrowolski, A and Lolkema, JS (2011) Cross-linking of dimeric CitS and GltS transport proteins. Mol. Membr. Biol. 28:243-53 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.3109/09687688.2011.581252 |
| Keywords |
Amino Acid Transport Systems, Acidic/chemistry; Amino Acid Transport Systems, Acidic/metabolism; Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Carrier Proteins/chemistry; Carrier Proteins/metabolism; Cross-Linking Reagents/metabolism; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Klebsiella pneumoniae/metabolism; Models, Biological; Protein Multimerization; Protein Structure, Tertiary; Protein Subunits/chemistry; Protein Subunits/metabolism; Recombinant Fusion Proteins/metabolism; Symporters/chemistry; Symporters/metabolism; Titrimetry |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
See also
References
See Help:References for how to manage references in EcoliWiki.