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Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum
- Source :
- Journal of molecular biology. 337(5)
- Publication Year :
- 2003
-
Abstract
- The high-affinity glycine betaine uptake system BetP, an osmosensing and osmoregulated sodium-coupled symporter from Corynebacterium glutamicum , was overexpressed in Escherichia coli with an N-terminal StrepII-tag, solubilized in β-dodecylmaltoside and purified by streptactin affinity chromatography. Analytical ultracentrifugation indicated that BetP forms trimers in detergent solution. Detergent-solubilized BetP can be reconstituted into proteoliposomes without loss of function, suggesting that BetP is a trimer in the bacterial membrane. Reconstitution with E. coli polar lipids produced 2D crystals with unit cell parameters of 182 A×154 A, γ=90° exhibiting p 22 1 2 1 symmetry. Electron cryo-microscopy yielded a projection map at 7.5 A. The unit cell contains four non-crystallographic trimers of BetP. Within each monomer, ten to 12 density peaks characteristic of transmembrane α-helices surround low-density regions that define potential transport pathways. Small but significant differences between the three monomers indicate that the trimer does not have exact 3-fold symmetry. The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation. The projection map of BetP shows no clear resemblance to other secondary transporters of known structure.
- Subjects :
- Osmosis
Stereochemistry
Protein Conformation
Recombinant Fusion Proteins
Trimer
Corynebacterium
medicine.disease_cause
Corynebacterium glutamicum
chemistry.chemical_compound
Betaine
Structural Biology
medicine
Cloning, Molecular
Protein Structure, Quaternary
Molecular Biology
Escherichia coli
Symporters
Cryoelectron Microscopy
Membrane Transport Proteins
Affinity Labels
Transmembrane protein
Membrane
chemistry
Symporter
Glycine
Liposomes
Crystallization
Subjects
Details
- ISSN :
- 00222836
- Volume :
- 337
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- Journal of molecular biology
- Accession number :
- edsair.doi.dedup.....934e1028d5ca46da2986f0b4e67b71c8