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Intracellular domains of the delta-subunits of Torpedo and rat acetylcholine receptors--expression, purification, and characterization

Kottwitz, Denise LU ; Kukhtina, Viktoria; Dergousova, Natalia; Alexeev, Timophey; Utkin, Yuri; Tsetlin, Victor and Hucho, Ferdinand (2004) In Protein Expression and Purification 2(38). p.237-247
Abstract
There are quite detailed structural data on the extracellular ligand-binding domain and the intramembrane channel-forming domain of the nicotinic acetylcholine receptors (nAChR). However, the structure of the intracellular domain, which has variable amino acid sequences in different nAChR subunits, remains unknown. We expressed in Escherichia coli the intracellular loops (between transmembrane fragments TM3 and TM4) of the δ-subunits from the Torpedo californica and Rattus norvegicus muscle nAChRs. To facilitate purification, (His)6-tags were attached with or without linkers, and the effects of protein truncations at C- or N-termini were examined. The proteins were purified from inclusion bodies under denaturing conditions by... (More)
There are quite detailed structural data on the extracellular ligand-binding domain and the intramembrane channel-forming domain of the nicotinic acetylcholine receptors (nAChR). However, the structure of the intracellular domain, which has variable amino acid sequences in different nAChR subunits, remains unknown. We expressed in Escherichia coli the intracellular loops (between transmembrane fragments TM3 and TM4) of the δ-subunits from the Torpedo californica and Rattus norvegicus muscle nAChRs. To facilitate purification, (His)6-tags were attached with or without linkers, and the effects of protein truncations at C- or N-termini were examined. The proteins were purified from inclusion bodies under denaturing conditions by Ni–NTA-chromatography. Molecular weight and peptide mass fingerprint was determined by MALDI mass spectrometry. Size-exclusion chromatography revealed that the Torpedo intracellular δ-loop refolded in an aqueous buffer was present in solution as a dimer. Phosphorylation of this protein with protein kinase A and tyrosine kinase (Abl) occurred at the same serine and tyrosine residues as in the native receptor. According to CD spectra, the secondary structure was not sensitive to phosphorylation. The rat intracellular loops could be solubilized only in the presence of non-ionic detergents or lipids. CD spectra indicate that the Torpedo and rat proteins have differences in their secondary structure. In the presence of dodecylphosphocholine, high concentrations (up to 6 mg/ml) of the Torpedo and rat intracellular loops were achieved. The results suggest that the spatial structure of the intracellular loops is dependent on environment and species, but is not changed significantly upon enzymatic phosphorylation. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Protein Expression and Purification
volume
2
issue
38
pages
237 - 247
publisher
Academic Press
external identifiers
  • scopus:8844242589
ISSN
1046-5928
DOI
10.1016/j.pep.2004.07.017
language
English
LU publication?
yes
id
c37821a9-5541-4bb9-a1f5-ca21c7a6832e (old id 1131085)
date added to LUP
2008-06-16 13:44:46
date last changed
2017-01-01 05:06:34
@article{c37821a9-5541-4bb9-a1f5-ca21c7a6832e,
  abstract     = {There are quite detailed structural data on the extracellular ligand-binding domain and the intramembrane channel-forming domain of the nicotinic acetylcholine receptors (nAChR). However, the structure of the intracellular domain, which has variable amino acid sequences in different nAChR subunits, remains unknown. We expressed in Escherichia coli the intracellular loops (between transmembrane fragments TM3 and TM4) of the δ-subunits from the Torpedo californica and Rattus norvegicus muscle nAChRs. To facilitate purification, (His)6-tags were attached with or without linkers, and the effects of protein truncations at C- or N-termini were examined. The proteins were purified from inclusion bodies under denaturing conditions by Ni–NTA-chromatography. Molecular weight and peptide mass fingerprint was determined by MALDI mass spectrometry. Size-exclusion chromatography revealed that the Torpedo intracellular δ-loop refolded in an aqueous buffer was present in solution as a dimer. Phosphorylation of this protein with protein kinase A and tyrosine kinase (Abl) occurred at the same serine and tyrosine residues as in the native receptor. According to CD spectra, the secondary structure was not sensitive to phosphorylation. The rat intracellular loops could be solubilized only in the presence of non-ionic detergents or lipids. CD spectra indicate that the Torpedo and rat proteins have differences in their secondary structure. In the presence of dodecylphosphocholine, high concentrations (up to 6 mg/ml) of the Torpedo and rat intracellular loops were achieved. The results suggest that the spatial structure of the intracellular loops is dependent on environment and species, but is not changed significantly upon enzymatic phosphorylation.},
  author       = {Kottwitz, Denise and Kukhtina, Viktoria and Dergousova, Natalia and Alexeev, Timophey and Utkin, Yuri and Tsetlin, Victor and Hucho, Ferdinand},
  issn         = {1046-5928},
  language     = {eng},
  number       = {38},
  pages        = {237--247},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Intracellular domains of the delta-subunits of Torpedo and rat acetylcholine receptors--expression, purification, and characterization},
  url          = {http://dx.doi.org/10.1016/j.pep.2004.07.017},
  volume       = {2},
  year         = {2004},
}