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  Structural characterization of neuronal voltage-sensitive K+ channels heterologously expressed in Pichia pastoris

Parcej, D. N., & Eckhardt-Strelau, L. (2003). Structural characterization of neuronal voltage-sensitive K+ channels heterologously expressed in Pichia pastoris. Journal of Molecular Biology, 333(1), 103-116. doi:10.1016/j.jmb.2003.07.009.

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 Creators:
Parcej, David N.1, Author           
Eckhardt-Strelau, Luise1, Author           
Affiliations:
1Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068291              

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Free keywords: channel; expression; electron microscopy; structure
 Abstract: Neuronal voltage-dependent K+ channels of the delayed rectifier type consist of multiple Kv α subunit variants, which assemble as hetero- or homotetramers, together with four Kvβ auxiliary subunits. Direct structural information on these proteins has not been forthcoming due to the difficulty in isolating the native K+ channels. We have overexpressed the subunit genes in the yeast Pichia pastoris. The Kv1.2 subunit expressed alone is shown to fold into a native conformation as determined by high-affinity binding of 125I-labelled α-dendrotoxin, while co-expressed Kv1.2 and Kvβ2 subunits co-assembled to form native-like oligomers. Sites of post-translational modifications causing apparent heterogeneity on SDS-PAGE were identified by site-directed mutagenesis. Engineering to include affinity tags and scale-up of production by fermentation allowed routine purification of milligram quantities of homo- and heteroligomeric channels. Single-particle electron microscopy of the purified channels was used to generate a 3D volume to 2.1 nm resolution. Protein domains were assigned by fitting crystal structures of related bacterial proteins. Addition of exogenous lipid followed by detergent dialysis produced well-ordered 2D crystals that exhibited mostly p121 symmetry. Projection maps of negatively stained crystals show the constituent molecules to be 4-fold symmetric, as expected for the octameric K+ channel complex.

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Language(s): eng - English
 Dates: 2003-07-302003-06-172003-07-312003-09-212003-10-10
 Publication Status: Published in print
 Pages: 16
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.jmb.2003.07.009
PMID: 14516746
 Degree: -

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Title: Journal of Molecular Biology
  Other : JMB
  Abbreviation : J. Mol. Biol.
Source Genre: Journal
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Publ. Info: Elsevier
Pages: - Volume / Issue: 333 (1) Sequence Number: - Start / End Page: 103 - 116 Identifier: ISSN: 0022-2836
CoNE: https://pure.mpg.de/cone/journals/resource/0022-2836