Purification and reconstitution of the connexin43 carboxyl terminus attached to the 4th transmembrane domain in detergent micelles

Admir Kellezi, Rosslyn Grosely, Fabien Kieken, Gloria E.O. Borgstahl, Paul L. Sorgen

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

In recent years, reports have identified that many eukaryotic proteins contain disordered regions spanning greater than 30 consecutive residues in length. In particular, a number of these intrinsically disordered regions occur in the cytoplasmic segments of plasma membrane proteins. These intrinsically disordered regions play important roles in cell signaling events, as they are sites for protein-protein interactions and phosphorylation. Unfortunately, in many crystallographic studies of membrane proteins, these domains are removed because they hinder the crystallization process. Therefore, a purification procedure was developed to enable the biophysical and structural characterization of these intrinsically disordered regions while still associated with the lipid environment. The carboxyl terminal domain from the gap junction protein connexin43 attached to the 4th transmembrane domain (TM4-Cx43CT) was used as a model system (residues G178-I382). The purification was optimized for structural analysis by nuclear magnetic resonance (NMR) because this method is well suited for small membrane proteins and proteins that lack a well-structured three-dimensional fold. The TM4-Cx43CT was purified to homogeneity with a yield of ∼6 mg/L from C41(DE3) bacterial cells, reconstituted in the anionic detergent 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], and analyzed by circular dichroism and NMR to demonstrate that the TM4-Cx43CT was properly folded into a functional conformation by its ability to form α-helical structure and associate with a known binding partner, the c-Src SH3 domain, respectively.

Original languageEnglish (US)
Pages (from-to)215-222
Number of pages8
JournalProtein Expression and Purification
Volume59
Issue number2
DOIs
StatePublished - Jun 1 2008

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Keywords

  • Cx43
  • Cx43 carboxyl terminal domain
  • Gap junctions

ASJC Scopus subject areas

  • Biotechnology

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