Purification and reconstitution of the connexin43 carboxyl terminus attached to the 4th transmembrane domain in detergent micelles.
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 approximately 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 alpha-helical structure and associate with a known binding partner, the c-Src SH3 domain, respectively.
Kellezi
Admir
A
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Grosely
Rosslyn
R
Kieken
Fabien
F
Borgstahl
Gloria E O
GE
Sorgen
Paul L
PL
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D018031:Connexin 43 / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D003902:Detergents / Q000737:chemistry; D008823:Micelles; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats
D018031:Connexin 43; D003902:Detergents; D008823:Micelles