Optimizing the solution conditions to solve the structure of the Connexin43 carboxyl terminus attached to the 4(th) transmembrane domain in detergent micelles.
pH-mediated gating of Cx43 channels following an ischemic event is believed to contribute to the development of lethal cardiac arrhythmias. Studies using a soluble version of the Cx43 carboxyl-terminal domain (Cx43CT; S255-I382) have established the central role it plays in channel regulation; however, research in the authors' laboratory suggests that this construct may not be the ideal model system. Therefore, we have developed a more 'native-like' construct (Cx43CT attached to the 4th transmembrane domain [TM4-Cx43CT; G178-I382]) than the soluble Cx43CT to further investigate the mechanism(s) governing this regulation. Here, we utilize circular dichroism and nuclear magnetic resonance (NMR) were used to validate the TM4-Cx43CT for studying channel gating and optimize solution conditions for structural studies. The data indicate that, unlike the soluble Cx43CT, the TM4-Cx43CT is structurally responsive to changes in pH, suggesting the presence of the TM4 facilitates pH-induced structural alterations. Additionally, the optimal solution conditions for solving the NMR solution structure include 10% 2,2,2 trifluoroethanol and removal of the 2nd extracellular loop (G178-V196).
Grosely
Rosslyn
R
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Kieken
Fabien
F
Sorgen
Paul L
PL
D000818:Animals; D002942:Circular Dichroism; D018031:Connexin 43 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014270:Trifluoroethanol / Q000737:chemistry
D018031:Connexin 43; D003902:Detergents; D008823:Micelles; D011994:Recombinant Proteins; D014270:Trifluoroethanol