The secondary structure of gap junctions. Influence of isolation methods and proteolysis.
Paired intercellular transmembrane channels, termed connexons, comprised of hexameric assemblies of gap junction protein, were isolated and purified from rat liver by exploiting their resistance to either Sarkosyl detergent solubilization or alkali extraction. The secondary structures of the gap junction proteins prepared by these methods were compared by circular dichroism (CD) spectroscopy. Both the spectra and the calculated net secondary structures of the proteins obtained by the two isolation methods were different. The protein isolated by the Sarkosyl treatment was found to be approximately 50% alpha-helical, while protein isolated by alkali extraction had a lower helix content (approximately 40%). In both types of preparations, however, the helical content of the gap junction protein was sufficiently large to be consistent with an all-helical model for the membrane-spanning parts of the structure. CD spectroscopy was also used to examine the effects of proteolytic digestion of the cytoplasmic domain on the net secondary structure of the detergent-treated gap junction protein. The membrane-bound fragments had a slightly higher proportion of their residues that were alpha-helical in nature, suggesting that the transmembrane and/or intra-gap domains are indeed enriched in this type of secondary structure. This information constrains the range of models which can be realistically proposed for the channel structure.
Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180.
M
M
Cascio
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E
Gogol
B A
BA
Wallace
D000818:Animals; D002942:Circular Dichroism; D003094:Collagen; D017630:Connexins; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D007365:Intercellular Junctions / Q000648:ultrastructure*; D008099:Liver / Q000648:ultrastructure; D008565:Membrane Proteins / Q000302:isolation & purification / Q000648:ultrastructure*; D008854:Microscopy, Electron; D011487:Protein Conformation; D051381:Rats; D012521:Sarcosine / Q000031:analogs & derivatives; D012697:Serine Endopeptidases
D017630:Connexins; D003902:Detergents; D008565:Membrane Proteins; C025231:sarkosyl; D003094:Collagen; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D012521:Sarcosine