DIFFRACTION DIAGNOSIS OF PROTEIN FOLDING IN GAP JUNCTION CONNEXONS

To diagnose the regular polypeptide conformation in gap junction membranes, the x-ray intensities diffracted from oriented specimens have been separated into a modulated component due to the coherently ordered portion of the channel-forming pairs of connexon hexamers and a diffuse component due to the disordered parts. The spherically averaged ordered protein diffraction, in the resolution range 15–4 A, was compared with intensity curves calculated from the Fourier transforms of proteins representative of the major tertiary structural classes. From this comparison the alpha-helical content of the ordered portion of the connexon was estimated to be approximately 60%. Calculation of cylindrically averaged patterns for oriented distributions of alpha-helical and beta-sheet proteins demonstrated that the ratio of the modulated diffracted intensity near 5 A spacing on the meridian and 10 A spacing on the equator observed from the gap junctions can be accounted for by alpha-helical segments inclined relative to the connexon axis. Model dimers of connexonlike hexamers were constructed from alpha-helix bundle proteins to correlate features in the calculated diffraction patterns with the model parameters. On the basis of these correlations, the ordered gap junction diffraction data indicate that alpha-helical segments centered at 38 A from the midplane of the gap have a mean radial location approximately 24 A from the hexamer axis, and an axial projected length of approximately 35 A. Thus, these alpha-helical segments traverse the hydrocarbon core of the lipid bilayer, as expected for the four hydrophobic sequences of the connexin molecule. © 1990, The Biophysical Society. All rights reserved.