Transmembrane alpha-helices in the gap junction membrane channel: Systematic search of packing models based on the pair potential function

Recent progress in the field of electron cryo-microscopy and image analysts has shown that there is an overwhelming need to interpret medium resolutio n (5 to 10 Angstrom) three-dimensional maps. Traditional methods of fitting amino acid residues into electron density using molecular modeling program s must be supplemented with further analysis. We have used a potential of m ean force (PMF) method, derived from Boltzmann statistics in protein struct ure, to generate models for the packing of oc-helices, using pairwise poten tials between amino acid residues. The approach was tested using the three- dimensional map of a recombinant cardiac gap junction membrane channel prov ided by electron cryo-crystallography (Unger et al., 1997; 1999a, 1999b) wh ich had a resolution of 7.5 Angstrom in the membrane plane and 21 Angstrom in the vertical direction. The dodecameric channel was formed by the end-to -end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which was consistent with an or-helical conformatio n for the four transmembrane domains of each connexin subunit. Based on the three-dimensional map and the amino acid sequence for the 4 transmembrane domains determined by hydropathy analysis, we used the modeling utility Sym Serv (Macke et al., 1998) to build hexameric connexons with 24 transmembran e a-helices. Canonical cc-helices were aligned to the axes of the rods of d ensity and translated along the density so that the center of masses coinci ded. The PMF function was used to evaluate 162,000 conformations for each o f the 24 possible or-helical packing models. Since the different packing mo dels yielded different energy distributions, the pair potential function ap pears to be a promising tool for evaluating the packing of oc-helices in me mbrane proteins. The analysis will be refined by energy calculations based on the expectations that the outer boundary of the channel will be formed b y hydrophobic residues in contact with the lipids. Microsc. Res. Tech. 52:3 44-351, 2001. (C) 2001 Wiley-Liss. Inc.