Twenty-Ninth Annual Meeting 24–28 February 1985 Convention Center, Baltimore, Maryland

1985 Biophysical Journal  
In X-ray diffraction patterns from oriented gap junction specimens, intensity on the equator at -lOA spacing and on the meridian at -4.7A spacing indicates a substantial proportion of the trans-membrane protein is in a a-sheet conformation, with the strands of the sheet running parallel to the membrane surface. The location and relative amount of S-sheet in the connexon protein spanning the bilayer has been determined from an analysis of the meridional intensity observed from several specimens.
more » ... A phase assignment was made using the minimum wavelength principle, assuming the connexon pair profile is centrosymmetric. The S-sheet is concentrated in two domains; a major domain running from '20-5oA from the center of the gap, and a minor domain running from the center of the gap to -15A from the center. The major domain has from 4 to 7 S-strands, the minor domain from 2 to 3 strands. This assignment of S-sheet protein correlates well with the 3-D gap-junction model deduced by Makowski et al. from low-resolution X-ray data. The magnitude of the electron density fluxuation in the S-strands has been scaled by comparing the 4.7A intensity with the low-resolution intensities, which were put on an absolute scale by solvent density contrast. Comparison with a 1-D profile calculated from a model S-sheet protein suggests that almost all the connexon protein in the region of the dominant domain may be in the S-sheet conformation. The amount of S-sheet estimated with this comparison does not depend on the choice of phases, although the positions of the S-sheet the transition is shifted upwards to 630C indicating that the phospholipid moiety stabilizes the native structure of the enzyme. The lipid bilayer environment contributes -10 kcal/mole to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome C oxidase is not a two-state process. Deconvolution analysis of the heat capacity function indicates that the overall curve is composed of four well defined sequential melting steps. The calorimetric experiments have been complemented with thermal gel electrophoresis experiments directed to identify the enzyme subunits involved in each melting step. According to these experiments, the first melting step, at 520C, involves subunits III and VIa (using the subunit nomenclature of Kadenbach et al., Anal. Biochem. (1983) 129, 517). This step is followed by two very closely spaced melting steps at 61 and 640C involving the bulk of the enzyme complex (subunits I, II, VIc, VII and remaining low molecular weight subunits). The other melting step probably involves subunit IV. The melting of subunits V and VIb could not be detected by gel electrophoresis and therefore their melting temperatures could not be assigned. The coat (gene 8) protein of the filamentous bacteriophage, M13, is an intrinsic membrane protein during phage infection of E. coli. Its 50 amino acids may be divided into three regions: an acidic N-terminus; a basic C-terminus; and a hydrophobic core. The hydrophilic and hydrophobic domains have been biosynthetically labelled with the 3-fluoro-analogs of phenylalanine (Fphe) and tyrosine (Ftyr), respectively. Structural information has been obtained by monitoring the motion and exposure of the Fphe and Ftyr residues of the protein bound by either deoxycholate (DOC) or phospholipid vesicles using 19F NMR. The exposure of the fluoro-residues of the labelled coat proteins in DOC micelles was determined from the results of proteolytic digestions, solvent effects and 9F photo-chemically induced dynamic nuclear polarization experiments; the motions of the Fphe and Ftyr rings were analyzed using a model dependent approach. Fphell was found to be outside the micelle, but, at least, partially buried in a hydrophobic pocket, Ftyr2l was at the solvent-micelle interface and Ftyr24 was inside the micelle. The exposure of the fluoro-residues of the labelled coat proteins reconstituted into phospholipid vesicles was determined by chymotryptic digestion and temperature studies; fluororesidue ring motion analyses were also done. The Fphe residues were outside the bilayer while the Ftyr residues were inside the bilayer. The Fphe residues were not much more mobile than the Ftyr residues, suggesting that the hydrophilic ends were structured. We have measured the phase behavior of mixed dipentadecanoyl phosphatidylglycerol (DC15PG)/dimyristoyl phosphatidylcholine (DMPC) small, unilamellar vesicles (SUV) in the presence of saturati~g (> 95% occupancy of binding sites) concentrations of bov1ne prothrombin fragment 1 and 5 mM Ca2 Binding of the fragment 1 peptide in the presence of Ca2 was verified by 900 light scattering. A stopped-flow technique which is based upon Cs-induced fluorescence intensity decay and allows one to measure kinetic parameters of acetylcholine receptor (AchR)-mediated ion translocation has been optimized for reconstituted Torpedo californica AchR vesicles. A fluorescent dye, 1,5anthraquinone disulphonate, was loaded into AchR vesicles by using a freeze-thaw method, and the dye outside the vesicles was removed by gel-chromatography. The optimum concentration of dye was found to be 10 mM. The total fluorescence decay increased with decreasing lipid-to-protein ratio, but with low lipid-to-protein ratios the rate of ion translocation by the AchR became too fast to be measured with the stopped-flow technique. A lipid-to-protein ratio of 1:20 was found to be optimum in terms of a good signal-to-noise ratio and a moderate reaction rate. Purification of the crude asolectin, which was used as lipid, was necessary in order to get a flat baseline. Crude asolectin gives a fluorescence decay without adding AchR agonists and this makes kinetic studies difficult. Using the stopped-flow technique, two rate coefficients have been measured over a range of acetylcholine concentration: J , the rate coefficient for ion translocation by the active state of AchR in the absence of inactivation (desensitization), and a, the rate coefficient for the fast activation of the receptor by acetylcholine. The method is being used to investigate the effect of inhibitors and chemical modification of the receptor on receptor function.
doi:10.1016/s0006-3495(85)83907-2 fatcat:vs62f7bkhbdn7nscuau5tgnlnq