Hydrogen-Deuterium Solvent Exchange in Gramicidin a Single Crystals
The structure of gramicidin A crystallized from ethanol (P212121: a = 24.6, b = 32.3, c = 32.5) remains unsolved because of a lack of isomorphous derivatives for phase determination. In such a case, neutron diffraction offers the possibility of approaching the phase problem by means of isomorphous deuterium-hydrogen substitution. The solvent (ethanol-water: 50-50) comprises about 15% of the mass of these crystals. Neutron diffraction data to 2-Å resolution have been recorded at Brookhaven’s High Flux Beam Reactor for crystals of gramicidin A containing either H2O/ethanol or D2O/deuteroethanol. The average difference in |I1/2| is 28% between these two data sets. Difference neutron Patterson maps show significant peaks in the region where a previous x-ray self Patterson analysis indicated there was a channel down the center of the cylindrical molecule. The strongest difference peak has been refined in three centrosymmetric projections, with use of the Dickerson phase refinement program. A single site having an occupancy of four deutrons refines to a centric R-factor of 0.39 at 8-Å resolution (0.46 at 5 Å). This site has been used to calculate (FgramD − FgramH − fsite) difference Fourier projections to locate additional sites, which were then refined and used in further difference Fourier calculations. The procedure was stopped after the difference data had been fitted with use of eight sites having occupancies ranging from 0.5 to 4 deuterons, the R-factor for the fit being 0.28 for three centric projections at 3-Å resolution. Single isomorphous replacement phases (mean figure of merit = 0.72 at 5 Å) were used to calculate projection and 3-dimensional Fourier maps at 5-Å resolution. The maps confirm previous indications concerning the cylindrical shape of gramicidin A in these crystals. We do not yet know whether it will be possible to trace the course of the polypeptide backbone by using these maps. We intend to resolve the single isomorphous phase ambiguity by using either a covalent deuterovaline derivative or a crystal soaked in H2O/deuteroethanol.