HIGH-RESOLUTION STRUCTURE AND DYNAMIC IMPLICATIONS FOR A DOUBLE-HELICAL GRAMICIDIN-A CONFORMER

The high-resolution structure of a dimeric conformer of gramicidin A, a 15-residue polypeptide, has been determined in the mixed-solvent sys tem of benzene and ethanol by 2D NMR techniques. NOEs, coupling consta nts and hydrogen-bond information were used to generate 744 experiment al constraints for the dimer. Stereoassignment of most beta-methylene groups was achieved by analysis of 3J(alphabeta), d(alphabeta)(i,i), d (Nbeta)(i,i) and d(Nbeta)(i+ 1,i) distances, and consideration of the initial backbone structure determinations. Stereoassignment of several leucine methyl groups was accomplished via a distance geometry/simula ted annealing routine, used for structure determination and refinement . The relatively static backbone structure was determined first and he ld rigid while side-chain conformations were calculated. This procedur e is evaluated versus standard NMR structure determination protocols. The backbone is an antiparallel intertwined double helix, with 5.6-5.7 residues per turn, a total dimer length of 36-37 angstrom, and a pore width of 2.5-3.0 angstrom (van der Waals to van der Waals). The struc ture and dynamics of the side chains are discussed in depth, with care ful attention for both the convergence of structures and the residual constraint violations per residue. Side-chain positions impart substan tial amphipathic character to the helix, which could influence the con formational change that takes place upon membrane insertion of this ch annel-forming polypeptide.