The crystal structure of the uncomplexed orthorhombic form of gramicidin A has been determined at 120 K and at 0.86 angstrom resolution. The pentadecapeptide crystallizes as a left-handed antiparallel double-stranded helical dimer with 5.6 amino acid residues per turn. The helix has an overall length of 31 angstroms and an average inner channel diameter of 4.80 angstroms. The channel of this crystalline form is void of ions or solvent molecules. The channel diameter varies from a minimum of 3.85 angstroms to a maximum of 5.47 angstroms and contains three pockets where the cross-channel contacts are 5.25 angstroms or greater. The range of variation seen for the phi and psi torsion angles of the backbone of the helix suggests that these potential ion binding sites can be induced to travel the length of the channel in a peristaltic manner by cooperatively varying these angles. The indole rings of the eight tryptophan residues of the dimer are overlapped in three separate regions on the outer surface of the helix when viewed down the barrel of the channel. This arrangement would permit long-chained lipid molecules to nest parallel to the outer channel surface between these protruding tryptophan regions and act like molecular splines to constrain helical twist deformations of the channel.

The presence of multiple a,a-dialkyl amino acids such as a-methylalanine (a-aminoisobutyric acid, Aib) leads to predominantly helical structures, either with a-helical or 310-helical hydrogen bonding patterns. The crystal structure of emerimicin-(1-9) benzyl ester (Ac-Phe-Aib-Aib-AibVal-Gly-Leu-Aib-Aib-OBzl) reported here shows essentially pure a-helical character, whereas other similar compounds show predominantly 310-helical structures. The factors that govern helical preference include the inherent relative stability of the a-helix compared with the 310-helix, the extra hydrogen bond seen with 310-helices, and the enhanced electrostatic dipolar interaction of the 310-helix when packed in a crystalline lattice. The balance of these forces, when combined with the steric requirements of the amino acid side chains, determines the relative stability of the two helical conformations under a given set of experimental conditions. The presence of a,a-dialkyl amino acids in microbial natural products, such as the peptaibol antibiotics, requires novel biosynthetic pathways to produce and incorporate these unusual amino acids in the face of the usual ribosomal mechanisms available for normal amino acids. This argues strongly for a special role related to function, one aspect of which may be their increased resistance to proteolytic degradation. Another aspect is the conformational restrictions imposed by these amino acids as first pointed out by Marshall and Bosshard (1, 2) and verified by others (3-7). While most work has focused on a-methylalanine (a-aminoisobutyric acid, Aib), a-ethylalanine (isovaline) has also been found to be a natural component of several peptaibol antibiotics (8,9). In addition, chiral a,a-dialkyl amino acids, such as amethylphenylalanine, have been incorporated into naturally occurring peptides in an effort to restrict their conformational freedom (10, 11).From the Ramachandran plots published by Marshall and Bosshard in 1972 (1), the presence of an additional alkyl substituent on the a-carbon severely restricted the values of the torsional variables 4 and qk as compared with those available to normal amino acids. While the two major allowed conformational areas were associated with either right-or left-handed helical conformations (both a and 310), the calculation also revealed other sets of energetically feasible values for 4 and q, adjacent to the a,a-dialkyl residue associated with extended structures as well as turns. The effect on conformation of alkyl groups larger than methyl as substituents in a,a-dialkyl amino acids has also been investigated (11,12). Despite the variety of conformations theoretically available to a,a-dialkyl amino acids, the impact of multiple substitutions ofthis type of amino acid on the overall conformation of a peptide is dramatic. The crystal structure of alamethicin (13), which contains 8 Aib residues out of 20, is predominantly a-helical, with NMR data (14) supporting a similar solution conformation in methanol. A review (15) of crystal structures of tr...

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The linear pentadecapeptide antibiotic, gramicidin D, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The x-ray crystal structures of the right-handed double-stranded double-helical dimers (DSDH ) reported here agree with 15 N-NMR and CD data on the functional gramicidin D channel in lipid bilayers. These structures demonstrate single-file ion transfer through the channels. The results also indicate that previous crystal structure reports of a left-handed double-stranded double-helical dimer in complex with Cs ؉ and K ؉ salts may be in error and that our evidence points to the DSDH as the major conformer responsible for ion transport in membranes.

The Shake-and-Bake method of structure determination is a new direct methods phasing algorithm based on a minimum-variance, phase invariant residual, which is referred to as the minimal principle. Previously, the algorithm had been applied only to known structures. This algorithm has now been applied to two previously unknown structures that contain 105 and 110 non-hydrogen atoms, respectively. This report focuses on (i) algorithmic and parametric optimizations of Shake-and-Bake and (ii) the determination of two previously unknown structures. Traditional tangent formula phasing techniques were unable to unravel these two new structures.