An Ornithine-Free Gramicidin S Analogue Using Norleucine, Cyclo(Val-Nle-Leu-D-Phe-Pro)2, Forms Helically Aligned ß-Sheets.

The structure of an ornithine (Orn)-free Gramicidin S (GS) analogue, cyclo(Val-Nle-Leu-D-Phe-Pro)2 (NGS), was studied. Its circular dichroism (CD) spectrum showed that NGS has a structure similar to GS, though the value of [θ] indicated smaller ß-turn and sheet populations. This is probably because the Nle side chain could not form intramolecular hydrogen bonds stabilizing the sheet structure. The chemical shift perturbation of αH and JNH-αH were similar in GS and NGS. Three independent NGS molecules formed intramolecular ß-sheet structures in crystal. The turn structures of D-Phe-Pro moieties were classed as type II' ß-turns, but one part was unclassed. The molecules were arranged in a twisting manner, which resulted in the formation of a helical sheet. Similar structural characteristics were observed previously in a Leu-type, Orn-free GS analogue and in GS trifluoroacetic acid salt.

[Leu2]Gramicidin S preserves the structural properties of its parent peptide and forms helically aligned ß-sheets.

For crystallographic analysis, Leu was substituted for Orn in Gramicidin S (LGS) to suppress interactions with hydrophilic solvent molecules, which increased the flexibility of the Orn side chains, leading to disorder within the crystals. The asymmetric unit (C62H94N10O10·1.296C3H8O·1.403H2O) contains three LGS molecules (A, B and C) forming ß-turn and intramolecular ß-sheet structures. With the exception of one motif in molecule C, D-Phe-Pro turn motifs (Phe is phenylalanine and Pro is proline) were classed as type II' ß-turns. The peptide backbones twist slightly to the right along the long axis of the molecule. The puckering of Pro is in a Cγ-endo or twisted Cγ-endo-Cß-exo form. Flanking molecules are arranged such that the angles (A...B = 104°, B...C = 139° and C...A = 117°) form helical ß-sheets. Solvent molecules interact with the peptide backbones supporting the ß-sheets. The forms of the replacement Leu side chains are consistent with the e-form of the Orn side chain in GS analogues. No hydrophilic region composed of solvent molecules, such as that observed in Gramicidin S hydrochloride (GS·HCl) crystals, was found. The perturbation of αH chemical shifts and coupling constants of CONH showed that the structural properties of GS·HCl and LGS are similar to each other in solution. CD spectra also supported the structural similarity. With the sequence cyclo(-Val-Leu-Leu-D-Phe-Pro-)2 (Val is valine and Leu is leucine), LGS lacks the amphiphilicity and antimicrobial activity of parental Gramicidin S (GS). However, the structure of LGS reflects the structural characteristics of GS and no disordering inconvenient for structural analysis was found. Thus, LGS could be a novel scaffold useful for studying ß-turn and sheet structures.