We report on the first chemical syntheses and structure-activity analyses of the cyclic lipopeptide battacin which revealed that conjugation of a shorter fatty acid, 4-methyl-hexanoic acid, and linearization of the peptide sequence improves antibacterial activity and reduces hemolysis of mouse blood cells. This surprising finding of higher potency in linear lipopeptides than their cyclic counterparts is economically beneficial. This novel lipopeptide was membrane lytic and exhibited antibiofilm activity against Pseudomonas aeruginosa, Staphylococcus aureus, and, for the first time, Pseudomonas syringe pv. actinidiae. The peptide was unstructured in aqueous buffer and dimyristoylphosphatidylcholine-polymerized diacetylene vesicles, with 12% helicity induced in 50% v/v of trifluoroethanol. Our results indicate that a well-defined secondary structure is not essential for the observed antibacterial activity of this novel lipopeptide. A truncated pentapeptide conjugated to 4-methyl hexanoic acid, having similar potency against Gram negative and Gram positive pathogens was identified through alanine scanning.
Designed octapeptide beta-hairpins containing a central (D)Pro-Gly segment have been used as a scaffold to place the aromatic residues Tyr and Trp at various positions on the antiparallel beta-strands. Using a set of five peptide hairpins, aromatic interactions have been probed across antiparallel beta-sheets, in the non-hydrogen bonding position (Ac-L-Y-V-(D)P-G-L-Y/W-V-OMe: peptides 1 and 2), diagonally across the strands (Boc-Y/W-L-V-(D)P-G-W-L-V-OMe: peptides 3 and 6), and along the strands at positions i and i + 2 (Boc-L-L-V-(D)P-G-Y-L-W-OMe: peptide 4). Two peptides served as controls (Boc-L-L-V-(D)P-G-Y-W-V-OMe: peptide 5; Boc-L-Y-V-(D)P-G-L-L-V-OMe: peptide 7) for aromatic interactions. All studies have been carried out using solution NMR methods in CDCl(3) + 10% DMSO-d(6) and have been additionally examined in CD(3)OH for peptides 1 and 2. Inter-ring proton-proton nuclear Overhauser effects (NOEs) and upfield shifted aromatic proton resonances have provided firm evidence for specific aromatic interactions. Calculated NMR structures for peptides 1 and 2, containing aromatic pairs at facing non-hydrogen bonded positions, revealed that T-shaped arrangements of the interacting pairs of rings are favored, with ring current effects leading to extremely upfield chemical shifts and temperature dependences for specific aromatic protons. Anomalous far-UV CD spectra appeared to be a characteristic feature in peptides where the two aromatic residues are spatially proximal. The observation of the close approach of aromatic rings in organic solvents suggests that interactions of an electrostatic nature may be favored. This situation may be compared to the case of aqueous solutions, where clustering of aromatic residues is driven by solvophobic (hydrophobic) forces.
Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro. Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)–carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline's unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics and NMR experiments. We therefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis.
Hybrid peptide segments containing contiguous alpha and gamma amino acid residues can form C(12) hydrogen bonded turns which may be considered as backbone expanded analogues of C(10) (beta-turns) found in alphaalpha segments. Exploration of the regular hydrogen bonded conformations accessible for hybrid alphagamma sequences is facilitated by the use of a stereochemically constrained gamma amino acid residue gabapentin (1-aminomethylcyclohexaneacetic acid, Gpn), in which the two torsion angles about C(gamma)-C(beta) (theta(1)) and C(beta)-C(alpha) (theta(2)) are predominantly restricted to gauche conformations. The crystal structures of the octapeptides Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (1) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (2) reveal two distinct conformations for the Aib-Gpn segment. Peptide 1 forms a continuous helix over the Aib(2)-Aib(6) segment, while the peptide 2 forms a beta-hairpin structure stabilized by four cross-strand hydrogen bonds with the Aib-Gpn segment forming a nonhelical C(12) turn. The robustness of the helix in peptide 1 in solution is demonstrated by NMR methods. Peptide 2 is conformationally fragile in solution with evidence of beta-hairpin conformations being obtained in methanol. Theoretical calculations permit delineation of the various C(12) hydrogen bonded structures which are energetically feasible in alphagamma and gammaalpha sequences.
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