Synthesis, microbicidal activity, and solution structure of the dodecapeptide from bovine neutrophils

Abstract

The dodecapepetide sequence R–L–C–R–I–V–V–I–R–V–C–R with a disulfide bridge between the cysteine residues found in bovine neutrophils was synthesized by solid-phase procedures. Its antimicrobial activity against oral microorganisms such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus gordonii was examined, and its structural features were examined by CD and determined by two-dimensional (2D) nmr. The strains P. gingivalis (W50 and 381), A. actinomycetemcomitans (Y4 and 67), S. gordonii (DL1), and S. mutans (GS5) are found to be highly sensitive to this peptide at 2–2.5 μM concentrations, suggesting that the dodecapeptide is a potent antibiotic for oral pathogens. The weak negative n–σ* band observed at ∼265–270 nm in the CD spectra of this peptide provides evidence for the presence of a disulfide bridge. The negative n–π* band at ∼200 nm and the positive π–π* band at 185 nm suggest a folded structure for this peptide. The negative n–π* shifts from 200 to 206 nm with an increase in intensity in dipalmitoylphosphotidylcholine vesicles, suggesting that the peptide might associate to form higher order aggregates in lipid medium. The assignment of backbone and side-chain proton resonances has been accomplished by the combined analysis of 2D total correlated and nuclear Overhauser effect spectroscopy. The temperature dependence of amide NH chemical shifts and 1H–2H exchange effect on amide NH resonances indicate the involvement of amide NH groups of Cys3, Ile5, Ile8, Val10, and Arg12 in intramolecular hydrogen bonding. The coupling constant (J) values, the set of medium-, short-, and long-range nuclear Overhauser effects, and the results of restrained structure calculation using the distance geometry algorithm for nmr applications provide evidence for a folded, loop-like structure with a type I (III) β-turn involving Ile5, Val6, Val7, and Ile8, and two antiparallel β-strands involving the N-terminal Arg1, Leu2, Cys3, and Val4 and the C-terminal Arg9, Val10, Cys11, and Arg12 residues. The structure of the dodecapeptide mimics the amphiphilic structure of large 30–35 residue defensins and the peptide appears to exhibit similar antimicrobial potency. © 2000 John Wiley & Sons, Inc. Biopoly 53: 281–292, 2000