Studies on the mechanism of aspartic acid cleavage and glutamine deamidation in the acidic degradation of glucagon.

In this study, the polypeptide hormone glucagon was used as a model to investigate the mechanisms of aspartic acid cleavage and glutaminyl deamidation in acidic aqueous solutions. Kinetic studies have shown that cleavage at Asp-21 occurred at significantly slower rates than at Asp-9 and Asp-15 while deamidation rates were similar at the three Gln residues. The role of side-chain ionization in the cleavage mechanism was investigated by determining the pK(a) values of the three Asp residues using TOCSY and NOESY NMR methods. The role of proton transfer was investigated using kinetic solvent isotope effect studies (KSIE). The pK(a) values for the sidechains of Asp-9, Asp-15, and Asp-21 were found to be 3.69, 3.72, and 4.05 respectively. No kinetic solvent isotope effect was observed for the cleavage reaction whereas an inverse effect was observed for deamidation. Based on the lack of sequence effects, pH-rate behavior, and KSIE, the deamidation mechanism was proposed to involve direct hydrolysis of the amide side-chain by water. Based on substrate ionization, pH-rate profiles, and KSIE, the proposed mechanism for Asp cleavage involved nucleophilic attack of the ionized side-chain carboxylate on the protonated carbonyl carbon of the peptide bond to give a cyclic anhydride intermediate.

Impact of single-residue mutations on the structure and function of ovispirin/novispirin antimicrobial peptides.

We studied three model antibacterial peptides that resembled the N-terminal 18 amino acids of SMAP-29, an alpha-helical, antimicrobial peptide of sheep. Although the parent compound, ovispirin-1 (KNLRR IIRKI IHIIK KYG), was potently antimicrobial, it was also highly cytotoxic to human epithelial cells and hemolytic for human erythrocytes. Single residue substitutions to ovispirin-1 yielded two substantially less cytotoxic peptides (novispirins), with intact antimicrobial properties. One of these, novispirin G-10, differed from ovispirin-1 only by containing glycine at position 10, instead of isoleucine. The other, novispirin T-7, contained threonine instead of isoleucine at position 7. We determined the three-dimensional solution structures of all three peptides by circular dichroism spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy. Although all retained an amphipathic helical structure in 2,2,2-trifluoroethanol, they manifested subtle fine-structural changes that evidently impacted their activities greatly. These findings show that simple structural modifications can 'fine-tune' an antimicrobial peptide to minimize unwanted cytotoxicity while retaining its desired activity.

SMAP-29 has two LPS-binding sites and a central hinge.

The CD spectra of SMAP-29, an antimicrobial peptide from sheep, showed disordered structure in aqueous buffers, and significant helicity in membrane-like environments, including SDS micelles, lipopolysaccharide (LPS) dispersions, and trifluoroethanol buffer systems. A structure determined by NMR in 40% perdeuterated trifluoroethanol indicated that residues 8-17 were helical, residues 18-19 formed a hinge, and residues 20-28 formed an ordered, hydrophobic segment. SMAP-29 was flexible in 40% trifluoroethanol, forming two sets of conformers that differed in the relative orientation of the N-terminal domain. We used a chromogenic Limulus assay to determine the EC50 of the peptide (the concentration that bound 50% of the added LPS). Studies with full-length and truncated SMAP-29 molecules revealed that each end of the holopeptide contained an LPS-binding domain. The higher affinity LPS-binding domain was situated in the flexible N-terminal portion. LPS binding to full-length SMAP-29 showed positive cooperativity, so the EC50 of the peptide (2.6 microm) was considerably lower than that of the individual LPS-binding domains. LPS-binding studies with a mixture of truncated peptides revealed that this cooperativity was primarily intramolecular (i.e. involving the N- and C-terminal LPS-binding sites of the same peptide molecule). CAP-18[106 -142], an antimicrobial cathelicidin peptide of rabbits, resembled SMAP-29 in that it contained N- and C-terminal LPS-binding domains, had an EC50 of 2.5 microm, and bound LPS with positive cooperativity. We conclude that the presence of multiple binding sites that function cooperatively allow peptides such as SMAP-29 and CAP-18 to bind LPS with high affinity.