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 EC 50 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 EC 50 of the peptide (2.6 lM) 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). , an antimicrobial cathelicidin peptide of rabbits, resembled SMAP-29 in that it contained N-and C-terminal LPS-binding domains, had an EC 50 of 2.5 lM, 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.

Human beta-defensin-2 (HBD-2) is a member of the defensin family of antimicrobial peptides. HBD-2 was first isolated from inflamed skin where it is posited to participate in the killing of invasive bacteria and in the recruitment of cells of the adaptive immune response. Static light scattering and two-dimensional proton nuclear magnetic resonance spectroscopy have been used to assess the physical state and structure of HBD-2 in solution. At concentrations of < or = 2.4 mM, HBD-2 is monomeric. The structure is amphiphilic with a nonuniform surface distribution of positive charge and contains several key structural elements, including a triple-stranded, antiparallel beta-sheet with strands 2 and 3 in a beta-hairpin conformation. A beta-bulge in the second strand occurs at Gly28, a position conserved in the entire defensin family. In solution, HBD-2 exhibits an alpha-helical segment near the N-terminus that has not been previously ascribed to solution structures of alpha-defensins or to the beta-defensin BNBD-12. This novel structural element may be a factor contributing to the specific microbicidal or chemokine-like properties of HBD-2.

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.

Rate-limiting steps and transition state structure for the acylation stage of acetylcholinesterase-catalyzed hydrolysis of (acetylthio)choline have been characterized by measuring substrate and solvent isotope effects and viscosity effects on the bimolecular rate constant k E (=k cat/K m). Substrate and solvent isotope effects have been measured for wild-type enzymes from Torpedo californica, human and mouse, and for various active site mutants of these enzymes. Sizable solvent isotope effects, D 2 O k E ∼ 2, are observed when substrate β-deuterium isotope effects are most inverse, βD k E = 0.95; conversely, reactions that have D 2 O k E ∼ 1 have substrate isotope effects of βD k E = 1.00. Proton inventories of k E provide a quantitative measure of the contributions by the successive steps, diffusional encounter of substrate with the active site and consequent chemical catalysis, to rate limitation of the acylation stage of catalysis. For reactions that have the largest solvent isotope effects and most inverse substrate isotope effects, proton inventories are linear or nearly so, consistent with prominent rate limitation by a chemical step whose transition state is stabilized by a single proton bridge. Reactions that have smaller solvent isotope effects and less inverse substrate isotope effects have nonlinear and upward bulging proton inventories, consistent with partial rate limitations by both diffusional encounter and chemical catalysis. Curve fitting of such proton inventories provides a measure of the commitment to catalysis that is in agreement with the effect of solvent viscosity on k E and with the results of a double isotope effect measurement, wherein βD k E is measured in both H2O and D2O. The results of these various experiments not only provide a model for the structure of the acylation transition state but also establish the validity of solvent isotope effects as a tool for quantitative characterization of rate limitation for acetylcholinesterase catalysis.