Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both lowand high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an ␣-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.The rapidly expanding prevalence of bacterial strains resistant to conventional antibiotics has prompted a search for new therapeutic agents, including various antimicrobial peptides of animal origin (15). Two broad classes of mammalian antibacterial peptides have been especially well studied: the cysteinerich ␣-and ␤-defensins and various cathelicidins (6,13,22,26,27,41,42). Both classes are produced as precursors that require proteolytic processing to generate the mature antimicrobial peptide. Cathelicidins contain an N-terminal domain called cathelin, for which no function has yet been ascribed, and a C-terminal domain that comprises an antimicrobial peptide (reviewed in references 41 and 42). While the cathelin domains are highly conserved across species, the C-terminal antimicrobial domains are structurally diverse. The first cathelicidin precursor to be described was rabbit CAP18 (20), and its mature peptide was shown to have broad-spectrum bactericidal activity (19). Homologs of CAP18 have since been identified in other species including humans (FALL39/LL37) (1, 19), mice (mCRAMP) (12, 30), rats (rCRAMP), and sheep (SMAP29 and SMAP34...

Relationships between protein structure and ionization of carboxyl groups were investigated in 24 proteins of known structure and for which 115 aspartate and 97 glutamate pK(a) values are known. Mean pK(a) values for aspartates and glutamates are < or = 3.4 (+/-1.0) and 4.1 (+/-0.8), respectively. For aspartates, mean pK(a) values are 3.9 (+/-1.0) and 3.1 (+/-0.9) in acidic (pI < 5) and basic (pI > 8) proteins, respectively, while mean pK(a) values for glutamates are approximately 4.2 for acidic and basic proteins. Burial of carboxyl groups leads to dispersion in pK(a) values: pK(a) values for solvent-exposed groups show narrow distributions while values for buried groups range from < 2 to 6.7. Calculated electrostatic potentials at the carboxyl groups show modest correlations with experimental pK(a) values and these correlations are not improved by including simple surface-area-based terms to account for the effects of desolvation. Mean aspartate pK(a) values decrease with increasing numbers of hydrogen bonds but this is not observed at glutamates. Only 10 pK(a) values are > 5.5 and most are found in active sites or ligand-binding sites. These carboxyl groups are buried and usually accept no more than one hydrogen bond. Aspartates and glutamates at the N-termini of helices have mean pK(a) values of 2.8 (+/-0.5) and 3.4 (+/-0.6), respectively, about 0.6 units less than the overall mean values.

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.

The structures of partially-folded states appearing during the folding of a (βα) 8 TIM barrel protein, the indole-3-glycerol phosphate synthase from S. solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō-model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (βα) 4 region, modest protection in the neighboring (βα) 1-3 and (βα) 5 β 6 segments and no significant protection in the remaining N-and Cterminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (βα) 2-5 β 6 region after 5 s of folding demonstrates that these species represent kinetically-distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C α Gō-model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of structure offering protection against exchange in the on-pathway intermediate (s). Because the native-centric Gō-model simulations do not explicitly include sequencespecific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō-model simulation.

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