Melittin, the principal toxic component of bee venom, is a cationic, amphipathic, linear peptide composed of 26 amino acids, which exhibits unique structural and biological characteristics. It has high antimicrobial activity but also has the very detrimental property of killing eucaryotic cells, as illustrated by the lysis of sheep red cells. Several attempts have been made through synthesis of replacement analogs to advance the molecular understanding of the cause of these effects. We have now synthesized retro melittin, an amphipathic α-helical analog with reversal of sequence and therefore of the positions of charged and apolar residues, notably, the cluster of basic residues Lys21-Arg-Lys-Arg24 near the C-terminus which is now located at positions 3−6 near the amino terminus. This peptide retained high antimicrobial activity against a range of test bacteria, but lost much of its hemolytic properties. Modification of the N-terminal positive charge by acetylation did not further alter the antibacterial activity or red cell lysis. The synthetic retroenantio melittin (all-d isomer) and its acetylated derivative both retained full antibacterial activity, but with complete elimination of the hemolytic effect. Therefore, the two effects of melittin have been separated. Melittin and these analogs promote electrical conductivity in lipid bilayers. Circular dichroism measurements showed that all of these peptidesnormal, enantio, retro, retroenantio, and their acetylated derivativeswere 80−100% helical in 12−20% hexafluoro-2-propanol, a structure inducing solvent, and they are thought to be helical in lipid bilayers and bacterial membranes. Nonhelical analogs are inactive. It is believed that the helix dipole plays a major part in orienting the peptides in membranes. Active sequences are not unique, but sequence plays a role in peptide conformation and activity. Chirality has virtually no role in the antibacterial activity of normal and retro melittin analogs, which leads to the conclusion that these peptides do not function via a receptor or by enzymatic processing, but by self-aggregation and formation of ion-conducting pores.
The basis for wavelength regulation in bacteriorhodopsin (BR) and retinylidene proteins in general has been studied for decades but is still only partially understood. Here we report the preparation and spectroscopic characterization of BR analogs aimed at investigating the existence of spectral tuning mechanisms other than the two widely accepted mechanisms, weakened counterion interactions and ring/chain coplanarization. We synthesized two novel retinal analogs containing a saturated 13-14 bond, which interrupts the interaction of the protein counterions with the chromophore conjugation system. Furthermore, one of the analogs has a planar polyene system so that the contribution to the red shift of BR by retinal ring/chain coplanarization is also absent. We incorporated these analogs into bacterioopsin and discovered a sizable amount of red shift, which can be accounted for by interactions between the polar or polarizable groups of the protein and the retinal polyene chain. Our results suggest that the wavelength regulation in BR is achieved by synergistic chromophore/protein interactions including ring/ chain coplanarization, excited state stabilization by polar or polarizable protein side chains located along the polyene chain, and weakened counterion interactions near the Schiff base positive charge. Bacterioopsin (BO)1 tunes the absorption maximum ( max ) of a retinal protonated Schiff base (PSB) from 440 nm in methanol to 568 nm in bacteriorhodopsin (BR). The mechanism of wavelength regulation in BR and in retinylidene proteins in general has been a subject of intensive investigation. Nakanishi et al.(1) introduced the term opsin shift (OS) to refer to the energy difference (in cm Ϫ1
The design of cecropin-melittin hybrid analogues is of interest due to the similarities in the structure of the antimicrobial peptides cecropin and melittin but differences in their lytic properties. We suspected that a hydrophobic residue in position 2 of milittin (Ile8 in the hybrid) plays an important role in the activity of the 15-residue hybrid, KWKLFKKIGAVLKVL-NH2, [CA(1-7)M(2-9)NH2] and have now examined its role in the analogue toward five test bacteria. Deletion of Ile8 reduced activity, and it was not restored by lengthening to 15 residues by addition of another threonine at the C-terminus. Replacement of Ile8 by a hydrophobic leucine maintained good activity and Ala8 was equally active for four organisms, although less active against Staphylococcus aureus. Replacement by the hydrophilic Ser8 strongly reduced potency against all five organisms. Deletion of Leu15 decreased activity, but addition of Thr16 maintained good activity. The presence of hydrophobic residues appears to have a significant effect on the process of antibacterial activity. These peptide analogues showed voltage-dependent conductance changes and are capable of forming ion-pores in planar lipid bilayers. The antibacterial action of the peptides is thought to be first an ionic interaction with the anionic phosphate groups of the membrane followed by interaction with the hydrocarbon core of the membrane and subsequent reorientation into amphipathic alpha-helical peptides that form pores (ion-channels), which span the membrane. The analogue also showed an increase in alpha-helicity with an increase in hexafluoro 2-propanol concentration.
Dedicated to Professor Koji Nakanishi on the occasion of his 70th birthday. Two antimicrobial peptides, cecropin P1 (CPl), with a C‐terminal carboxyl group, and PR‐39, with an amidated C‐terminus, are found in the small intestine of the pig. Each is active against both Gram‐positive and Gram‐negative bacteria. We have synthesized these peptides and several analogs, including the d‐enantiomers and the retro sequences, each with a free or acetylated amino terminus. The CPI amide was also prepared. The retro CP1 peptides were much less active than the parent CPl peptide, confirming the importance of sequence or the amide bond and helix dipole direction, and the Nα‐acetyl peptides were also less active, indicating that a free amino terminus is essential for high activity. The ratio of the lethal concentration of L/D isomers of CP1 is less than 1 for Gram‐negative, but greater than 1 for Gram‐positive bacteria. PR‐39 showed no significant chiral selectivity toward Escherichia coli, Bacillus subtilis and Streptococcus pyogenes, but the l/d ratio was high for Pseudomonas aeruginosa (66), and very high for Staphylococcus aureus (>1000). In the latter case the lethal concentration for the d‐isomer was 0.57 μ whereas this organism was quite resistant to the l‐isomer (>600μ). Thus the enantiomers of CP1 and PR‐39 are not equally active for all species. In a plate assay with a very small log‐phase inoculum of Staph. aureus, D‐PR‐39 produced a clear zone of killing surrounded by a zone of stimulated growth. After prolonged incubation the two zones became one clear zone. Addition of D‐PR‐39 to the wells of a dense turbid plate of growing cells showed a cleared zone for each of the test organisms, indicating that PR‐39 lyses the bacteria rather than simply inhibiting their multiplication. © Munksgaard 1997.
In our effort to understand the structural requirements for the antimicrobial activity of cecropin A (CA) and melittin (M), we synthesized the normal, enantio, retro and retroenantio hybrid analogs; we related activity to their sequence, chirality, amide bond direction (helix dipole) and end group charges. To compare the effect of the end groups, each of these analogs was synthesized both with an acid and an amide C‐terminus and also with and without an Nα‐acetyl N‐terminus. The all‐l‐ and all‐d‐enantiomers of several cecropin‐melittin hybrids were previously found to be equally potent against several bacterial species, and no chiral effect was observed. This general rule has now been confirmed and extended. However, two exceptions have been found. All‐l‐CA(1‐13)M(1‐13) acid was 5 times and 9 times less potent than the all‐d‐analog, respectively, toward Gram‐positive Staphylococcus aureus and Gram‐negative Pseudomonas aeruginosa. A11‐l‐CA(1‐7)M(2‐9) acid was 5 times and 14 times less active against S. aureus and P. aeruginosa, respectively, than its all‐d acid isomer. The corresponding d‐ and l‐retro analogs differed only marginally. A role for proteolytic enzymes has been implicated as a cause for these differences in the activities of l‐ and d‐enantiomers. In all cases, blocking the α‐amine by acetylation had no significant effect on potency. The retro and retroenantio analogs of CA(1‐13)M(1‐13) acid were as potent as their normal and enantio analogs against all the test bacteria. The C‐terminal amides also showed similar potency against four test bacteria. It should be noted that the negative end of the helix dipole of a normal peptide points toward the C‐terminus, whereas it points away in the case of a retro derivative when viewed in the direction of the normal sequence.
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