No abstract

An amphipathic model peptide, KLALKLALKALKAAKLA-NH2, and its complete double D-amino acid replacement set was used to analyze the process of peptide binding at lipid vesicles of different surface charge and to determine the structure of the lipid-bound peptides using CD spectroscopy. The relationship between peptide helicity, model membrane permeability, and biological activity has been studied by dye release from liposomes and investigation of antibacterial and hemolytic activity. The accumulation of cationic KLAL peptides at and the membrane-disturbing effect on bilayers of high negative surface charge were found to be dominated by charge interactions. Independent of any structural propensity, the cationic peptide side chains bind to the anionic phosphatidylglycerol moieties. The charge interactions hold the peptides at the bilayer surface, where they may disturb preferentially lipid headgroup organization by formation of peptide-lipid clusters. In contrast, KLAL peptide interaction with bilayers of low negative surface charge is highly dependent on peptide helicity. With decreasing amounts of anionic phosphatidylglycerol in the bilayer the membrane-disturbing effect of KLAL and other helical analogs substantially increases despite drastically reduced binding affinity. Less helical peptides exhibit reduced bilayer-disturbing activity, showing that the hydrophobic helix domain is decisive for binding at and inducing permeability in membranes of low negative surface charge. It is suggested that hydrophobic interactions drive the penetration of the amphipathic peptide structure into the inner membrane region, thus disturbing the arrangement of the lipid acyl chains and causing local disruption. On the basis of the proposed model for membrane disturbance, interactions modulating antibacterial and hemolytic activity are discussed.

Human lumbar CSF patterns of Ab peptides were analysed by urea-based b-amyloid sodium dodecyl sulphate polyacrylamide gel electrophoresis with western immunoblot (Ab-SDS-PAGE/immunoblot). A highly conserved pattern of carboxyterminally truncated Ab1-37/38/39 was found in addition to Ab1-40 and Ab1-42. Remarkably, Ab1-38 was present at a higher concentration than Ab1-42, being the second prominent Ab peptide species in CSF. Patients with Alzheimer's disease (AD, n ¼ 12) and patients with chronic inflammatory CNS disease (CID, n ¼ 10) were differentiated by unique CSF Ab peptide patterns from patients with other neuropsychiatric diseases (OND, n ¼ 37). This became evident only when we investigated the amount of Ab peptides relative to their total Ab peptide concentration (Ab1-x%, fractional Ab peptide pattern), which may reflect diseasespecific c-secretase activities. Remarkably, patients with AD and CID shared elevated Ab1-38% values, whereas otherwise the patterns were distinct, allowing separation of AD from CID or OND patients without overlap. The presence of one or two ApoE e4 alleles resulted in an overall reduction of CSF Ab peptides, which was pronounced for Ab1-42. The severity of dementia was significantly correlated to the fractional Ab peptide pattern but not to the absolute Ab peptide concentrations. Keywords: Alzheimer's disease (AD), b-amyloid protein precursor/metabolism, biological markers, cerebrospinal fluid, 2D-PAGE, western immunoblot.

Investigation of magainin II amide analogs with cationic charges ranging between +3 and +7 showed that enhancement of the peptide charge up to a threshold value of +5 and conservation of appropriate hydrophobic properties optimized the antimicrobial activity and selectivity. High selectivity was the result of both enhanced antimicrobial and reduced hemolytic activity. Charge increase beyond +5 with retention of other structural motifs led to a dramatic increase of hemolytic activity and loss of antimicrobial selectivity. Selectivity could be restored by reduction of the hydrophobicity of the hydrophobic helix surface (H hd ), a structural parameter not previously considered to modulate activity. Dye release experiments with lipid vesicles revealed that the potential of peptide charge to modulate membrane activity is limited: on highly negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol bilayers, reinforcement of electrostatic interactions had an activity-reducing effect. On neutral 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, the high activity was determined by H hd . H hd values above a certain threshold led to effective permeabilization of all lipid systems and even compensated for the activity-reducing effect of charge increase on highly negatively charged membranes. ß

The hydrophobicity (H), hydrophobic moment (|i) and the angle subtended by the positively charged helix face () of a set of model and magainin 2 amide peptides with conserved charge and helix propensity have been shown to be effective modulators of antibacterial and haemolytic activity. Except peptides of low hydrophobicity which are inactive, changing the parameters has little influence on the activity against Gramnegative bacteria, thus revealing the dominance of electrostatic interactions for the effect. However, the increase of H, |i and <5> substantially enhances haemolytic and Gram-positive antibacterial activity and is related to a reduction of peptide specificity for Gram-negative bacteria.

Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).

Arginine- and tryptophan-rich motifs have been identified in antimicrobial peptides with various secondary structures. We synthesized a set of linear hexapeptides derived from the sequence AcRRWWRF-NH(2) by substitution of tryptophan (W) by tyrosine (Y) or naphthylalanine (Nal) and by replacement of arginine (R) by lysine (K) to investigate the role of cationic charge and aromatic residues in membrane activity and selectivity. A second set of corresponding head-to-tail cyclic analogues was prepared to analyze the role of conformational constraints. The biological activity of the linear peptides followed the order Nal- >> W- > Y-containing compounds and slightly decreased upon R-K substitution. A pronounced activity-improving and bacterial selectivity-enhancing effect was found upon cyclization of the R- and W-bearing parent peptide, whereas the activity-modifying effect of cyclization of Y- and Nal-containing peptides was low. The analysis of the driving forces of peptide interaction with model membranes showed that the activities correlated with the partition coefficients and the depths of peptide insertion into neutral and negatively charged lipid bilayers. Spectroscopic studies, RP-HPLC, and titration calorimetry implied that the combination of cationic and aromatic amino acid composition and conformational rigidity afforded a membrane-active, amphipathic structure with a highly charged face opposed by a cluster of aromatic side chains. However, threshold values of low and high hydrophobicity seemed to exist beyond which the activity-enhancing effect of cyclization was negligible. The results suggest that cyclization of small peptides of an appropriate amino acid composition may serve as a promising strategy in the design of antimicrobial peptides.

The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an alpha-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic analogs for negatively charged membranes. Likewise, the peptide affinity to PC-rich membranes increases in the order of hydrophobicity. Correlation of peptide binding and membrane permeabilization of PC/PG (3:1) vesicles revealed that the observed differences in peptide activity on membranes of low negative surface charge are mainly caused by the different binding affinities. The antibacterial and hemolytic activity of the peptides increases with enhanced hydrophobicity. A strong correlation was found between the hemolytic effect and the bilayer-permeabilizing activity against PC-rich vesicles. Whereas the antibacterial specificity of the more hydrophobic analogs is retained for Escherichia coli, the specificity for Pseudomonas aeruginosa decreases with increasing hydrophobicity.