In this paper, we set out to discover peptides that disturb membranes in a pH-dependent manner by screening an M13 12-mer filamentous phage library, Ph.D.-12, for pH-dependent liposome binding. Two different liposome types were used: one that represents the aliphatic lipid bilayer core, POPC MLVs, and the second mimicking the endosomal membrane, ELM MLVs. In this context, we have identified 19 distinct peptide sequences, of which three peptides PC1, PC2, and PC4, are able to induce pH-dependent calcein leakage to a similar extent as that of the positive control peptides SFP3 and INF7* ().
In contrast to peptides identified from phage screens against receptors or antibodies (50
), BLAST analysis showed that our panel of active peptides do not share significant homology with any proteins described. Instead, our peptides have varied sequences without an obvious consensus motif. This lack of a consensus sequence is unsurprising, since secondary (12
) and multi-dimensional (44
) homologies seem to be more important in defining membrane activity than their primary sequence.
On the amino acid level, compositions of the discovered peptides reflect the nature of the lipids against which the screen has been conducted, aromatic groups against isoelectric POPC liposomes, and basic and less hydrophobic residues against negatively charged ELM liposomes. We have also enriched for histidines, a titratable amino acid in the pH-range of the screen.
Against our expectations, aliphatic amino acids (with the exception of PC2) have not been enriched in our screens. Aromatics, on the other hand, were highly enriched in our POPC screen. Two of the membrane-active peptides — namely PC1 and PC4 — have a striking number of tryptophans. In contrast, trypthophan occurred only once in the 13 sequences discovered in the ELM screen and was completely absent from all the inactive peptides isolated from the POPC screen ().
When peptide activity was determined in vitro
using the calcein leakage assay (26
), we found that PC1, PC2 and PC4 were active both on electroneutral POPC as well as negatively charged ELM liposomes. These results imply that the calcein leakage activity of peptides is not dependent on ionic interaction with the lipid headgroups, but is instead governed by the ability of the peptides to interact with the hydrophobic core of the lipid bilayer.
For PC1 and PC4, it is likely that the multiple tryptophans play an important role in this putative interfacial insertion into the bilayer. According to the Wimley-White interfacial hydrophobicity scale (51
), aromatic and hydrophobic aliphatic residues (with the exception of valine) favor partitioning into a lipid bilayer (). Tryptophan not only favors partitioning into bilayers more than aliphatics, but it is — unlike, for example, leucine — energetically undesirable to be located in the center of a transmembrane domain (16
). The enrichment of tryptophans in the membrane-proximal domain has been implicated in the membrane fusogenic activity of viral fusion proteins (53
). The reason for the partial bilayer insertion of PC2, on the other hand, probably lies in its tendency to form an amphipathic helix as seen in classical viral fusion peptides (54
). In a helical conformation, the acidic glutamates and the hydrophobic amino acids (leucine, isoleucine and methionine) of PC2 lie on opposite faces of the helix.
Another factor that can influence both membrane activity of peptides as well as the angle of peptide insertion into membranes is peptide length. Peptide length is often cited as a reason for diminished membrane activity of shorter peptides. While there are 6-mers (55
) that have been shown to be membrane-active and 8 amino acid peptides are reported to interact stably with membranes (56
), the membrane-active peptides studied in most detail are helices of a length of approximately 20 amino acids. One particularly well-studied peptide is the N-terminus of the influenza fusion protein hemagglutinin. In this model, using a host-guest system, increased peptide length increases both the depth and angle of membrane insertion as well contents leakage from red blood cells (57
). Given this information, it is remarkable that the 13 amino-acid-long peptides in our study enable contents leakage to a similar extent as the 24 amino-acid-long influenza fusion peptide analog INF7* (), which has been optimized for its lytic activity (28
Together these results suggest that the propensity of the membrane-active peptides to insert shallowly into the membrane is important for their lytic activity. The ability to penetrate deep into the hydrophobic core, on the other hand, may be less significant. The data are indeed consistent with a model in which a shallow interaction with the hydrophobic core of a bilayer is sufficient to induce content leakage.
Most membrane-interacting synthetic peptides are more active as dimers (58
) and oligomers (60
) or when anchored to a membrane (40
). This principle also holds true for two of our leakage active peptides, PC2 and especially PC1 (). PC1 conjugated to poly(L-Lysine) by disulfide bonds, a multimeric presentation, is considerably more active in the calcein leakage assay than when the peptide was released from the oligomer in presence of 1 mM DTT.
As stated in the introduction, membrane binding is obligatory, though not necessarily sufficient, for a peptide to be membrane-active. True to this prediction, peptides identified in our screen that lost the ability to bind stably to membranes are not lytic. The fact that in the context of the phage the peptides are both C-terminally anchored to a protein as well as being displayed in a pentameric form likely influences their ability to interact with membranes. Especially, because oligomeric presentation of the peptides almost certainly increases avidity, it is not surprising that some peptides — PC3, PC5 and PC6 as well as ELM1, ELM2 and ELM3 — lost the ability to bind stably to liposomes in their monomeric form.
PC1 binds to both POPC and ELM liposomes. This is not unexpected since, as stated above, the interaction of PC1 with membranes is less likely to be influenced by the surface charge, but more likely by insertion of the peptide into the aliphatic core of the bilayer. Interestingly, the membrane-active peptides PC1, PC2 and PC4 bind to MLVs at both pHs. This appears to conflict both with the phage screen, which was based on the elution of the phages at pH 7.5, as well as the calcein leakage results. The simplest explanation for this behavior lies in the different natures of the liposome-binding assay and the phage screen and calcein leakage assay. In the phage screen, the eluted phages are amplified in each round of screening. Even if only a small amount of bound phage is eluted at pH 7.5, the bulk of the phage remaining bound to MLVs, it will be amplified and thus recovered in the screen. The liposome-binding assay, on the other hand, measures binding of the bulk of peptides.
Despite the pH-independent binding of the peptides to MLVs, the lytic activity of all active peptides is strongly pH-dependent. Apparently, the lytic activity of our peptides is influenced by additional factors — such as pH-dependent structural changes — not just binding to liposomes. Alternatively, the reason for the difference between binding and lytic activity might lie in the different peptide and lipid concentrations used in the two assays. Because of the detection limit in the liposome binding assay, both the lipid and the peptide concentrations were substantially higher in the binding than in the leakage assay.
The secondary structure of peptides has been instrumental in further understanding the interaction peptides with membranes. Many of the membrane-interacting components of viruses form amphipathic helices (12
). In line with these observations, we find that two of our peptides, PC2 and PC4, despite their short length, show helical propensities in 50% TFE (); although taking into account PC4′s highly aromatic amino acid composition, the “helical” structure must be confirmed by other methods (49
). The helicity of PC2 in aqueous solution shows a pH- and lipid-dependent increase, reminiscent of the conformational change and the resulting deeper membrane insertion of influenza fusion domain in acidic pH (61
). Thus, a model in which this change in helicity plays a role in the lytic activity of PC2 is particularly attractive.
The fact that PC4 tends to aggregate in solution might also play an important role in the leakage potential of this peptide. Interestingly, aggregation of PC4 in solution was not strongly dependent on pH, in contrast to the pH-dependent leakage induced by PC4. This disparity suggests that differences exist in peptide organization in the absence or presence of membranes. These results are consistent with other studies showing that peptide assembly on membrane templates and aggregation has an important role in membrane activity (57
PC4 is of further interest because it possesses pH-dependent lytic activity in the absence of any titratable groups. One possible reason for the pH-dependency of PC4 is the presence of two prolines. Prolines can confer pH sensitivity by isomerizing in a pH-dependent manner (62
). The possible importance of prolines for the pH-dependent lytic activity of PC4 is highlighted by the fact that the probability of the double prolines at position 9 and 10 to occur by random chance in our screen is less than 10-7
(see Materials and Methods). This indicates that these prolines are a result of the selective pressure in this screen for pH-dependent phage binding to the membranes. These findings resonate with the fact that centrally located prolines (63
), and their ability to change between secondary structures (55
), play an integral role in pH-dependent membrane destabilization induced by other peptides.
Our work demonstrates that peptides that are membrane-active in a pH-dependent fashion can be identified by phage-display. The study also demonstrates that the choice of lipids used in the screen is important in selecting for activity. When the screen is performed with electroneutral PC liposomes that mimic the aliphatic core, 50% of the sequences discovered on the screen produced pH-dependent lytic peptides. Furthermore, membrane-active peptides — as opposed to their inactive counterparts — contain a remarkably high content of tryptophan, reminiscent of antimicrobial peptides (66
), suggesting a crucial role of this amino acid in the lytic behavior of these peptides.
Future studies aimed at deciphering the mechanism of membrane activity will help us to put these newly identified peptides into the context of both naturally occurring, as well as synthetic lytic peptides. The present approach may also open new avenues to gain a deeper understanding of peptide-lipid interactions and their role in cell biological and pathological processes, with the potential of yielding new strategies for therapeutic applications.