Abstract
In this chapter the mechanism of action of the peptide trichogin GAIV (GAIV henceforth) will be investigated.
1 Membrane Thickness and the Mechanism of Action of Trichogin GAIV
1.1 Trichogin GAIV
In this chapter the mechanism of action of the peptide trichogin GAIV (GAIV henceforth) will be investigated.
GAIV belongs to the family of peptaibols (or peptaibiotics), i.e. AMPs produced by the Trichoderma fungi. This class of pore-forming, bactericidal peptides, was actually discovered well before cationic AMPs [136]. They are produced non-ribosomally by fungi, are characterized by a C-terminal 1, 2-amino alcohol and a high content of non proteinogenic residues, most notably α-aminoisobutyric acid (Aib), and are usually acetylated or acylated at the N-terminus. Peptaibiotics are usually helical both in solution and when membrane-bound, and, unlike cationic AMPs, their content of charged residues is very low or even absent altogether. The best characterized member of this family (and the second to be identified, in 1967) is alamethicin (Alm) [76]. For this peptide it has been conclusively demonstrated through a combination of a large set of biophysical techniques that it forms pores through the “barrel-stave” mechanism [8, 19, 22, 36, 42, 74, 126]. This model was first proposed in 1974, but until now it has been convincingly demonstrated only for Alm.
Alm comprises 19 amino acids and its helix has a length corresponding almost exactly to the thickness of biological membranes (Fig. 4.1). However, it is also one of the longest members of the peptaibiotic family, which contains peptides going from 21 amino acids (SCH 643432) to just 4 (peptaibolin) [136]. Considering the closely related amino acid composition and physico-chemical properties of all members of the peptaibiotic family, it is conceivable that they could form pores in a similar way. However, an important drawback seems to preclude the formation of transmembrane pores to shorter peptides: how could such short helices form channels spanning through the whole bilayer?
Comparison of the length of the Alm and GAIV helices with the thickness of a POPC bilayer. Phosphorus, nitrogen and oxygen atoms of phospholipids are shown as gold, blue and red spheres, respectively, while water oxygen atoms are colored in pink. The acyl chains of the phospholipids are omitted for the sake of clarity. Peptides are represented as ribbons. The N-terminal acyl and C-terminal amino alcohol groups are shown in a “stick” representation [Reproduced from [15] with permission]
A well characterized, medium-length peptaibiotic is GAIV, whose sequence is
where nOct is n-octanoyl, and Lol is leucinol. This 10-mer peptide was isolated from Trichoderma longibrachiatum in 1992 [6] and since then it was studied both in solution and in model membranes by a number of physico-chemical techniques [108] including NMR [6, 54], X-ray crystallography [137], EPR [88–90, 114, 130, 131] fluorescence [45, 86, 126, 140–142] electrochemistry [10, 123], and molecular dynamics (MD) simulations [17]. Its 3D-structure is helical, with a flexible hinge in the central part, formed by two consecutive Gly residues [6, 137, 141, 138]. In this conformation, its length is only about half the normal thickness of a biological membrane (Fig. 4.1).
For this reason, different models were proposed to explain its pore-forming activity, including the SMH mechanism [38, 54, 90] and a carrier function [88, 89]. However, several evidences would favor a barrel-stave structure for its pores, just like Alm. In this connection, by combining several different fluorescence experiments it has been shown that, above a threshold concentration, GAIV inserts deeply into the hydrophobic core of the membrane and forms aggregates. These inserted, aggregated species are responsible for membrane leakage [45, 85, 126] Later on, these findings were confirmed by EPR measurements [114, 129, 130]. Other studies established that GAIV-induced membrane permeability is ion-selective and depends on the sign and magnitude of the transmembrane potential, exactly like that of Alm channels [10, 68, 122].
One hypothesis that was put forward to solve the apparent contradiction between these data and the short length of the GAIV helix is formation of head-to-head dimers able to span the entire bilayer [130, 137]. However, this hypothesis seems not to be supported by the voltage dependence of GAIV-induced membrane permeability, which would require a co-linear alignment of the helices forming the channel, to generate an overall dipole that could sense the transbilayer potential [10, 68] In a previous study [17] GAIV location inside a lipid bilayer was investigated by MD simulations using a self-assembling approach [20, 39] that has been defined as “minimum bias” [18, 99]. In this method, the simulation starts from a random mixture of lipids and water, containing one peptide molecule. During the simulation, a lipid bilayer self-assembles spontaneously, but in the initially highly fluid environment the peptide is able to attain its most favorable position in the membrane. This study [17] indicated a possible solution to the problem of the mismatch between membrane thickness and peptide length. In two out of three such simulations with GAIV, the peptide positioned close to the membrane surface, parallel to it, without significantly perturbing the membrane. However, in a third simulation it inserted into the bilayer in a transmembrane orientation. This simulation showed a bilayer that, near the peptide, was significantly thinned, so that the short GAIV helix was able to span completely the membrane. Nevertheless, this preliminary result could not rule out the possibility that the observed effect was an artifact of the way the bilayer formed, since in this simulation GAIV was initially inserted in a local defect of the membrane, which healed only after an extensive simulation. The short length of the equilibrated segment of that simulation (10 ns) left open the possibility that the observed bilayer structure was just a transient, metastable state, which would eventually relax. More importantly, this purely computational but intriguing indication needed an experimental verification.
In this chapter, combined experimental and simulative data will be reported, supporting the possibility for GAIV to form barrel-stave channels. Neutron reflectivity studies were used to experimentally verify the effects of GAIV on bilayer thickness. Moreover, vesicle leakage experiments were exploited to determine the influence of membrane thickness on GAIV activity. In addition, previous MD simulation studies were significantly extended, to confirm the stability of the transmembrane orientation, even when starting from a preformed bilayer, and to verify the cumulative effects of multiple membrane-inserted peptide chains. Overall, these data indicate that GAIV might be able to form barrel-stave channels by causing a significant thinning of the bilayer to a thickness comparable with the length of its helix (Fig. 4.1).
Schematic representation of the bilayer model used for data analysis
1.2 Neutron Reflectivity Experiments
Specular neutron reflectivity measurements were carried out on planar POPC bilayers supported by a silicon crystal and submerged in a water phase, to which increasing GAIV concentrations were added. To increase the reflectivity contrast, in a first set of experiments, fully hydrogenated peptide was added to a chain-deuterated POPC bilayer (Fig. 4.3), while in a second set, partially deuterated GAIV (Fig. 4.4) was added to a normal POPC bilayer (Fig. 4.3) (See Table 4.1). Four different concentrations of peptide were used, namely C0 (bare lipid bilayer), C1 (4.5 μM), C2 (15 μM), and C3 (30 μM). All of the peptide-lipids-solvent combinations exploited are reported in Table 4.1. The data analysis has been carried out dividing the membrane into three sublayers, i.e. the outer headgroups region, the tails region, and the inner headgroup region. A further region is represented by the thin SiO2 layer that covers the silicon support, with its hydration water (Fig. 4.2).
Structures of hydrogenated (top) and deuterated (bottom) POPC
Structure of the deuterated GAIV
Each layer is characterized by defined values of SLD, thickness (d), and roughness (σ). The SLD of each layer will provide information about the species present in the considered region.
The cited parameters are used to calculate a model for reflectivity profile. The theoretical profile obtained by this model is then compared to the experimental one, and the quality of the fit is assessed calculating the χ2 parameter in the least-squares method (as described in Sects. 3.11 and 3.12). The SLD and d for each layer can be varied until the optimum fit is found. All the reflectivity profiles obtained for the same peptide concentration under different contrast conditions were fitted simultaneously.
1.2.1 Bare Substrates Characterization
Two different Si substrates were used for the POPC and (d31)POPC bilayers. Before membrane deposition, these substrates were characterized using three different water compositions (Table 4.1). For that used in combination with hydrogenated POPC, the dioxide layer was characterized by a thickness t ox = 15.8 ± 0.1 Å, a volume fraction of hydrating water f ox = 0.15 ± 0.05 and a roughness σ = 2.2 ± 0.2 Å. The block used for supporting deuterated lipids was characterized by the parameters tox = 15.5 ± 0.1 Å, f ox = 0.12 ± 0.05, and σ = 2.4 ± 0.3 Å. These values were kept fixed during the modeling of the sample data.
1.2.2 Bare POPC Bilayer Characterization
The simultaneous fits on all of the available datasets for a pure lipid bilayer are shown in Fig. 4.5. The parameters obtained are in complete agreement with those already reported in the literature [69]. In both cases, the parameters of the pure lipid bilayer were indicative of a symmetric bilayer, with a total thickness of 48 ± 3 Å. The inner and outer headgroup regions were 10 Å thick (t h = 10 ± 1 Å), with a volume fraction of water f h = 0.5 ± 0.1. The hydrophobic core of the bilayer was 28 Å thick (2xt t = 28 ± 1 Å) with no water penetration, which suggests an almost perfect coverage of the substrate surface [28]. The roughness of all interfaces was similar and close to 2 Å. From these values the SLD profiles along the normal of the bilayer were evaluated (Fig. 4.5, bottom panel).
Panel A: neutron reflectivity profiles for POPC and (d31)POPC bilayers in different media. From top to bottom POPC in H2O (violet), D2O (red), SMW (green) and (d31)POPC in H2O (violet), D2O (red) and SMW (green). Global fits to these data are reported as solid or dotted lines, for (d31)POPC and POPC data, respectively. The curves have been rescaled to improve the visibility. Panel B: SLD profiles for POPC and (d31) POPC bilayers in different media. The color code is the same as in panel A [Reproduced from [15] with permission]
1.2.3 Peptide Effects
In Fig. 4.6 the curves of the POPC bilayer in D2O at the different concentrations of deuterated peptide investigated are compared. Two main features are visible. From the pure lipid bilayer to the C3 sample a decrease in reflectivity in the mid-Q region (0.05–0.15 Å−1) is observed that could be interpreted as a change of contrast between the sample and the solvent. Indeed, the addition of deuterated peptide molecules into a hydrogenated bilayer would lead to a total SLD closer to that of D2O. It has to be reminded that the contrast is the difference between the SLD of the sample and that of the medium. The lower is this value, the weaker is the reflectivity (or scattering) signal.
Reflectivity profiles of POPC in D2O. Violet no peptide; Blue C1; Green C2, Red C3 [Reproduced from [15] with permission]
The second feature clearly visible is a shift of the main minimum of the profiles towards higher Qz values. For the pure POPC bilayer it was located around Qz = 0.20 Å−1 and it shifted to higher values after peptide insertion. This is a clear indication of thinning of the overall thickness of the deposition.
From the simultaneous fits according to the Parrat’s recursive formula, the sample at the four different peptide concentrations was characterized in a clear and unambiguous way. The resulting parameters are listed in Table 4.2. From these parameters we were able to determine the main structural changes induced by the peptide inclusion into the lipid bilayer. First of all, the peptide is present only in the hydrophobic layer, as described by the volume fractions f ph and f pt , representing the volume fraction of peptide molecules occurring in the headgroup and tail regions, respectively. f ph is zero for all of the investigated samples. Instead, the quantity of peptide inserting into the hydrophobic layer is clearly concentration dependent. From the peptide volume fraction f pt , and from the molecular volumes of GAIV and of the lipid tails the inserted peptide to lipid ratio at the different concentrations (N pt /N l ) has been evaluated. Unfortunately, within the experimental accuracy, it was not possible to distinguish whether the peptide molecules are located in a specific part of the tail region or with a specific orientation with respect to the bilayer normal z.
The second important information arising from the modeling is that the inclusion of the peptide produces a thinning of the bilayer, especially because of a thickness decrease of the tail region. Actually, within the experimental accuracy, the headgroup thickness t h is stable at all concentrations, while a decrease is observed in the t t parameter (t t is the thickness of the hydrophobic region of a single leaflet). The overall hydrophobic region is “compressed” by ~7 Å going from the pure lipid system to that with the highest amount of peptide included. This thinning affects the overall bilayer thickness, decreasing form 48 ± 3 to 39 ± 3 Å (Fig. 4.7). All of these structural changes are detectable also from a comparison of the SLD profiles, as shown in Fig. 4.8.
Hydrophobic thickness versus peptide concentration. The blue line represents GAIV length [15]
1.3 Molecular Dynamics Simulations
In order to understand at the molecular level the structural changes observed in the bilayer in the NR experiments, MD simulations of GAIV-membrane systems were performed in the research group where this thesis was carried out. In a previous computational study [17] it has been shown that when the peptide is associated to the membrane parallel to its surface it does not cause any significant bilayer perturbation. Therefore, in the present study the stability of a transmembrane peptide orientation and its effects on the bilayer has been investigated.
The peptide was inserted in the bilayer according to a protocol developed specifically for this purpose [15, 16]. After GAIV insertion and an appropriate 10 ns equilibration period (during which the peptide was position-restrained), the simulations were continued for 100 ns more. A total of 7 simulations was performed: in one of them, only one peptide molecule was inserted in the bilayer (comprising 128 POPC lipid molecules), in three other simulations 4 peptide molecules were inserted, and in further three simulations the peptides in the bilayer were 8. In most cases, the peptide molecules maintained a transmembrane orientation for the whole trajectory, and quickly (i.e. in about 10–30 ns) some lipid headgroups in the region above and below the peptide were drawn deeper in the membrane, due to the interaction with the free peptide NH and CO groups at the two termini of the helices. A few structures representative of the time-evolution of the simulations are shown in Fig. 4.9, and the structures at the end of the simulations are illustrated in Fig. 4.10.
Snapshots showing the evolution of the structures during the simulation with four GAIV molecules [Reproduced from [15] with permission]
Final structures obtained from simulation with, 1, 4 or 8 peptide molecules [Reproduced from [15] with permission]
To better define the interactions responsible for the observed bilayer deformation an analysis was carried out of the interactions of the peptide NH and CO groups not involved in intramolecular H-bonds, and of the OH group of the C-terminal amino alcohol, with different parts of the lipid molecules or with water. This analysis was limited to the trajectory segment following the formation of the bilayer thinning, i.e. from 30 ns onward (Fig. 4.11). As expected, the NH groups at the N-terminus interact mainly with the oxygen atoms of the phosphate and glycerol groups, while the C-terminal CO groups were almost invariably associated with the quaternary ammonium of the lipid choline group. The OH group of Lol interacts mostly with the glycerol moiety. All three peptide groups (NH, CO and OH) also interact with water molecules. These data indicate that the bilayer deformation is driven by electrostatic and H-bonding interactions, which are enhanced in the low-dielectric constant environment of the hydrophobic membrane core. These interactions lead to thinning by drawing phospholipid headgroups deep into the membrane, thus inducing an increase in the number of gauche conformations in the lipid tails, as shown by a progressive decrease in their order parameters, as the number of peptides in the bilayer increases (Fig. 4.12).
Statistics of the interactions of selected peptide groups during the equilibrated segments of the MD trajectories with 4 GAIV molecules (30–110 ns). For each trajectory frame, the lipid or water atom closest to the different peptide groups (N-terminal NH, C-terminal CO and amino alcohol OH) was identified [Reproduced from [15] with permission]
Order parameters for the C–C bonds of the palmitic chains, calculated with respect to the bilayer normal, on the last 10 ns of the trajectories. Black: no peptide (black), blue:1, green: 4, red: 8 peptide molecules [Reproduced from [15] with permission]
To further confirm the stability of the transmembrane state, the simulation previously obtained by using the “minimum bias approach”, whose equilibrated segment was initially just 10 ns long, was extended by further 40 ns. During this time, no significant modification in the peptide location, nor in the bilayer structure in its surroundings was observed (data not shown). The same system was simulated at 350 K for 65 ns without any evidence of destabilization of the transmembrane conformation.
Overall, these simulations strongly support the possibility of a transmembrane orientation for GAIV and a significant thinning effect on the bilayer, so that the short peptide helix could span it from one side to the other. However, in order to assess the reliability of the MD results, it was essential to evaluate their agreement with the experimental data.
1.3.1 Comparison with the NR Data
Neutron SLD profiles can be calculated from the MD trajectory and compared with the experimental data (Sect. 3.9). However, in order to perform a meaningful comparison, it was necessary to take into account the fact that in our experiments the lipid bilayer was supported on a silicon crystal, while it was free-standing in water in the simulations. It was previously demonstrated that GAIV very quickly partitions in both layers of the membrane [85] and this should lead to a symmetrical SLD profile for a trichogin-containing bilayer in water. Therefore, the SLD profile of a free-standing membrane was obtained from the experimental profiles by reflecting the section of the profile corresponding to the water-facing side of the bilayer with respect to the center of the hydrophobic region. The comparison of the resulting profiles, reported in Fig. 4.13, shows a good qualitative agreement, regarding both the thickness of the bilayer and the increase in SLD due to peptide insertion in the membrane. This comparison confers a good confidence to the atomic-level picture provided by the MD simulations.
Comparison between the neutron SLD profiles calculated from the MD trajectories of a peptide-free POPC bilayer (pink dotted line) and in the presence of 8 trichogin molecules (violet dotted line, peptide to lipid ratio 0.0625) with the symmetrical profiles obtained from the experimental neutron data of the peptide-free bilayer (pink solid line) and in the presence of deuterated GAIV at concentration C1 (violet solid line, estimated inserted peptide to lipid ratio 0.07 ± 0.02) [Reproduced from [15] with permission]
1.4 Vesicles Leakage Experiments
Both the simulations and the neutron reflectivity experiments indicate the possibility for GAIV to span the bilayer entirely, by causing a significant thinning of the membrane. However, this bilayer deformation requires a free energy cost. Therefore, if a transmembrane orientation is involved in the GAIV pore-formation process, the membrane-perturbing activity of this peptide should increase significantly in thinner membranes, which require a smaller deformation or no thinning at all. To verify this point, peptide-induced vesicle leakage experiments have performed with liposomes formed by lipids with different chain lengths and bilayer thicknesses [55] (Figs. 4.14 and 4.15). For comparison, the same experiments were carried out also with the much longer peptaibol Alm (Fig. 4.16). As shown in Figs. 4.15 and 4.16, the activity of GAIV increases dramatically with decreasing the bilayer thickness, while that of Alm is affected only marginally. In bilayers with a thickness comparable to that of biological membranes (i.e. with a number of carbon atoms of 16–18) the activity of GAIV is much lower than that of Alm. However, in the thinner membranes (whose hydrophobic core has a size comparable to that of the GAIV helix), the activity of the two peptides becomes comparable (Fig. 4.17). It is also worth mentioning that the curves of peptide-induced leakage as a function of GAIV concentration (Fig. 4.15) are steeply sigmoidal in membranes with high thickness, indicating a strong cooperativity of the pore formation process, but this cooperativity decreases drastically in thinner membranes.
Structures of the lipids used for the leakage experiments
Leakage curves of GAIV (A) in vesicles with a different hydrophobic thickness: Red di22:1 PC (1, 2-dierucoyl-sn-glycero-3-phosphocholine); orange di20:1 PC (1, 2-dieicosenoyl-sn-glycero-3-phosphocholine); green di18:1 PC (1, 2-dioleoyl-sn-glycero-3-phosphocholine); blue di16:1 PC (1, 2-dipalmitoleoyl-sn-glycero-3-phosphocholine); violet di14:1 PC (1, 2-dimyristoleoyl-sn-glycero-3-phosphocholine) [Reproduced [15] with permission]
Membrane-permeabilizing peptide activity as a function of the hydrophobic thickness of the bilayer. The peptide concentration is required to induce the leakage of 50 % of liposome contents is reported on y axis [15]
1.5 Discussion
Both NR data and MD simulations show a peptide-induced thinning of the membrane. The effect of the insertion of a peptide/protein in a bilayer with an equilibrium hydrophobic thickness differing from that of the inclusion has been rationalized in terms of hydrophobic mismatch [63, 92]. The free energy cost of exposing to water hydrophobic groups of the protein or lipids, due to their different thickness, is higher than that of distorting the lipids from their equilibrium conformation. As a consequence, the membrane thickness locally adapts to the size of the inclusion, although other effects are also possible [64]. The case of trichogin falls under the category termed negative mismatch, where the inclusion is shorter than the membrane thickness. A systematic study on model peptides demonstrated that hydrophobic mismatch is sufficient to drive membrane thickness adjustments comparable to those that would be necessary for barrel-stave pore formation by trichogin [67]. However, the MD results indicate that, in the case of trichogin, membrane thinning might be driven also by specific interactions between the peptide N- and C-termini and the phospholipid headgroups. In this respect, it is important to note that we have previously shown that in solvents of low polarity trichogin binds cations with a very high affinity [142]. This result might reflect what is happening in the interaction between the peptide and lipid headgroups in the low dielectric environment of the bilayer core.
A completely different interpretation for peptide-induced membrane thinning has been provided by Huang [58] and used to describe the mechanism of action of both cationic AMPs and peptaibiotics. According to his model, binding of an amphipathic molecule to the bilayer surface at the water-lipid chain interface leads to an increase in the interfacial area, and thus to a decrease in the hydrocarbon thickness, due to the very low volume compressibility of the lipid chains. This deformation has an elastic energy penalty, and thus, at a threshold bound peptide concentration, a transmembrane orientation becomes favored. In this model, membrane thinning is due to the surface-bound peptide, rather than to the transmembrane-inserted molecules. Unfortunately, previous works showed that in both orientations the peptide is located essentially in the hydrophobic core of the membrane [17, 85, 126] and therefore the NR data do not allow us to discriminate between the Huang hypothesis and thinning due to transmembrane inserted peptides. However, several indications suggest that the Huang mechanism is unlikely for trichogin: insertion of this highly hydrophobic peptide in the phospholipid tail region, parallel to the membrane surface, can be easily accommodated, without causing a significant increase in the interfacial area nor a perturbation of membrane order, as also shown by previous simulations and experiments performed by our research group [17].
Whatever the driving force of the trichogin-induced membrane thinning might be, this effect makes possible for the peptide to span the bilayer from one side to the other, and also provides a rationalization to a previous observation regarding this peptaibol. Fluorescence experiments demonstrated that membrane-inserted trichogin exists essentially in an aggregate state [85, 126]. Clearly, the free energy cost of membrane deformation can be significantly reduced by peptide aggregation (just like in the hydrophobic effect aggregation of apolar molecules in water reduces the entropic cost of water structuring around them). Therefore, a monomeric transmembrane mismatched inclusion is significantly unstable [64]. This is probably the main driving force for the highly cooperative oligomerization in membrane-permeabilizing channels. This interpretation would also explain the observation that curves of membrane-perturbing activity as a function of peptide concentration are highly cooperative in thick membranes, while this cooperativity decreases significantly in thinner membranes (Fig. 4.15).
Alm is one of the longest peptaibiotics, but many “medium”- or “short”-length peptides of this class do exist [30, 136]. Therefore, the present findings might be relevant for a rather wide class of peptides. Longer peptaibols are much more active than shorter ones [46], but barrel-stave channel formation has been hypothesized for some of the latter, like the 16 residue long antiamoebin [37]. Solid-state NMR measurements have also shown that such relatively short peptides, like the 14 residue ampullosporin or the 15 residue zervamicin II, attain a predominantly transmembrane orientation when the membrane thickness is comparable to their length, while they are largely parallel to the surface in thicker bilayers [9, 115]. Therefore, it has been proposed that all peptaibiotics might act according to the barrel-stave mechanism, the lower activity of the shorter ones being due to the lowest fraction of peptide molecules in a transmembrane orientation [115].
Some other findings reported in the literature further support the possibility for a peptaibiotic to form barrel-stave channels in a membrane thicker than its length. For instance, Alm is able to form pores even in artificial membranes formed by diblock copolymers whose hydrophobic region is much thicker than the peptide length [143], thus paralleling the situation of trichogin in biological membranes. In addition, our findings regarding the membrane thickness dependence of peptide’s activity nicely parallel the observation that in a series of short-chain trichogin analogs the activity significantly and progressively decreased with the shortening of the peptide chain [41].
No electrophysiology measurements of the conductance of single trichogin channels in planar membranes, which would provide a conclusive confirmation of a barrel-stave mechanism, have been reported to date. However, experiments on liposomes have shown that the pores formed by this peptide are ion selective and that their conductance is voltage-dependent, as it would be expected for Alm-like barrel-stave pores [68]. The voltage-gated nature of the trichogin channels was recently confirmed also by electrochemical measurements on Hg-supported tethered bilayer lipid membranes [10]. Overall, the present data indicate that formation of transmembrane barrel-stave channels might indeed be possible even for short peptaibiotics.
2 Selectivity
2.1 Role of Pro Residues in AMPs Sequences
In order to be able to design new peptide-based antibiotic drugs, it is essential to define the peptide characteristics that are responsible for AMPs selectivity.
The importance for selectivity of some AMPs molecular characteristics, such as charge and amphiphilicity, for their selectivity has been discussed in the Chap. 1. In the present chapter the role of another feature, common to many AMPs, will be discussed, i.e. the presence of a Pro or Gly residue in the amino acidic sequence. It is a conserved feature, present in several peptides [138, 157]. A statistical analysis quantitatively demonstrated the frequent presence of Gly residues close to the peptide center [138], but a similar statistical evaluation has not been reported for Pro. Nevertheless, several examples of Pro-containing helical AMPs or membrane-active toxins are described in the literature: Alm [127], BMAP-27 and BMAP-28 [147, 151], caerin [147], cecropin [156], fowlicidin [148], gaegurin [129], indolicin [151, 152], maculatin [26], melittin [31], pardaxin [146], pin2 [113], PMAP-23 [99], SMAP-29 [132], temporin A [23], tripticin [149], XT-7 [128], and an antimicrobial lysozyme fragment [60], among others. For many of these a Pro-induced kink in the helical structure has been demonstrated by NMR studies in solution or in membrane-mimicking media.
An analysis carried out using the AMPs database APD2 (http://aps.unmc.edu/AP/main.php), showed that in AMPs with less than 25 residues, and a single Pro residue, this amino acid is mainly found in proximity of the sequence center, with a preference for position closer to the N-terminus (Fig. 4.18).
Statistics of Pro position in helical AMPs, 11–25 residues long, containing a single Pro residue [Reproduced from [16] with permission]
The possible role of Pro in AMPs activity or selectivity is less easy to understand, with respect to the positive charge, or amphipaticity, which can be immediately related to the association to the lipid membranes. To study the effect of the presence and of the position of Pro in AMPs sequences, the P5 peptide was chosen.
2.2 The P5 Peptide
P5 is a model peptide, designed with the aim to obtain new synthetic peptides with a strong antibacterial activity. It was first derived from an hybrid parent peptide, CA-MA [104]. It is a Leu-Lys rich peptide, with a strong antibacterial activity and high selectivity, being nontoxic for erythrocytes up to the maximum investigated concentration, corresponding to 100 μM, i.e. 100-fold higher than the one required to kill bacteria [104]. The sequence of P5 is the following:
The peptide sequence clearly shows some of AMPs characteristics, like the positive net charge. If a helical structure is assumed for P5, we can notice how the amphipathic sequence has been designed to obtain two portions of a perfect amphipathic helix.
2.3 Pro Presence and Function in Proteins
The conformational effects induced by Pro in helical conformations are due to its particular structure: Pro is the only N-substituted residue, with a cyclic side chain. Due to this characteristics, Pro has a restricted torsional space, and its backbone amide, lacking a proton, cannot act as a donor in H-bond; thus, a Pro in i position will break the i to i + 4 H-bond usually formed in alpha-helices, and also the i + 1 to i − 3, for steric reasons [111]. Furthermore, it has been shown that Pro perturbs the conformational space of the preceding residue [59, 70, 118]. For all these reasons, it can dramatically affect the stability of helical structures, when located in the middle of the amino acidic sequence.
As a consequence, in soluble proteins Pro can often be found in proximity of the termini, but rarely in the helix interior [70, 111]. It has been estimated that only 5 % of total Pro residues are found inside helices, and that in this case they induce a kink [7, 80], conferring high flexibility to the protein structure.
Since in helical regions of soluble proteins Pro is extremely rare, and it is often located in hydrophilic regions, it would not be expected to be found in transmembrane helices. By contrast it surprisingly represents about 3–4 % of TM helical residues [77, 116], and, in addition, it is preferentially located close to the center of the helix [27, 117, 139]. Like in soluble proteins, also in the membrane environment Pro induces a kink [135] and indeed about 64 % of TM helices are kinked. Pro is usually 2 or 3 residues in the C-terminal direction with respect to the bend [73]. Pro residues are usually rather conserved in TM helices, hinting to a possible functional role [21, 117, 139]: it has been hypothesized that the Pro-induced kink could be needed to have free CO groups inside membrane channels, that could participate in ligand binding, or to gate channels with cis/trans isomerization or Pro-induced flexibility [21, 35, 78].
In the case of AMPs, no clear explanation has been proposed for the presence of the central kink. P5 is an excellent model system to study the role of Pro residues in AMPs.
2.4 P5 Analogues
In order to perform a systematic and complete study, a series of analogues were designed and synthesized starting from P5. In every analogue, Pro was moved towards the peptide termini, in positions which would keep the amphipathic character of the two helix portions, or removed altogether. In this case, a perfect amphipathic helix was obtained (Fig. 4.19).
Helical wheel projections of P5 and P5Del [16]
The structures of P5 analogues are summarized in Table 4.3.
2.5 Antimicrobial and Hemolytic Activity
The antimicrobial activity of P5 and its analogues was tested against different Gram-positive and Gram-negative bacteria. The bactericidal activity is expressed as minimum inhibitory concentration, or MIC, defined as “the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation” [4].
All analogues featured a good antimicrobial activity, even though the Pro displacement caused a slight increase in MIC values, that are shown in Fig. 4.20.
Antibacterial activity of P5 analogues on different bacterial strains
The hemolytic activities of the analogues, instead, are dramatically affected by the modification of Pro position: the results show a strong increase in the peptides’ toxicity as Pro is shifted from the sequence center or removed altogether. P5 is not hemolytic up to the higher tested concentration, 100 μM [104]. By contrast, analogues have an increasing toxicity, which becomes stronger as the Pro residue is shifted along the sequence. The most toxic analogue is P5Del, which has a hemolytic activity comparable to that of melittin (a potent bactericidal peptide derived from the bee venom, which is also dramatically hemolytic) as shown in Fig. 4.21.
Hemolytic activity of the analogues
The dramatic differences in peptides selectivity could depend by a different affinity for membranes of different composition, or by a different perturbing activity towards different membranes. Studies on model membranes were performed to clarify this point.
P5, the parent peptide, and P5Del, the Pro-lacking peptide. show the greatest differences in hemolytic activity. Therefore, these two AMPs have been chosen to perform experiments on model membranes.
2.6 Membrane-Perturbing Activity of P5 and P5Del
2.6.1 Peptide-Induced CF Leakage
To perform carboxyfluorescein leakage, to investigate the membrane-perturbing ability of P5 and P5Del [18], 0.03 μM peptide was added to various liposome solutions of different lipid concentrations.
In ePC/ePG (2:1 molar ratio) liposomes P5 and P5Del show a very similar activity (Fig. 4.22), causing a strong membrane perturbation and both reaching a value close to 100 % of leakage in a similar range of P/L values.
CF leakage from ePC/ePG vesicles. Red, filled symbols: P5. Blue, empty symbols: P5Del [16]
In ePC/cholesterol (1:1 molar ratio), on the other hand, P5Del resulted to be dramatically more active than P5 (Fig. 4.23).
CF leakage from ePC/cholesterol vesicles. Red, filled symbols: P5. Blue, empty symbols: P5Del [16]
To investigate the role of cholesterol, which has been suggested as a possible cause for the low activity of AMPs towards eukaryotic membranes, as discussed in Sect. 1.4 of Chap. 1 [84], the same experiments were performed also using vesicles composed only by ePC: also in this case the activity of P5 was significantly lower than that of P5Del (Fig. 4.24). This result proved that the differences in activities are due to the bilayer negative charge, given by ePG phospholipids, and not to the presence of cholesterol, which has only a slight influence on P5 behavior. The leakage data are in good agreement with the findings obtained by the hemolytic and antibacterial activity assays, in which the two peptides showed a similar bactericidal power, but only P5Del resulted to have hemolytic properties.
CF leakage from ePC vesicles. Red, filled symbols: P5. Blue, empty symbols: P5Del [16]
These experiments also allowed to point out that the distinct biological activities of the two analogues derive from their different ability to perturb membranes of different composition.
The effect of cholesterol resulted to be negligible; for this reason, all the following experiments on peptide-induced membrane-perturbation were performed using PC/PG and PC/cholesterol liposomes only.
2.6.2 Vesicles Aggregation
Liposomes-induced aggregation was investigated measuring the light scattering increment at 400 nm. The experiments were performed titrating P5 or P5Del (at 1 μM concentration) with increasing lipid amounts. If liposomes start to aggregate, an increase in the turbidity of the suspension occurs. P5 and P5Del are able to induce vesicle aggregation, as measured by the turbidity of liposome suspensions. For both P5 and P5Del, the turbidity of a peptide-containing suspension of charged vesicles is significantly higher than that of peptide-free liposomes, indicating peptide-induced aggregation (Fig. 4.25). Interestingly, this phenomenon is largely reversible after a given lipid to peptide ratio is reached, indicating that association, rather than fusion, of vesicles is taking place. The maximum in vesicle aggregation takes place at a peptide to lipid ratio that corresponds approximately to membrane neutrality. This behavior is consistent with what has been reported for other peptides [14, 83, 109, 161]. With neutral membranes, on the other hand, significant aggregation is induced only by P5Del, while the effect of P5 is negligible (Fig. 4.26).
Peptide-induced ePC/ePG vesicles aggregation. Red: P5, blue, P5Del, grey: no peptide [16]
Peptide-induced ePC/cholesterol vesicles aggregation. Red: P5, blue, P5Del, grey: no peptide [15]
2.6.3 Effect of the Peptide in the Thermotropic Phase Transition of Liposomes
For this experiment DPH-labeled DMPC vesicles in a 10 μM concentration were used, in the presence of 5 μM peptide. DPH (Fig. 4.27) inserts into the hydrophobic core of the membrane, essentially parallel to the lipid chains. For this reason, its fluorescence anisotropy reports on the membrane order and dynamics, and represents a measure of the membrane fluidity. Above the phase transition temperature, when the lipids are in the liquid-crystalline state, DPH anisotropy is very low, due to the fact that the probe is quite free to rotate around its axis [18]. Under the transition temperature, on the other hand, DPH anisotropy reaches a value around 0.35, because its mobility is largely inhibited when the lipids are in the ordered gel phase. The solution temperature was varied between 10 and 45 °C; DMPC vesicles showed the very nice liquid-to-gel phase transition at 24 °C. P5 had only a very slight effect on the membrane order and dynamics, even though it was present in a large excess (Fig. 4.28); P5Del, on the other hand, had a dramatic effect on the vesicles fluidity, with a strong broadening of the transition curve; in the presence of P5Del, indeed, the anisotropy values of DPH in the liquid and in the gel phase are much more similar than in the curve obtained without the peptide (Fig. 4.28).
DPH structure
Thermotropic gel to liquid crystalline phase transition in DMPC vesicles. [Lipid] = 10 μM; [peptide] = 5 μM. Red: P5, blue, P5Del, grey: no peptide [16]
The effects of the peptides on DPH anisotropy were investigated also in ePC vesicles at 25 °C; in these conditions, ePC lipids are in the physiological fluid state. A 10 μM solution of liposomes was titrated with increasing amounts of peptide, showing that also in this case P5Del has a stronger effect on DPH anisotropy, which increases in a concentration-related manner (Fig. 4.29).
peptide concentration dependence of DPH anisotropy in ePC vesicles. [Lipid] = 10 μM; T = 25 °C. Grey symbols: peptide-free vesicles; red symbols: vesicles with P5; blue symbols: vesicles with P5Del
Overall, these data concur to show that P5Del strongly perturbs both charged and neutral membranes, while P5 has a much higher activity on anionic vesicles than on neutral ones.
2.7 Water-Membrane Partition Experiments
The different perturbing activity of P5 and P5Del on neutral membranes could be due to a different affinity for them, or to a different behavior of the peptides once bound to membranes with a similar affinity. To answer to this question, experiments were performed to investigate the peptide water-membrane partition equilibria.
The peptide intrinsic fluorescence, due to the presence of a Trp residue in the sequence, allowed us to perform fluorescence experiments.
2.7.1 Average Wavelength and Anisotropy Measurements
Peptide emission spectra were collected titrating a 1 μM solution of P5 or P5Del with increasing amounts of liposomes.
When the peptide is bound to the membrane, the Trp emission maximum is shifted towards lower wavelengths (i.e. the so-called blue shift happens), because the fluorophore is located in a less polar environment [18]. The extent of spectral changes was obtained calculating the average wavelength parameter
in the spectral interval between 320 and 420 nm.
In ePC/ePG liposomes, both P5 and P5Del exhibit a significant change in their emission spectrum, that is an evidence that the association is taking place (Fig. 4.30). In ePC/cholesterol liposomes, instead, only P5Del showed a spectral shift in its Trp maximum, while no significant change is shown by P5 (Fig. 4.31).
Peptide water-membrane partition as measured from the blue-shift in the emission spectrum of the Trp residue in the presence of ePC/ePG (2:1) liposomes. [Peptide] = 1 μM. Red symbols: P5; blue symbols: P5Del [16]
Peptide water-membrane partition as measured from the blue-shift in the emission spectrum of the Trp residue in the presence of ePC/cholesterol (1:1) liposomes. [Peptide] = 1 μM. Red symbols: P5; blue symbols: P5Del [16]
This result does not necessarily mean that P5 does not bind to neutral membranes, because there is the possibility for the peptide to attain a membrane-bound conformation which leaves the Trp residue exposed to the aqueous phase, so that its spectrum is not affected by the binding to the bilayer. To exclude this possibility, fluorescence anisotropy experiments have been performed. When free in solution, the peptide rotational diffusion is very fast, and in this case Trp anisotropy is very low. When the peptide is associated to the membrane, on the other hand, its mobility is inhibited, and the anisotropy value increases [18]. These experiments were carried out with the same peptide and lipid concentrations used for the fluorescence spectra and showed very similar results: both peptides revealed affinity for negatively charged membranes (Fig. 4.32), while P5 does not bind to neutral bilayers (Fig. 4.33).
Peptide water-membrane partition as measured from the peptide fluorescence anisotropy in the presence of ePC/ePG (2:1) liposomes. [Peptide] = 1 μM. Red symbols: P5; blue symbols: P5Del [16]
Peptide water-membrane partition as measured from the peptide fluorescence anisotropy in the presence of ePC/cholesterol (1:1) liposomes. [Peptide] = 1 μM. Red symbols: P5; blue symbols: P5Del [16]
Fluorescence anisotropy measurements were also performed with pure ePC vesicles, to find out if cholesterol plays a role also in the association phenomenon: P5, even in this case, did not show any evidence of binding (Fig. 4.34), proving that the lack of positive charge is the main factor inhibiting P5’s binding, rather than cholesterol presence.
Peptide water-membrane partition as measured from the peptide fluorescence anisotropy in the presence of ePC liposomes. [Peptide] = 1 μM. Red symbols: P5; blue symbols: P5Del [16]
From these measurements it was also evident that the binding of P5 and P5Del with charged membranes occurs at relatively low peptide to lipid ratio, indicating a strong affinity of the two peptides for this kind of bilayers. The apparent partition constants K p were calculated from the fluorescence anisotropy data according to the following equation:
where r f and r b are the Trp anisotropy values related to the free and membrane-bound peptide.
The resulting K p are summarized in Table 4.4.
2.7.2 FRET Measurements-NBD
FRET (Sect. 2.1) of Chap. 2 can be used to determine the association of a peptide to a membrane. In the case of P5 and P5Del, the first fluorophore, the donor, is the Trp of our peptide. The second fluorophore, the acceptor, will be located within the membrane. FRET occurs, and can be detected, when the distance between the two fluorophores is comparable with the Förster radius. Under these conditions, the intensity of the donor fluorescence spectrum (Trp in this case) decreases, while the acceptor intensity increases.
A first set of experiments was performed using the fluorescent lipid NBD-PC. A peptide solution (1 μM) was titrated with ePC liposomes, labelled with NBD-PC (5 %) and the Trp and NBD fluorescence was detected. If FRET occurs, the intensity of NBD signal, located around 525 nm, increases. For P5Del, FRET occurs both in neutral and charged membranes, while for P5 a significant change in the NBD signal was observed only with ePC/ePG liposomes (Figs. 4.35 and 4.36).
NBD emission spectrum of labeled ePC/cholesterol liposomes in the absence (yellow) or in the presence of P5 (red) or P5Del (blue). [Peptide] = 1 μM, [Lipid] = 10 μM
NBD emission spectrum of labeled ePC/ePG liposomes in the absence (yellow) or in the presence of P5 (red) or P5Del (blue). [Peptide] = 1 μM, [Lipid] = 10 μM
2.7.3 FRET Measurements-DPH
Further FRET experiments were performed with DPH as acceptor; the Förster distance of the pair Trp-DPH is longer than that for Trp-NBD (Förster distances for the pairs are 40 and 16 Å, respectively) [71]. In this case it is possible to determine a peptide-membrane association even if the fluorophore of the peptide remains exposed to the aqueous phase, and thus the binding is not detectable with the experiments illustrated until now. This further experiment has been carried out to determine whether P5 actually does not bind to neutral membranes.
In this case, DPH excitation spectra are reported: if FRET occurs, the Trp band appears in the spectrum. Once again, in neutral vesicles there is evidence of FRET only for P5Del, and for both peptides in neutral membranes (Figs. 4.37 and 4.38).
DPH excitation spectra of ePC/cholesterol liposomes in the absence (green) or in the presence of P5 (red) or P5Del (blue). [Peptide] = 1 μM, [Lipid] = 10 μM
DPH excitation spectra of ePC/ePG liposomes in the absence (green) or in the presence of P5 (red) or P5Del (blue). [Peptide] = 1 μM, [Lipid] = 10 μM
All the experiments indicate that P5 and P5Del exhibit a similar affinity for anionic model membranes. On the other hand, only P5Del significantly interacts with neutral membranes. Thus, the different peptides’ activities are due to their different degree of interaction with lipid bilayers of different composition.
2.8 Peptide Structures in Water and Membranes
The affinity of the peptides for neutral membranes resulted dramatically affected by Pro removal. The explanation for this result is not obvious. Indeed, in practically all hydrophobicity scales, Pro contribution to the peptide preference for water or apolar phases is substantially negligible. For instance, in the Wimley-White water-octanol partition scale [145] the Pro contribution to the free energy of partition is just 0.14 ± 0.11 kcal/mol, to be compared with 2.80 ± 0.11 kcal/mol for Lys, −1.25 ± 0.11 kcal/mol for Leu and −2.09 ± 0.11 kcal/mol for Trp. Therefore, Pro omission per se cannot be responsible for the different behavior of the two analogues. However, these calculations do not take into account peptide conformation. CD spectra showed that P5 and P5Del attain different conformations in water: P5 is essentially unstructured, while P5Del features a significant amount of helical structure (Fig. 4.39). This result is consistent with the helix-breaking character of Pro in water. In a membrane-like environment, i.e. SDS or TFE both peptides showed a predominantly helical conformation (Figs. 4.40 and 4.41).
CD spectra of P5 and P5Del in phosphate buffer 10 mM, 140 mM NaCl, pH 7.4; [peptide] = 10 μM. Red: P5; Blue: P5Del [16]
CD spectra of P5 and P5Del in 50 % TFE. [Peptide] = 30 μM; Red: P5; Blue: P5Del [16]
CD spectra of P5 and P5Del in 30 mM SDS. [Peptide] = 30 = μM. Red: P5; Blue: P5Del [16]
The relatively high helicity content in water of a quite short peptide such as P5Del might seem surprising. One possibility is that this structure is induced by aggregation. Incidentally, differences in aggregation have been sometimes invoked to explain differences in selectivity among AMP analogues [5, 66, 151, 152], although without providing a clear mechanistic explanation of the link between these two properties. To investigate this possibility, several concentration-dependent studies were performed. The fluorescence spectra did not show any significant variation (Fig. 4.42); thus, the hypothesis of aggregation can be excluded, at least in the low micromolar concentration range used in this study.
Average wavelength of the peptide emission spectrum, as a function of peptide concentration. The error bars have been calculated repeating the experiment three times
Probably, the lack of aggregation at relatively low concentrations is due to electrostatic repulsion caused by the high overall peptide net charge. At the same time, the helix stabilization of monomeric P5Del is provided by interactions between its hydrophobic residues, which in a helical conformation stack one on top of the other. Quantitative studies indicate that hydrophobic interactions between side chains located four residues apart in a helical peptide account for a stabilizing free energy difference that is 3–4 times the destabilizing effect due to the repulsion between two Lys residues [34]. Consequently, it is an established strategy in the design of helical peptides to integrate both charged and hydrophobic residues (but located on two different faces of the helix), to increase water solubility and secondary structure stability, respectively [2, 158].
The different conformations the two peptides attain in water have a dramatic effect on their effective hydrophobicity. A reliable measurement of this property is provided by the retention time in RP-HPLC. Several studies concur to show that this parameter is a good measure of peptide hydrophobicity, and takes into account also conformational effects [29, 47, 56, 82, 106, 153, 154, 159]. The observed retention time was 19 min for P5 and 25 min for P5Del. Based on the range of retention times observed for peptides of varying hydrophobicities [47, 106, 153], this is a very large difference, indicating that the P5Del is much more hydrophobic than P5.
2.9 Molecular Dynamics Simulations
In order to better elucidate the conformational effects of the Pro residue, MD simulations were performed for P5 and P5Del in the research group where this thesis was carried out. Five simulations, each 120 ns long, were performed for the two peptides in water, starting from a helical structure. In the initial stages of the simulations a conformational transition took place for both peptides, and equilibration was reached after simulation times ranging from about 5 to 70 ns, as judged from the matrixes of root mean square distances (RMSD) between trajectory structures [125]. The final, equilibrated 50 ns of the 5 trajectories of the two peptides were subjected to a cluster analysis, to find the predominant conformations, as reported in Table 4.5, and in Fig. 4.43. In the case of P5Del, all structures were very similar: one cluster (Fig. 4.43e) contained about 60 % of the structures, and the first 3 clusters contained almost 100 % of them (Fig. 4.43e–g). In all these structures P5Del maintained an essentially helical conformation, with some fraying at the termini. By contrast, simulations of P5 indicated a much higher conformational flexibility: in this case the first cluster represented only 40 % of the structures (Fig. 4.43a), and the first three only 76 % (Fig. 4.43a–c). In these structures the peptide attained a compact conformation, allowed by a break in the helix caused by the Pro residue, where the hydrophobic residues were partially shielded from the solvent. In addition, the remaining 24 % of the P5 structures were not included in any cluster of significant size, and correspond to largely disordered conformations (Fig. 4.43d).
Cluster analysis of MD simulations in water. Overlap of 50 structures, reported as a ribbon representation of the backbone, belonging to the clusters of the MD simulations of P5 (left, a–d) and P5Del (right, e–g) in water. The clusters are reported in order of relative population, from top to bottom. The last image for P5 (d) represents the unclustered structures. For the most populated cluster (top, a and e) the most representative structure (i.e. the structure with the smallest RMSD distance to the others) is also shown: the backbone is reported as a green ribbon, and side chains are shown as sticks (red: Lys; blue: Leu; yellow: Trp; cyan: Pro) [Reproduced from [16] with permission]
Conformational clusters obtained analyzing the Cα coordinates sampled each 250 ps in the last 50 ns of 5 independent 120 ns long simulations of P5 and P5Del in water. Only clusters containing more than 10 % structures are reported in the table. MLP values are reported with their standard errors.
These findings are in qualitative agreement with the CD spectra, which indicated a much higher helicity for P5Del, although the simulations overestimate the degree of structuring of P5, possibly as a result of biasing from the starting conformation. Anyway, the MD results provide an explanation for the different hydrophobicities and RP-HPLC retention times of the two analogues: the compact or bent conformations favored by the presence of Pro in P5 allow a partial shielding of the hydrophobic residues from the water phase. A rough but conformation-dependent assessment of peptide hydrophobicity in the simulated structures can be obtained with the molecular lipophilicity potential (MLP) [44]. The values reported in Table 4.5. confirm that the conformations sampled by P5 during the simulations are less hydrophobic than those of P5Del. This is particularly true for the unstructured P5 conformations, which, based on the CD data, are probably more populated than what our simulations show.
In principle, in addition to the peptide conformation in water, another property possibly causing the lack of P5 binding to neutral membranes could be its inability to attain a correct amphiphilic conformation in this membrane. Indeed, CD shows that P5 is largely helical once membrane bound, and, as shown in Fig. 4.19, a perfectly helical conformation would not give rise to the amphiphilic arrangement of the side chains needed for binding with high affinity to the bilayer. Obviously the free energy change associated with P5 binding to neutral membranes depends both on the starting and final states (P5 in water and in a PC bilayer, respectively). However, the latter condition is not attainable experimentally. Fortunately, MD simulations are particularly suited to investigate complex systems under experimentally inaccessible conditions. A simulation of P5 in the presence of POPC lipids was performed by using the so-called “minimum bias” approach [39, 99] in which the simulation starts from a random mixture of peptide, lipids and water (Fig. 4.44). In this way, a bilayer forms spontaneously during the simulation, but the peptide is free to find its minimum free energy position with respect to the lipids in the very dynamic environment of the initial stages of the simulation. As the starting structure for the peptide, a representative conformation of the most populated cluster obtained from the simulations in water was used. The lipid and water molecules present in the simulation were 128 and 7500, respectively, in a volume of about 390 nm3 and this would correspond to a lipid concentration of about 0.55 M, i.e. probably high enough to ensure partition into the neutral membrane even for a peptide with a very low affinity such as P5. This is indeed what was observed: as shown in Fig. 4.44, P5 inserted into the membrane, lying parallel to the plane of the bilayer, just below the polar headgroups, in a position similar to that previously observed for other analogous peptides [99]. Notwithstanding the presence of the Pro residue, and in agreement with the CD findings, in the membrane P5 attained a helical conformation, although partially distorted with respect to the ideal geometry. The arrangement of intramolecular hydrogen-bonds was the canonical i to i + 4 for the N-terminal segment, then switched to i to i + 5 starting from the CO of residue 5, so that it was engaged in an H-bond notwithstanding the missing NH of Pro9. Eventually the H-bond pattern changed again to i to i + 3 at the N-terminus. This last shift left the CO groups of residues 12 and 13 lacking an intramolecular partner. However, their position in the membrane was rather superficial, thus allowing them to form H-bonds with water molecules, and to interact electrostatically with the headgroups of phospholipids. An important consequence of these deviations of the backbone dihedrals from the ideal helical angles (and probably the main driving force for these distortions of the helix) was the achievement of a perfect amphiphilic arrangement of the side-chains, with those of the hydrophobic residues embedded in the apolar core of the membrane. These findings suggest that the conformational properties of P5 in the membrane probably do not contribute to its low affinity for neutral bilayers.
MD simulations of P5 in the membrane. Structures at the beginning and end of the simulation of P5 are shown in the left and right panel, respectively. The peptide backbone is reported as a green ribbon, and side chains are shown as sticks (red: Lys; blue: Leu; yellow: Trp; cyan: Pro). For phospholipids, phosphorus and nitrogen atoms are represented as spheres (grey and blue, respectively), while the bonds as sticks. Water molecules are not shown, to simplify the image [Reproduced from [16] with permission]
2.10 Discussion
All the data clearly demonstrate a dramatic difference in the affinity of P5 and P5Del for neutral membranes. The main origin of this difference seems to be the peptides conformation in water.
In order to bind efficiently to membranes, AMPs have a sequence that allows them to attain an amphiphilic helical structure, with the hydrophobic residues on the same side of the helix. This alignment of hydrophobic residues is reminiscent of “Leu zippers” [1, 5, 101], and this structure is also present in these peptides. Hydrophobic interactions between Leu residues can stabilize helical conformations even in water, leaving a large exposed apolar surface, and favoring interaction with neutral membranes and toxicity. The data presented here indicate that the role of Pro residues in the sequence of AMPs is to destabilize these helical conformations, thus reducing the hydrophobic driving force for membrane binding. It is important to note that, on the other hand, peptide structure in the membrane is not very sensitive to amino acidic substitutions, and is usually helical [155]. Even peptides with a high content of Pro [99] or D-residues [97], or cyclized peptides [98] have been shown to be largely helical when bound to membranes, due to the lack of competition between intramolecular and intermolecular H-bonds, and to the free-energy cost of introducing a non-H-bonded amide bond in the membrane environment [145, 153, 154]. This is also what has been observed in the present study for the Pro-containing P5. Therefore, the most relevant factor in determining peptide toxicity is the helix stability in the water phase [138]. Destabilization of the helical structure in water reduces peptide affinity for neutral membranes by allowing a reduction in peptide hydrophobicity, due to the shielding of apolar residues, and also by introducing an entropic penalty for the membrane-binding process, due to the membrane-induced coil to helix transition. Indeed, a general correlation between peptide helicity and toxicity has been observed [25, 62, 81, 107, 138, 153–155] and some selective AMPs are imperfectly amphipathic [99, 107]. These findings were tentatively explained by proposing that helicity influences the peptide propensity to aggregate, or to interact with other components of the medium, which can reduce its activity by sequestering it [66]. However, aggregation was not relevant in the present instance. Therefore, it can be supposed, that a higher hydrophobicity, amphipathicity, affinity for neutral membranes, toxicity and tendency to aggregate are all possible consequences of a stable helical structure in water.
Several studies investigated the effect of substituting or inserting Pro residue on the activity and selectivity of specific AMPs, and provide further support to the present findings, and to the proposed interpretation. In most cases, substitution of Pro with another amino acid led to increased toxicity. For instance, this was observed for melittin [31], pardaxin [122, 134], tripticin [150], PMAP-23 [151, 152], temporin A [23] and model peptides [121]. By contrast, insertion of a Pro in peptides that did not contain it, usually led to the opposite effect, reducing peptide toxicity [12, 23, 124, 126, 157]. For instance, toxic peptides pisicidin [12], temporin L [23] and the scorpion toxin IsCT [75] became selective once a Pro residue was inserted close to the center of their sequence. In some instances, it was shown that the Pro-induced changes in toxicity were mediated by an effect on the peptide affinity for neutral membranes [75, 124, 126, 151, 152]. Some exceptions to these general rules do exist: in some cases Pro insertions/substitution did not have an effect on hemolytic activity [13, 57], while in other cases they also affected significantly peptide activity against bacteria [3, 26, 110, 112, 157]. However, generally speaking, Pro insertion can be considered an effective method to reduce peptide toxicity, usually without perturbing significantly the antimicrobial activity. As a consequence of this, other similar approaches aiming at the destabilization of helical structures have been tested with success. Effects similar to those caused by Pro insertion/substitution were observed for Gly [24], another helix-destabilizing residue often found at the center of AMPs sequences. In addition, Shai and co-workers showed clearly that insertion of D-amino acids is a promising strategy to decrease peptide toxicity, and this approach was successfully applied to melittin, pardaxin, and model peptides [96, 102, 120]. Other investigators employed peptoid residues with the same goal [65]. However, peptides comprising D-residues or peptoids can be more immunogenic than sequences containing natural amino acids only, and are problematic for biotechnological production [11, 84]. Therefore Pro or Gly residues might be preferable to these alternatives.
In conclusion, these data provide a clear hypothesis for the role of Pro residues usually present in the sequence of AMPs: they are essential for peptide selectivity, by destabilizing helical conformations in the water phase, thus allowing conformations in which the apolar side-chains are partially buried, and reducing the hydrophobic driving force for binding to neutral membranes. These findings will be critical in the design of artificial molecules with a selectivity comparable to that of natural AMPs.
3 Tuning the Biological Activity
3.1 Cell-Penetrating Peptides
In this Chapter the possibility will be explored, to tune the biological activity of a peptide by limited modifications to its sequence. A case will be presented in which an AMP will be obtained from a cell-penetrating peptide (CPP).
CPPs are molecules endowed with the capability to cross eukaryotic cell membranes, and to deliver hydrophilic and macromolecular cargoes without causing membrane leakage [48, 72]. In some cases, it is possible for a CPP to deliver a molecule 100 fold bigger than its own weight [52, 79]. Therefore, they reveal a huge potential for gene and drug delivery applications [40], since in the last years protein or peptide-based drugs have been developed, but these molecules still feature important limitations, such as poor stability and low capability to reach their targets [48]: CPPs could represent a good solution to the problem of drug delivery.
CPPs have been firstly derived from proteins. In 1988 it was shown that the HIV-Tat protein is able to permeate the cell membrane, and to be internalized into the nucleus [43]; 10 years later, the Lebleu group demonstrated that that the minimal sequence needed for translocation was a 11-mer peptide (YGRKKRRQRRR) [144]. In 1991, Joliot et al. demonstrated that the Drosophila Antennapedia transcription factor [61] was also internalized by cells. The isolated domain, a 16-mer peptide (RQIKIYFQNRRMKWKK), was called penetratin, and it is now referred as pAntp [32, 79]. In the last twenty years, many chimeric or synthetic CPPs were obtained, also using unnatural amino acids to improve their systemic stability [48].
The mechanism of CPP internalization in many cases remains to be clarified. One of the most interesting hypotheses is that different mechanisms can compete for the uptake, also depending on the structural features of the peptide [48].
CPPs and AMPs have been designed, tested and analyzed as two distinct classes of peptides, due to their very different origin, and biological activities [51]. However, in many cases they share some common characteristics: many CPPs are short, amphiphilic and cationic, and they often attain a helical structure when associated to membranes, just like AMPs. The antibacterial activity of AMPs is strictly correlated with their structural features; thus, it is not a surprise, if some CPPs were shown also to be able to penetrate bacterial membranes [94, 100], and resulted to be, in this case, membranolytic.
For instance, the CPP pVEC was shown to kill bacteria by permeabilizing their membranes [100], and also transportan 10 (TP10) induces permeabilization of model membranes [149] and is bactericidal [94]. Both penetratin and Tat peptide were shown to be antimicrobial, with a MIC in the micromolar range. Similar results were obtained for the artificial peptide MAP [100]. On the other hand, while most AMPs exert their activity by perturbing the bacterial membrane, some of them are known to penetrate inside bacterial cells and to act on intracellular targets. The most prominent example of these AMPs is buforin [103], but many other exist [95]. However, even membrane-perturbing AMPs, such as magainin, were shown to penetrate into eukaryotic cells [133].
For these reasons, it is possible to ask which characteristics are responsible for different activities, or if there is really an effective separation between CPPs and AMPs. Since the activity of both CPPs and AMPs is strictly related to their structure, it is possible to hypothesize that it can be finely tuned with appropriate substitutions in the amino acidic sequence to switch between the two classes, i.e. it might be possible to obtain an AMP from a CPP, and vice versa. Such a result could be of great importance, because in this case the bactericidal activity could also be coupled with the ability to deliver a cargo in the cell compartment.
To clarify at least some of these points, the CPP called Pep-1 was chosen as a test case. Pep-1 (KETWWETWWTEWSQPKKKRKV), also called “Chariot” for its ability to transport cargoes inside cells, was designed by combining three domains [91]. The first is a hydrophobic, Trp-rich segment, KETWWETWWTEW, which is mainly responsible for peptide-protein interactions, that play a key role in the peptide carrier function. The second domain is constituted by a Pro-containing spacer, SQP, which gives flexibility to the peptide structure. Finally, a hydrophylic domain, crucial for the membrane translocation, is present (KKKRKV). The amphiphilic composition of Pep-1 allows it to interact with lipid bilayers [53], and to form non-covalent aggregates with cargo molecules, stabilized by both hydrophobic and electrostatic interactions [93].
Pep-1 exhibits a strong cell-permeating capability, but its translocation mechanism is still debated. The most promising hypothesis seems to involve a non-endocytotic pathway; furthermore, it has been proved that Pep-1 is able to perturb membrane permeability to translocate across the bilayer, and that a transmembrane potential is needed for activity [33, 50, 105, 160]. On the other hand, Pep-1 shows only a slight antimicrobial activity, at very high concentrations, for which it becomes also toxic for erythrocytes.
A Pep-1 analogue, called Pep-1-K, was designed, in which all negatively charged residues where replaced by Lys, with the aim to obtain a peptide more similar to AMPs, with a higher cationic charge [160]:
Indeed, Pep-1-K exhibited a strong antimicrobial activity, with MICs in the low μM range [160], as reported in Table 4.6. Pep-1-K has a bactericidal activity similar to that of melittin, without being toxic for erythrocytes.
Studies on Staphylococcus aureus cultures have shown that the antibiotic activity of Pep-1-K is related to its capability to cause the cell depolarization, reaching almost 100 % of depolarization at a peptide concentration of 64 µM [160].
These intriguing results raise a number of questions: do the sequence differences between Pep-1 and Pep-1-K influence only the antimicrobial activity of the two peptides or also their cell penetrating properties? What is the mechanism of the antibacterial activity of Pep-1-K? More specifically, is membrane depolarization caused directly by Pep-1-K association to the bacterial surface or is it just a consequence of some other effect of the peptide on the cell metabolism? Last, but not least, what are the causes of the switch in activity between Pep-1 and Pep-1-K?
To address these issues, the interaction of Pep-1 and Pep-1-K with cells and model membranes has been characterized.
3.2 Membrane-Perturbing Effects
3.2.1 Peptide-Induced Vesicle Leakage Assay
The membrane-perturbing activity of a peptide can be measured by its capability to cause the leakage from dye-loaded vesicles. For this purpose, ePC/ePG CF-loaded liposomes were used (200 μM, as described in Sect. 3.7) of Chap. 3 with the addition of several peptide concentrations.
The peptide was not able to cause the CF release, even at high concentration (Fig. 4.45).
Peptide-induced release from CF-loaded liposomes. Peptide concentration = 10 μM; lipid concentration: 200 μM [14]
3.2.2 Peptide-Induced Ion Leakage
The ability of Pep-1-K to cause the depolarization of bacterial membranes could be due to the formation of defects or pores in the membrane, too small to cause the leakage of a molecule like CF, but sufficiently large to allow the release of ions. To clarify this point, a sodium-sensing dye (SBFI, Fig. 4.46) was entrapped inside ePC/ePG lipid vesicles, which were then diluted them in a NaCl-containing buffer, while only KCl was present inside the vesicles. SBFI is a fluorescence probe, which excitation spectrum changes in the presence of sodium, due to the complex formation. The liposomes solution was titrated with increasing amounts of Pep-1-K, up to 10 μM.
SBFI probe structure
The addition of Pep-1-K to a liposome solution caused Na+ entry into the vesicles, as shown by the change in the SBFI excitation spectrum. The intensity of the signal increased (Fig. 4.47), and the maximum was blue-shifted (Fig. 4.48). By contrast, Pep-1 was not able to cause such leakage at any concentration tested (up to 10 μM).
Excitation spectra of SBFI in the presence of increasing amounts of Pep-1-K [14]
Intensity increase of the SBFI spectrum at λ = 335 nm as a function of peptide concentration. Full circles: Pep-1-K; empty circles: Pep-1 [14]
3.2.3 Peptide-Induced Vesicle Aggregation
Another important evidence of the membrane-perturbing activity of Pep-1-K is its ability to induce vesicles aggregation, which was detected by measuring the light scattering at 400 nm of a peptide solution, which was titrated with increasing amounts of ePC/ePG liposomes. Pep-1-K induced aggregation was reversible: the apparent absorbance reached a maximum around a lipid to peptide ratio of about 80, and then decreased (Fig. 4.49). No aggregation effect was detectable in the presence of Pep-1 (Fig. 4.49).
Peptide-induced vesicle aggregation, as measured by the sample turbidity at 400 nm. Empty circles: peptide-free sample; full circles: Pep-1-K containing sample; crosses: Pep-1 containing sample. [Peptide] = 1 μM. ePC/ePG 2:1 (mol/mol) vesicles [14]
The phenomenon can be explained in terms of electrostatic attraction: when the peptide is bound to the anionic membranes, its positive charges progressively neutralize the negative charges of the lipids, favoring the formation of aggregates. When more liposomes are added, the peptide molecules are distributed over more vesicles, and therefore the total negative charge in each liposome increases again, causing a repulsion between vesicles, and a reduction in the aggregation. The reversibility of the increase in light scattering indicates that Pep-1-K causes vesicle aggregation rather than fusion, and therefore this process is probably not the origin of Pep-1-K induced ion leakage.
3.2.4 Effect of the Peptide in the Thermotropic Phase Transition of Liposomes
Another evidence of the peptide perturbing effect was obtained by measuring the phase transition temperature of the membrane. The experiment was carried out as described in Sect. 4.2.6.3).
As shown in Fig. 4.50, Pep-1-K binding significantly modifies the dynamics of a DMPC/DMPG bilayer both above and below the thermotropic phase transition, while no peptide-induced membrane perturbation was observed for Pep-1 in the physiologically relevant fluid state. Therefore peptide-induced membrane perturbation could be the basis of the Pep-1-K induced ion leakage.
Effect of peptide–membrane interaction on the thermotropic phase transition of DMPC/DMPG vesicles, as followed by the fluorescence anisotropy of DPH. Empty circles: peptide-free vesicles; filled circles: Pep-1-K associated vesicles; crosses: Pep-1 associated vesicles DMPC/DMPG 2:1 (mol/mol), DPH 1 %, [lipid] = 50 μM; [Peptides] = 10 μM [14]
3.3 Water-Membrane Peptide Partition
3.3.1 Steady-State Fluorescence
The origin of the increased membrane-perturbing activity of Pep-1-K, as compared to Pep-1, might be related to a different affinity of Pep-1-K for anionic lipid bilayers. To study the water-membrane partition equilibria, liposomes mimicking the composition of bacterial membranes (ePC/ePG in 2:1 molar ratio) were used. The intrinsic fluorescence of the peptide, which features 5 Trp residues in its structure, can be exploited to perform static or dynamic fluorescence studies. From the fluorescence spectra, it is possible to evaluate the affinity of the peptide towards lipid vesicles, from the calculation of the average wavelength for every lipid concentration, as already illustrated for P5. The average wavelength shift for both Pep-1-K and Pep.1 is shown in Fig. 4.51.
Water to membrane partition of Pep-1 (empty symbols) and Pep-1-K (filled symbols), followed by the shift in the fluorescence emission spectrum. [Peptide] = 1 μM, ePC/ePG (2:1 mol/mol) vesicles (λ exc. = 280 nm (λ em. = 320–420 nm [14]
In the case of Pep-1-K and Pep-1, membrane binding caused a significant blue-shift in the emission spectrum of both peptides, but Pep-1-K exhibited a significantly higher affinity than Pep-1 for this kind of membranes. On the other hand, the spectral shift caused by membrane association was similar for both peptides, suggesting that they have a similar position and orientation in the membrane.
These findings indicate that the higher antibacterial activity of Pep-1-K is likely due, at least in part, to its higher membrane affinity.
3.3.2 Time-Resolved Fluorescence
The same peptide titration was performed also measuring the fluorescence decay lifetimes of Pep-1-K. The Trp decay profile in the presence of increasing amounts of liposomes was fitted with a triple-exponential function; from the data analysis it was possible to obtain the average lifetime value, \( \langle \tau \rangle \) (Fig. 4.52).
\( \langle \tau \rangle \) variation of Pep-1-K as a function of lipid concentration. Experimental conditions: (λ ex = 298 nm (λ em = 343 nm; [Peptide] = 1 μM
From the dynamic fluorescence data the partition constant can be derived, according to the equation
where \( \langle \tau \rangle \) [L = 0] e \( \langle \text{T} \rangle \) [L = ∞] are the values of the average lifetimes for the peptide alone and in the presence of lipids in saturation conditions, respectively. This equation has been used in the hypothesis that only two species are present, the free and the bound peptide. The partition constant derived from this expression resulted to be Kp = 5.0 × 106.
The partition constant has been derived from time-resolved measurements and not from steady-state measurements because the former are not affected by experimental problems, like the tendency of the peptide to attach to the cuvette quartz walls.
3.3.3 Giant Unilamellar Vesicles
Direct observation of Pep-1-K association to membranes was possible using a fluorescein-labeled analogue and giant unilamellar vesicles (GUVs), labelled with Rho-PE (1 %). The peptide was added to a GUVs solution in 3 μM concentration.
Pep-1-K is located almost exclusively on the membrane surface, with negligible peptide fluorescence in the water phase both outside and inside GUVs, indicating that translocation does not occur (Fig. 4.53). This is probably due to the absence of a transmembrane potential, which was needed also for Pep-1 cell internalization [49].
Giant unilamellar vesicles images in the presence of Pep-1-K: panel A, vesicels fluorescence; panel B, peptide fluorescence; panel C, merge of the two signals. A and B. Image size 37.5 × 75 μm. GUV composition: ePC/ePG/Rho-PE 66:33:1 (molar ratios) [Reproduced from [14] with permission]
Surprisingly, Pep-1-K did not associate homogeneously to all vesicles, and its fluorescence is concentrated only on certain GUVs, while does not appear on others. Anyway, this could be due to the impossibility of stirring the sample when the peptide was added in the observation chamber.
3.4 Peptide Location Inside the Bilayer
Depth-dependent quenching experiments were carried out to determine the position of Pep-1-K inside the lipid membrane. For this purpose, liposomes labelled with the nitroxyl group at different position of their acyl chain, or on the polar headgroups, were used. More precisely, 5-, 7-, 10-, 12-, 14-, 16-doxyl-PC and TEMPO-PC were used. Pep-1-K fluorescence spectra were collected in the presence of each type of liposomes, and also in a solution of unlabelled vesicles. All these experiments were performed at the same peptide and lipid concentration.
The nitroxyl moiety acts as a quencher of Trp fluorescence; thus, when the fluorophore is located at the same depth of the quencher group, its fluorescence will decrease. This is a short-range effect, so the quenching efficiency will rapidly decrease with the distance. From these experiments a quenching profile as a function of the distance from the bilayer center was obtained [99].
The quenching profile (Fig. 4.54) showed that Pep-1-K is located next to the membrane surface, right beneath the polar headgroups. The profile was rather well-defined, indicating that all the 5 Trp residues present in the peptide sequence are located at the same depth in the bilayer: the peptide lies parallel to the membrane surface, and its orientation does not change with peptide concentration (within the range investigated). This position and orientation suggest a mechanism of membrane perturbation that could be described according to the “carpet” model [119].
Depth-dependent quenching experiment to determine the position of Pep-1-K in the membrane. [Peptide] = 1 μM (full symbols) or 10 μM (empty symbols), [lipid] = 200 μM. F and F0 are the fluorescence intensities measured for the peptide associated to doxyl-labeled and unlabeled membranes, respectively. (λ exc. = 280 nm (λ em. = 320–420 nm. ePC/ePG (2:1 mol/mol) vesicles, doxyl labeled lipid content 7 % [14]
3.5 Cell-Penetrating Properties
To determine whether the cell-penetrating properties are retained by Pep-1-K, FITC-labeled analogues of Pep-1 and Pep-1-K were synthesized. The two peptides were then incubated with both bacterial and eukaryotic cells, and cell cultures were thereafter visualized by confocal microscopy. The collected images (Figs. 4.55 and 4.56) showed that both peptides were internalized in the cells, indicating that the Glu to Lys substitutions in Pep-1-K do not abolish the cell-penetrating activity.
Confocal laser-scanning and differential interference contrast (DIC) microscopy images (left and right panels, respectively) of E. coli treated with FITC-labeled peptides. Cells were treated with 10 μg/ml of FITC-labeled Pep-1 (a, b and c, representative images of different samples) or FITC-labeled Pep-1-K (d, e and f, representative images of different samples) [Reproduced from [14] with permission]
Confocal laser-scanning and DIC microscopy images (left and right panels, respectively) of HeLa cells treated with FITC-labeled peptides. Cells were treated with FITC-labeled Pep-1 (a, b and c; 10, 5 and 2.5 μg/ml peptide concentration, respectively) and FITC-labeled Pep-1-K (d, e and f; 10, 5 and 2.5 μg/ml peptide concentration, respectively) [Reproduced from [14] with permission]
3.6 Discussion
The data reported here indicate that the main difference between Pep-1 and Pep-1-K is in their relative affinities towards bacterial membranes: Pep-1-K binds to anionic bilayers more strongly, due to its higher cationic charge, and this appears to be the main reason for its strong bactericidal activity. This conclusion is in agreement with the correlation recently shown between water-membrane partition constants and MIC values of AMPs [87]. In the “carpet” model of peptide-induced membrane perturbation, AMPs need to reach a threshold of membrane-bound peptide concentration before they can cause the formation of defects or pores resulting in membrane leakage [119]. Therefore, it is evident that the higher the peptide affinity towards bacterial membranes, the lower is the concentration needed to reach this threshold. The reported data are consistent with a “carpet” model of membrane perturbation by Pep-1-K: it binds to the membrane surface, and perturbs the order of the bilayer. This leads to the leakage of ions, but not of larger molecules, at least in the concentration range investigated.
Apparently the change in membrane affinity caused by the Glu to Lys substitutions in Pep-1-K, while increasing its membrane-perturbing activity, does not inhibit its cell-penetrating properties. This is not surprising, since also the high translocation efficiency of Pep-1 itself has been shown to be linked to its strong affinity towards cellular membranes [53].
In conclusion, the example of Pep-1 and Pep-1-K clearly illustrates that cell-penetrating and antimicrobial peptides are not two separate classes, since subtle modifications can determine which of the two activities predominates.
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Bobone, S. (2014). Results and Discussion. In: Peptide and Protein Interaction with Membrane Systems. Springer Theses. Springer, Cham. https://doi.org/10.1007/978-3-319-06434-5_4
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