project is about peptide HHC-36 want introduction part Peptide HHC-36:-5000 words if you nee subtitle i can write .example i need to including in the introductionn about mechanism of action,

Stractural feature, specific peptide HHC-36, amino acid sequence and antimicrobial activity/n Downloaded via LONDON METROPOLITAN UNIV on August 16, 2022 at 09:54:49 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ACCOUNTS of chemical research pubs.acs.org/accounts Advances in Molecular Understanding of a-Helical Membrane- Active Peptides Ivo Kabelka and Robert Vácha* Cite This: Acc. Chem. Res. 2021, 54, 2196-2204 1. KEY REFERENCES ACCESSI CONSPECTUS: Biological membranes separate the interior of cells or cellular compartments from their outer environments. This barrier function of membranes can be disrupted by membrane- active peptides, some of which can spontaneously penetrate through the membranes or open leaky transmembrane pores. However, the origin of their activity/toxicity is not sufficiently understood for the development of more potent peptides. To this day, there are no design rules that would be generally valid, and the role of individual amino acids tends to be sequence-specific. In this Account, we describe recent progress in understanding the design principles that govern the activity of membrane-active peptides. We focus on a-helical amphiphilic peptides and their ability to (1) translocate across phospholipid bilayers, (2) form components. transmembrane pores, or (3) act synergistically, i.e., to produce a significantly more potent effect in a mixture than the individual We refined the description of peptide translocation using computer simulations and demonstrated the effect of selected residues. Our simulations showed the necessity to explicitly include charged residues in the translocation description to correctly sample the membrane perturbations they can cause. Using this description, we calculated the translocation of helical peptides with and without the kink induced by the proline/glycine residue. The presence of the kink had no effect on the translocation barrier, but it decreased the peptide affinity to the membrane and reduced the peptide stability inside the membrane. Interestingly, the effects were mainly caused by the peptide's increased polarity, not the higher flexibility of the kink. Flexibility plays a crucial role in pore formation and affects distinct pore structures in different ways. The presence of a kink destabilizes barrel-stave pores, because the kink prevents the tight packing of peptides in the bundle, which is characteristic of the barrel-stave structure. In contrast, the kink facilitates the formation of toroidal pores, where the peptides are only loosely arranged and do not need to closely assemble. The exact position of the kink in the sequence further determines the preferred arrangement of peptides in the pore, i.e., an hourglass or U-shaped structure. In addition, we demonstrated that two self-associated (via termini) helical peptides could mimic the behavior of peptides with a helix-kink-helix motif. Finally, we review the recent findings on the peptide synergism of the archetypal mixture of Magainin 2 and PGLa peptides. We focused on a bacterial plasma membrane mimic that contains negatively charged lipids and lipids with negative intrinsic curvature. We showed that the synergistic action of peptides was highly dependent on the lipid composition. When the lipid composition and peptide/lipid ratios were changed, the systems exhibited more complex behavior than just the previously reported pore formation. We observed membrane adhesion, fusion, and even the formation of the sponge phase in this regime. Furthermore, enhanced adhesion/partitioning to the membrane was reported to be caused by lipid-induced peptide aggregation. In conclusion, the provided molecular insight into the complex behavior of membrane-active peptides provides clues for the design and modification of antimicrobial peptides or toxins. Metrics & More • Tuerkova, A.; Kabelka, I.; Králová, T.; Sukeník, L.; Pokorná, S; Hof, M.; Vácha, R. Effect of helical kink in antimicrobial peptides on membrane pore formation. eLife 2020, 9, e47946.¹ ACS Publications © 2021 The Authors. Published by American Chemical Society Read Online 2196 AⒸⒸ Ac (cc Article Recommendations Received: January 26, 2021 Published: April 12, 2021 Article ACCOUNTS https://doi.org/10.1021/acs.accounts.1c00047 Acc. Chem. Res. 2021, 54, 2196-2204 Accounts of Chemical Research • Brožek, R.; Kabelka, I.; Vácha, R. Effect of Helical Kink on Peptide Translocation across Phospholipid Mem- branes. J. Phys. Chem. B 2020, 28, 5940-5947.² ● Kabelka, I.; Vácha, R. Optimal Hydrophobicity and Reorientation of Amphiphilic Peptides Translocating Through Membrane. Biophys. J. 2018, 115 (6), 1045- 1054. • Kabelka, I.; Pachler, M.; Prévost, S.; Letofsky-Papst, I.; Lohner, K.; Pabst, G.; Vácha, R. Magainin 2 and PGLa in Bacterial Membrane Mimics II: Membrane Fusion and Sponge Phase Formation. Biophys. J. 2020, 118 (3), 612-623. 2. INTRODUCTION Antibiotic-resistant bacterial strains present a global threat to public healthcare.$ Infections caused by resistant bacteria are responsible for the death of hundreds of thousands of people each worldwide. Hospital-acquired, or nosocomial, year, infections are becoming more frequently caused by bacteria resistant to at least one antibiotic. Such infections are associated with prolonged treatment, increased medical expenditures, and higher mortality. Several peptide classes, sometimes collectively referred to as membrane-active peptides, are promising alternatives to traditional antibiotics. One of the classes are antimicrobial peptides (AMPs), which are produced by many organisms. AMPS include both (1) host-defense peptides, i.e., ubiquitous components of the innate immune system, and (2) toxins with antimicrobial activity (e.g., melittin). These peptides can act both directly in killing the pathogens or indirectly as immunomodulators. Cell-penetrating peptides (CPPs) are molecules that are able to pass through the cellular protective barriers including the plasma membrane and get internalized into cells by either endocytotic or non-endocytotic pathways. Due to the similarities in their properties (e.g., composition, length, charge) and activity, the classification of these peptides can vary. 8 9 6 In general, the membrane-active peptides are positively charged, amphiphilic, relatively short peptides. Such peptides are commonly unstructured in solution and gain a secondary structure upon interaction with a lipid membrane. The spatial distribution of residues in the folded peptide can form discrete surfaces with either hydrophobic or hydrophilic properties. These secondary-amphiphilic¹0 peptides (amphiphilic in their secondary structure) are commonly a-helical in the membrane environment and can be conveniently represented using helical-wheel projection; see Figure 1. Peptide surface properties can then be represented by circular sectors. While the net positive charge of peptides is responsible for their increased selectivity toward specific membranes, their amphiphilic character enables their interaction with the membrane hydrophobic core. Note that there are exceptions and, e.g., CPPs also include cyclic or purely hydrophilic peptides such as polyarginines.¹¹ Furthermore, peptides with multiple arginines show different behavior, sometimes called "magic", and have been recently reviewed elsewhere.¹2 In this Account, we focus on linear amphiphilic peptides with a small positive charge that become helical upon interacting with the membrane. 11 Membrane-active peptides are also diverse in terms of their mechanisms of action." 7,14 The peptide activity may include (see Figure 2) the following: (a) disruption of the lipid packing 2197 pubs.acs.org/accounts (L15 L18) (L14) L8 S3 oooo L21 (L19) L1 L12 S10 S17 L5 (S16) S9 S6 S2 (520) (S13) LSSLLSLLSSLLSLLSSLLSL 13 Figure 1. Helical-wheel projection of a synthetic pore-forming amphiphilic peptide.¹3 Hydrophilic and hydrophobic resididues are shown in green and gray, respectively. Circular sectors represent the peptide's amphiphilic properties. a b C с de f Article Figure 2. Illustration of selected molecular mechanisms of membrane- active peptides. (a) Disruption of the lipid packing and membrane thinning in the vicinity of peptides.3,15 (b) Translocation across cell membrane. 16 (c) Pore formation.¹7 (d) Detergent-like membrane solubilization at high peptide concentrations (also called carpet model).¹7(e) Agglutination of the pathogens.¹8 (f) The formation of inverted micelles. Peptide helices are represented as cylinders with hydrophilic and hydrophobic surfaces shown as green and gray surfaces, respectively. Lipid head and tail groups are shown as orange spheres and black lines, respectively. 17 18 19 and membrane thinning in the vicinity of peptides.3,15 (b) translocation across the cell membrane,¹6 (c) pore formation, (d) detergent-like membrane solubilization at high peptide concentrations (also called carpet model), (e) the aggregation of membranes and agglutination of the patho- gens, and (f) the formation of inverted micelles. However, the peptide activity can be very sensitive to changes in the peptide sequence, and only a single-residue mutation can have a profound effect on the activity and the mechanism of action. ¹,20 We will focus on membrane-active peptides that directly interact with lipid bilayers and alter or even breach its https://doi.org/10.1021/acs.accounts.1c00047 Acc. Chem. Res. 2021, 54, 2196-2204 Accounts of Chemical Research protective function, leading to an uncontrolled transport of matter in and out of cells or cellular compartments. Cell membranes are mainly composed of lipids organized into two leaflets. The membrane core is a thin hydrophobic barrier preventing the permeation of polar molecules. Bio- logical membranes are frequently mimicked with just lipid bilayers, since lipids and more specifically glycerophospholi- pids, sphingolipids, and sterols are their major components. However, we should be aware that proteins also carry out important functions of cell membranes and amount to roughly 50% of their mass and can cover 30-55% of the membrane 21 22 area. The precise membrane lipid composition varies greatly in different organisms, tissues, cell types, compartments, or organelles. Moreover, the lipid species (varying in their head group types and tail saturation/length) are asymmetrically distributed between the membrane leaflets. The difference in lipid composition can be used for the selective targeting of peptide activity on specific membranes. Developing a resistance against such action is possible²³ but difficult, because maintaining the lipid composition is important for cell homeostasis, and the altered composition or even the loss of asymmetry is associated with pathological states or cell death.24 25 Due to the small size of peptides and transient structures formed during the membrane disruption, a high spatial and temporal resolution is needed to reveal the details of the disruption mechanism. Computer simulations can provide such resolution, including atomistic details, but they are limited to microsecond time scales. By sacrificing some of the interaction details, more complex systems and longer time scales (dozens of microseconds or even milliseconds) are accessible using coarse-grained models. In such models, groups of atoms are treated as a single particle to smoothen the potential energy landscape and drastically reduce the amount of interactions that have to be calculated. Indeed, a considerable number of studies on peptide-induced membrane disruption employed computational methods. 2,3,26-31 3. TRANSLOCATION OF PEPTIDES ACROSS MEMBRANE Classical textbook knowledge is that only small uncharged molecules can spontaneously pass through lipid membranes. 32 However, it has been shown that even large molecules such as polymers or peptides can spontaneously translocate across the membrane if they possess the right amphiphilicity. 33,34 Once inside the cell, a peptide can interfere with intracellular processes, resulting in cell death.³5 The advantage of these peptides is their activity even at low concentrations, when they are predominantly monomers. However, the right properties to allow the peptides to translocate across the membranes remain unclear. 36 The simplest model to evaluate the peptide propensity for translocation is based on the difference in the peptide partitioning between the membrane surface and its hydro- phobic core. In this model, the energy of peptide adsorption at the membrane surface is calculated via the Wimley-White interfacial hydrophobicity scale derived for 1-palmitoyl-2- oleoyl-glycero-3-phosphocholine (POPC)³7 lipids. The pep- tide energy in the membrane core is approximated by the full insertion of a peptide in octanol. The model was validated using fluorescence experiments, where the helical peptides were observed to translocate when the energy difference in the model was below 20 kcal mol-1.36 However, it is unclear what 36 38 2198 pubs.acs.org/accounts to use for different lipid compositions and how to advance the model to take into account the order of amino acids. Until recently, it was very challenging to obtain more detailed information about the peptide translocation process. Major progress has been achieved by computer simulations, which benefit from advances in both software and hardware. First, the insertion of mostly hydrophobic peptides (mimicking transmembrane domains of membrane proteins) into their stable transmembrane state was studied. Later, this was followed by the translocation of a few specific AMPs and general amphiphilic peptides, providing details on the translocation pathway." 39,40 2,3,26,41,42 The translocation pathway of secondary amphiphilic¹0 and mostly hydrophobic helical peptides is as follows: (0) starting from peptide in solution with random orientation to (1) adsorbed state on the membrane surface with orientation parallel to the membrane plane, followed by (2) partial insertion of a peptide terminus into the membrane (usually translocation barrier) leading to (3) full peptide insertion in the membrane hydrophobic core and adoption of a trans- membrane state with perpendicular orientation to the membrane plane.³ Note that primary-amphiphilic43 peptides (amphiphilic in their primary structure) could behave differently, because amphiphilic molecules such as cholesterol and other lipids follow a different translocation path. In this path, the translocating molecules change their orientation from perpendicular to parallel to the membrane plane, as their polar part moves from the membrane interface toward the middle of the membrane. 44 We performed simulations with a highly coarse-grained model (with side-chain properties averaged into hydrophobic/ hydrophilic surfaces) to elucidate the behavior of amphiphilic helices. Figure 3 illustrates the key findings and the change in behavior of membrane-spanning amphiphilic helices with increasing hydrophobicity. A highly hydrophilic peptide remains in solution (Figure 3a). Increasing peptide hydro- a o bo CO AG Peptide distance f AG AG 80000000 999doood g AG Peptide distance لام aaaaaaa TOT AG DEN Peptide distance h AG Peptide Peptide Peptide distance distance distance do e AG m Article Peptide distance (D AG AG SK Peptide distance Peptide distance Figure 3. Schematic representation of the effect of (a-e) peptide amphiphilicity on peptide-membrane interactions and (f-h) membrane composition, at high dilution. Helical segments are represented as cylinders. Hydrophilic/hydrophobic surfaces of peptides are shown in green/gray and as circular sectors above the figure. Lipid head groups are shown in red (conical), orange (cylindrical), and green (inverted conical) color. Lipid tails are shown as black lines. The effect of two cationic residues at the peptide termini on the barrier state is shown with a more detailed model (i). Blue arrows highlight the orientation of residues to opposite leaflets, causing a local membrane thinning in the barrier state of Buforin II (marked with a red circle on the free energy profile). https://doi.org/10.1021/acs.accounts.1c00047 Acc. Chem. Res. 2021, 54, 2196-2204 Accounts of Chemical Research phobicity (b) promotes binding on the membrane surface and subsequently (c) insertion into the hydrophobic core until (d) a highly hydrophobic peptide becomes confined in the membrane environment unable to desorb. Amphiphilic helices inserted into the membrane create transient defects that 3,45 facilitate lipid flip-flop, i.e., the so-called slip-pop mecha- nism. Such activity of the peptides in their transmembrane state could alter lipid homeostasis. Fully hydrophobic peptides (e) remain in the transmembrane orientation. Note that fully hydrophobic peptides shorter than the membrane hydrophobic thickness (i.e., ~10 residues long) remain perpendicular to the membrane plane, stabilized by hydrogen bonds with the backbone ends. Based on the hydrophobic mismatch, such peptides decrease the membrane thickness. Even shorter peptides (~5 residues long) remain below the phosphate region in a parallel orientation relative to the membrane plane.³ 3,46 We have shown that by tuning the peptide amphiphilicity, it is theoretically possible to obtain a peptide that does not have a preference for either the membrane or the solution. For efficient translocation, the free energy profile should be as flat as possible, with no significant barriers.³ In other words, the difference between the free energy minimum (typically adsorption) and maximum (barrier or solution state) should be below the thermal energy. The barrier was found to be predominantly affected by the polarity of peptide termini, with hydrophobic ends providing a lower barrier. This finding is in line with more detailed simulations (see Figure 3).26,42 Note that amidation at the C-terminus was shown to increase the antimicrobial activity, and almost half of known helical AMPS possesses this modification. Amidation could thus play a role in decreasing the insertion barrier into the membrane, but there are other possible explanations such as protection from degradation. The optimal peptide was found to have a length corresponding to the membrane thickness and hydrophobic ends and be roughly half hydrophobic.³ The peptide translocation is dependent not only on the peptide properties but also on the membrane lipid composition. We demonstrated that lipids with positive intrinsic curvature (inverted conical shape) facilitated the translocation, while lipids with negative intrinsic curvature (conical shape) hinder the peptide translocation compared to cylindrical lipids with zero intrinsic curvature; see Figure 3f-h. These results are in line with experimental observations.48 The main limitation of the employed highly coarse-grained model is its lack of direct connection to specific lipids and peptides sequences. However, the model provides robust general information, which we verified experimentally or computa- tionally with a more detailed model using a few specific sequences. 2,3,48 The calculation of the translocation free energy profile for peptides with specific sequences using a more detailed models is not straightforward. Only very recently, we developed a new collective variable/reaction coordinate, which can capture the translocation of AMPS across the membrane correctly." 42 We showed that the collective variable should utilize a local rather than global center of mass of the membrane as the reference position. Local center of mass prevents large-scale deformations of the membrane during the simulations of peptide insertion. Moreover, the movement of charged residues/groups through the hydrophobic core of the membrane is associated with a significant free energy barrier, and has to be included in the collective variable (description of 2199 pubs.acs.org/accounts the translocation) explicitly. The cationic residues close to peptide termini were also previously suggested to stabilize the peptide in the middle of the membrane due to the formation of salt bridges with the lipid-phosphate groups. Our simulations 49 are in line with such stabilization of the transmembrane state. However, the same salt bridges were also observed in the barrier state of Buforin II, where one peptide terminus was partially inserted in the membrane and two cationic residues at this terminus interacted with lipid phosphates on opposite leaflets causing a local membrane thinning; see the simulation snapshot in Figure 3i.42 Therefore, both the position of cationic residues in the sequence and their mutual orientation plays a complex roles during the peptide translocation and need to be captured in the collective variable. With such a collective variable, it is possible to calculate the translocation of AMPS reliably and obtain the translocation free energy profile, which could be used for sequence optimization. For example, we used it to elucidate the effect of peptide flexibility (presence of kink in the helical peptides); see the section on peptide flexibility. 42 The described progress represents the first steps toward fully understanding the role of residues at specific positions in the sequence and membrane properties in peptide translocation. Article 4. PORE FORMATION Another common mechanism of AMPs is the formation of a leaky transmembrane pore. This mechanism usually requires higher concentrations of peptides than translocation, because pores are typically formed by several peptides acting together. However, the concentrations for pore formation are lower than in the carpet mechanism, where the whole membrane is covered with peptides and becomes disintegrated as in the presence of surfactants. 50 There are two well-established pore structures: a barrel-stave and a toroidal pore. In the barrel-stave pore structure, all peptides are tightly packed together into a bundle surrounding a water channel. Lipid head groups do not participate directly in the pore structure, and the peptides orient roughly perpendicular to the membrane plane.1,51 In contrast, the presence of lipid head groups inside the pore is characteristic of toroidal pores, where peptides are only loosely arranged if they interact at all. ¹,28 Two prototypical peptides forming barrel-stave pores are alamethicin5¹ and LS3 peptide,¹3 while a large range of peptides were reported to form toroidal pores. 1,27,28,30,50 A number of variants of toroidal pores were reported; see Figure 4.¹ In disordered toroidal pores, peptides are only partially inserted into the pore and oriented parallel or only slightly tilted with respect to the membrane plane.²7 In contrast, peptides in the more prevalent toroidal pore variants are roughly perpendicular to the membrane plane. In the hourglass toroidal pore, the peptides span the membrane, and their kink in the membrane center creates the shape of an hourglass.52 In a U-shaped pore, peptides also have a kink in the middle of the membrane, but remain confined within one membrane leaflet (both termini are in the lipid head groups on one side of the membrane). The even more complex structure of toroidal pores was reported to be formed by Magainin 2 and PGLa peptides, with Magainin 2 inside the pore and PGLa at the edge of the pore. In contrast, maculatin peptides were shown to form an ensemble of structurally diverse membrane pores rather than a single well- defined toroidal structure." Therefore, obtaining detailed 53 54,55 25 https://doi.org/10.1021/acs.accounts.1c00047 Acc. Chem. Res. 2021, 54, 2196-2204 Accounts of Chemical Research (Disordered) toroidal pore AG↓ +kink U-shaped Hourglass Figure 4. Schematic representation of simplified/idealized peptide- membrane interactions, as determined by peptide amphiphilicity. Helical segments are represented as cylinders. Lipid head and tail groups are shown as orange spheres and black lines, respectively. Hydrophilic and hydrophobic surfaces of peptides are shown as green and gray surfaces, respectively. information about the pore structure, which is important for the understanding of peptide behavior, could require extensive simulations and is very challenging for experiments. 50,56 Without peptides, pore formation is an energetically demanding process. Pore opening proceeds by the formation of (1) an initial water defect, (2) followed by a water-wire (single-molecule-thick water channel), (3) and finally a water channel.30 Peptides bind more strongly to membrane pores, possibly due to the looser lipid packing at the pore edge. Therefore, peptides could stabilize the pore by fully (barrel- stave pore) or partially (toroidal pore) covering the membrane edge at the rim of the pore. Therefore, the higher the peptide concentration, the easier the pore nation, and the stronger the stabilization of the membrane edge. Moreover, nonequilibrium conditions could further reduce the free energy of the initial opening of the pore. For example, asymmetric peptide distribution/adsorption could induce a membrane tension/strain, facilitating pore formation. 51,57,58 Once the pore is opened, there is an exchange of material through the pore between the leaflets, which speeds up the equilibration. A large transient pore can be initially formed and subsequently become more stable as it shrinks. 50,51,57 5. EFFECTS OF PEPTIDE FLEXIBILITY We found the reports of the role of peptide flexibility to be contradictory, in particular, the presence of proline and glycine residues, which can induce the formation of a kink (i.e., sharp bend) in the regular a-helical structure. Peptide flexibility/kink was reported to both enhance and reduce antimicrobial effects. 1,2 We combined coarse-grained simulations with two inde- pendent models to provide a molecular understanding of the peptide kink. First, a highly coarse-grained phenomenological model was used to study the effect of peptide flexibility independently of peptide sequence, and second, the MARTINI coarse-grained model enabled decoupling the effect of peptide flexibility (secondary structure) and polarity (kink-forming residues in the sequence). ¹,2 Thus, we were able to determine which effects are mainly caused by the increased peptide polarity or the increased peptide flexibility. For a single 2200 pubs.acs.org/accounts peptide, we demonstrated that the presence of a kink (1) reduces the peptide affinity to the membrane, (2) has no effect on the peptide insertion barrier, and (3) destabilizes the transmembrane state; see Figure 5. Moreover, the kink AG Linear Kink r Article -Z +Z Peptide distance from membrane center Affinity ↑ || Stability Barrier Figure 5. Schematic depiction of the effect of the proline/glycine- induced kink on the free energy of insertion. Free energy profile for a fully helical peptide translocation across the membrane is in black, while the gray line is the free energy profile for a peptide with the kink. Adsorbed states are in the z-distance from the membrane center. Helical segments are represented as cylinders. Hydrophilic and hydrophobic surfaces of peptides are shown as green and gray surfaces, respectively. Adapted with permission from ref 2. Copyright 2020 American Chemical Society. facilitates the formation of toroidal pores, but destabilizes barrel-stave pores.¹,² The pore formation behavior could be rationalized by geometrical means, because the peptide flexibility hinders the necessary tight packing of the peptides in the barrel-stave bundle, while flexible peptides can more easily adapt to the curved catenoid shape of the pore rim in the toroidal pores.¹ The additional degree of freedom from the peptide flexibility entropically favors the solution state, and decreases the peptide adsorption to the membrane; however, the change in peptide polarity associated with the kink mutation could dominate this effect.2,60 We verified the key findings with fluorescence leakage experiments on large unilamellar vesicles (LUVs).¹ We investigated the interaction details the of adsorption of Buforin II P11L and WT (proline is in the middle of the sequence) peptides on a lipid membrane and the subsequent translocation. In simulations, we decoupled the effects of the kink flexibility (conformational freedom) and polarity (back- bone hydrogen bonding and side-chain properties). Unexpect- edly, the largest effect of the kink was in decreasing the peptide adsorption, mainly due to the increased polarity of the leucine to proline mutation. The flexibility of the kink did not have a significant effect. The increased peptide polarity also significantly decreased the population of the transmembrane state during the translocation. The translocation barrier remained the same, despite the additional degree of freedom. The reason for this is that the height of the barrier is determined by the insertion of the charged groups (peptide terminus and side-chains) in the hydrophobic core of the membrane. 2,3,26 These findings are in agreement with our results using a phenomenological model, and thus robust and more generally applicable.² For pore formation, the effect of the kink is more complex. Barrel-stave-pore-forming peptides without a kink had lower free energy of pore formation than their more flexible (with kink) counterparts. The investigated peptides include amphiphilic phenomenological helices with a large hydro- phobic patch and a-helical variants (P11L, P11A, and WT in a https://doi.org/10.1021/acs.accounts.1c00047 Acc. Chem. Res. 2021, 54, 2196-2204