Molecular mechanism of antimicrobial peptides: the origin of cooperativity

Based on very extensive studies on four peptides (alamethicin, melittin, magainin and protegrin), we propose a mechanism to explain the cooperativity exhibited by the activities of antimicrobial peptides, namely, a non-linear concentration dependence characterized by a threshold and a rapid rise to saturation as the concentration exceeds the threshold. We first review the structural basis of the mechanism. Experiments showed that peptide binding to lipid bilayers creates two distinct states depending on the bound-peptide to lipid ratio P/L. For P/L below a threshold P/L*, all of the peptide molecules are in the S state that has the following characteristics: (1) there are no pores in the membrane, (2) the axes of helical peptides are oriented parallel to the plane of membrane, and (3) the peptide causes membrane thinning in proportion to P/L. As P/L increases above P/L*, essentially all of the excessive peptide molecules occupy the I state that has the following characteristics: (1) transmembrane pores are detected in the membrane, (2) the axes of helical peptides are perpendicular to the plane of membrane, (3) the membrane thickness remains constant for P/L> or =P/L*. The free energy based on these two states agrees with the data quantitatively. The free energy also explains why lipids of positive curvature (lysoPC) facilitate and lipids of negative curvature (PE) inhibit pore formation.     

The cooperative behaviour of antimicrobial peptides in model membranes

A systematic analysis of the hypothesis of the antimicrobial peptides' (AMPs) cooperative action is performed by means of full atomistic molecular dynamics simulations accompanied by circular dichroism experiments. Several AMPs from the aurein family (2.5,2.6, 3.1), have a similar sequence in the first ten amino acids, are investigated in different environments including aqueous solution, trifluoroethanol (TFE), palmitoyloleoylphosphatidylethanolamine (POPE), and palmitoyloleoylphosphatidylglycerol (POPG) lipid bilayers. It is found that the cooperative effect is stronger in aqueous solution and weaker in TFE. Moreover, in the presence of membranes, the cooperative effect plays an important role in the peptide/lipid bilayer interaction. The action of AMPs is a competition of the hydrophobic interactions between the side chains of the peptides and the hydrophobic region of lipid molecules, as well as the intra peptide interaction. The aureins 2.5-COOH and 2.6-COOH form a hydrophobic aggregate to minimize the interaction between the hydrophobic group and the water. Once that the peptides reach the water/lipid interface the hydrophobic aggregate becomes smaller and the peptides start to penetrate into the membrane. In contrast, aurein 3.1-COOH forms only a transient aggregate which disintegrates once the peptides reached the membrane, and it shows no cooperativity in membrane penetration.     

Action mechanism and structural requirements of the antimicrobial peptides, gaegurins

Gaegurins (GGNs) are a family of cationic, α-helical, antimicrobial peptides that were isolated from a Korean frog, Glandirana emeljanovi (formerly classified as Rana rugosa) and represent one of the structurally well-characterized groups. Among six gaegurins, gaegurin 4 (renamed herein esculentin-2EM), gaegurin 5 (brevinin-1EMa), and gaegurin 6 (brevinin-1EMb) have been investigated comprehensively in terms of structure-activity relationships. In this paper, we first suggest renaming of gaegurins according to a recently raised rule of systematic nomenclature. Then, the current understanding of gaegurins is reviewed by summarizing their structure-activity relationships. In particular competing arguments on gaegurins are synthetically inspected. Finally their action mechanism and structural requirements will be discussed.     

Analysis of the segmental stability of helical peptides by isotope-edited infrared spectroscopy

Isotope-edited infrared spectroscopy has the ability to probe the segmental properties of long biopolymers. In this work, we have compared the infrared spectra of a model helical peptide ((12)C) Ac-W-(E-A-A-A-R)(6)-A-NH(2), described originally by Merutka et al. (Biochemistry 1991;30:4245-4248) and three derivatives that are (13)C labeled at the backbone carbonyl of alanines. The locations of six isotopically labeled alanines are at the N-terminal, C-terminal, and the middle two repeating units of the peptide. Variation in temperature from 1 degrees to 91 degrees C transformed the peptides from predominantly helical to predominantly disordered state. Amplitude and position of the infrared amide I' absorption bands from (12)C- and (13)C-labeled segments provided information about the helical content. Temperature dependence of infrared spectra was used to estimate segmental stability. As a control measure of overall peptide stability and helicity (independent of labeling), the temperature dependence of circular dichroism spectra in the far-UV range at identical conditions (temperature and solvent) as infrared spectra was measured. The results indicate that the central quarter of the 32 amino acids helix has the maximal helicity and stability. The midpoint of the melting curve of the central quarter of the helix is 5.4 +/- 0.8 degrees C higher than that of the termini. The N-terminal third of the helix is more helical and is 2.0 +/- 1.4 degrees C more stable than the C-terminus.     

The pH dependent properties of metallotransferrins: a comparative study

The dependence on pH of the absorption and circular dichroic spectra of iron(III), cobalt(III) and copper(II) transferrins has been (re)investigated. In the alkaline region, the CD profiles of iron(III) and cobalt(III) transferrin are essentially pH independent up to pH 11; only for very high pH values (pH > 11) is breakdown of the cobalt(III) and iron(III) transferrin derivatives observed, without evidence of conformational rearrangements. By contrast, the CD profiles of copper transferrin show drastic changes in shape around pH 10; these spectral changes, which are fitted to a pK_a of ~10.4, are interpreted in terms of a substantial rearrangement of the local environment of the copper ions at high pH. Although the CD spectra of copper transferrin at alkaline pH strictly resemble those observed upon addition of modifier anions, the mechanism of site destabilization in the two cases is different; at variance with the case of modifier anions, our results suggest that the high pH form of copper transferrin still contains the synergistic anion. A¹³C NMR experiment has confirmed this view. In the acidic region, iron(III) and cobalt(III) transferrins are stable down to pH ~6. For lower pH values progressive metal detachment is observed without evidence of conformational changes; around pH 4.5 most bound metals are released. In the case of the less stable copper-transferrin, metal removal from the specific binding sites is already complete around pH 6.0; in concomitance with release from the primary sites, binding of copper ions to secondary sites is observed. Additional information has been gained from CD experiments in the far UV. The pH dependent properties of iron(III), cobalt(III) and copper(II) transferrin are discussed in the frame of the present knowledge of transferrin chemistry, particular emphasis being attributed to the comparison between tripositive and bipositive metal derivatives.     

Solution structures of stomoxyn and spinigerin, two insect antimicrobial peptides with an alpha-helical conformation

Stomoxyn and spinigerin belong to the class of linear cysteine-free insect antimicrobial peptides that kill a range of microorganisms, parasites, and some viruses but without any lytic activity against mammalian erythrocytes. Stomoxyn is localized in the gut epithelium of the nonvector stable fly that is sympatric with the trypanosome vector tsetse fly. Spinigerin is stored and secreted by hemocytes from the fungus-growing termite. The structure of synthetic stomoxyn and spinigerin in aqueous solution and in TFE/water mixtures was analyzed by CD and NMR spectroscopy combined with molecular modeling calculations. Stomoxyn and spinigerin adopt a flexible random coil structure in water while both assume a stable helical structure in the presence of TFE. In 50% TFE, the structure of stomoxyn is typical of cecropins, including an amphipathic helix at the N-terminus and a hydrophobic C-terminus with helical features that probably fold in a helical conformation at higher TFE concentration. In contrast to stomoxyn, spinigerin acquires very rapidly a helical conformation. In 10% TFE the helix is highly bent and the structure is poorly defined. In 50% TFE, the helical structure is well defined all along its sequence, and the slightly bent alpha-helix displays an amphiphilic character, as observed for magainin 2. The structural similarities between stomoxyn and cecropin A from Hyalophora cecropia and between spinigerin and magainin 2 suggest a similar mode of action on the bacterial membranes of both pairs of peptides. Our results also confirm that TFE induces helix formation and propagation for amino acids showing helical propensity in water but also enhances the helix propagation propensity of nonpolar beta-branched residues.     

Design and synthesis of cationic Aib-containing antimicrobial peptides: conformational and biological studies.

Development of antimicrobial peptides has attracted considerable attention in recent years due to the excessive use of antibiotics, which has led to multiresistant bacteria. Cationic amphiphilic Aib-containing peptide models Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 1-4, and sequential cationic polypeptides (Arg-X-Gly)(n), X = Ala, Val, Leu, were prepared and studied for their antimicrobial and hemolytic activity, as well as for their proteolytic stability. Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 2, 3 and the polypeptide (Arg-Leu-Gly)(n) exhibited significant antimicrobial activity, and they were nontoxic at their MIC values and resistant, in particular the Aib-peptide models, to enzymatic degradation. The conformational characteristics of the peptide models were studied by circular dichroism (CD). Structure-activity relationship studies revealed the importance of the amphipathic alpha-helical conformation of the reported peptides in inducing antimicrobial effects. It is concluded that peptide models comprising cationic amino acids (Arg), helicogenic and noncoding residues (Aib) and/or hydrophobic and helix-promoting components (Leu) may lead to the development of antimicrobial therapeutics.     

The dermaseptin superfamily: A gene-based combinatorial library of antimicrobial peptides

Skin secretions of hylid frogs show amazing levels of interspecific and intraspecific diversity and are comprised of a cocktail of genetically-related, but markedly diverse antimicrobial peptides that are grouped into a superfamily, termed the dermaseptins, comprising several families: dermaseptins (sensu stricto), phylloseptins, plasticins, dermatoxins, phylloxins, hyposins, caerins, and aureins. Dermaseptin gene superfamily evolution is characterized by repeated gene duplications and focal hypermutations of the mature peptide coding sequence, followed by positive (diversifying) selection. We review here molecular mechanisms leading to these vast combinatorial peptide libraries, and structural and functional properties of antimicrobial peptides of the dermaseptin and plasticin families, as well as those of dermaseptin S9, an amyloidogenic peptide with antimicrobial and chemoattractant activities.     

The RH 421 styryl dye induced,pore model-dependent modulation of antimicrobial peptides activity in reconstituted planar membranes

Background

Antimicrobial agents, with different pore-formation mechanisms, may be differently influenced by alteration of the dipolar electric field of a lipid membrane.

Methods

By using electrophysiological measurements on reconstituted lipid membranes, we used alamethicin, melittin and magainin to report on how controlled manipulation of the membrane dipole potential by the styrylpyridinium dye RH 421 affects the kinetic and transport features of peptides within membranes.

Results

Our data demonstrate that the increase of the membrane dipole potential caused by RH 421 decreases the activity and single-channel conductance of alamethicin. Surprisingly, we found that RH 421 increases the activity of melittin and magainin, suggesting that RH 421 may contribute via electrostatic repulsions, among others, to an increase in the monolayer spontaneous curvature of the membrane. We propose that RH 421-induced dipole potential and membrane elasticity changes alter the peptide-induced channel dynamics, and the prevalence of one mechanism over the other for particular classes of peptides is dictated by the electrical and mechanical interactions which rule the pore-formation mechanism of such peptides.

General significance

These results point to a novel paradigm in which electrical and mechanical effects promoted by chemicals which preferentially alter the electrostatics of the membrane, may be employed to help distinguish among various pore-formation mechanisms of membrane-permeabilizing peptides.     

The inherent flexibility of peptides and protein fragments quantitized by CD in conjunction with CCA+

ECD spectroscopy is traditionally used for rapid, non‐atomic level structure analysis of natural products such as peptides and proteins. Unlike globular proteins, peptides less frequently adopt a single 3D‐fold in a time average manner. Moreover, they exhibit an ensemble of conformers composed of a multitude of substantially different structures. In principle, both ECD‐ and vibrational circular dichroism (VCD)‐spectroscopy are sensitive enough to pick up structural information on these dynamic ensembles. However, the interpretation of the raw spectral data of these highly dynamic molecular systems can be cumbersome. The herein presented Convex Constraint Analysis Plus method, or CCA+ for short ( http://www.chem.elte.hu/departments/protnmr/cca/ ), provides a unique opportunity for spectral ensemble analysis of peptides, glycopeptides, peptidomimetics, and other foldamers. The precision and accuracy of the approach is presented here through different peptide model systems. An interesting temperature and pH dependent folding and unfolding of a miniprotein (e.g. Tc5b variant) is also described. Analysis of CD spectra sets strongly affected by solvent and ion type is also introduced to account for severe environmental‐induced structure influencing effect(s). The deconvolution makes always possible the quantitative data analysis even when the interpretation of the deconvolution resulted in pure CD curves is complex. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The search for a deterministic origin for the presence of nonracemic amino-acids in meteorites: a computational approach

Amino-acid enantiomeric excesses (ee's) have been detected in different types of carbonaceous chondrites, all in favor of the L enantiomer. In this article, we discuss possible deterministic causes to the presence of these amino-acid ee's in meteorites and evaluate in particular enantioselective photolysis by circularly polarized light (CPL). The electronic circular dichroism spectra of a set of amino- and hydroxy-acids, all detected in chondritic matter but some with ee's and others without ee's, were calculated and compared. The spectra were calculated for the most stable conformation(s) of the considered molecules using quantum mechanical methods (density functional theory). Our results suggest that CPL photolysis in the gas phase was perhaps not at the origin of the presence of ee's in meteorites and that the search for another, but still unknown, deterministic cause must be seriously undertaken.     

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: A case study in latarcins

In silico structural analyses of sets of α-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.     

Spectroscopic properties of the nonplanar amide group: a computational study

Experimental studies suggest that amide bond may significantly deviate from planar arrangement even in linear peptides and proteins. In order to find out the extent to which such deviation may influence principal amide spectroscopic properties, we conducted a computational study of nonplanar N-methylacetamide (NMA) conformers. Vibrational absorption, Raman, and electronic spectra including optical activity were simulated with ab initio and density functional theory (DFT) methods. According to the results, small nonplanarity deviations may be detectable by nonpolarized spectroscopic techniques, albeit as subtle spectral changes. The optical activity methods, such as the vibrational circular dichroism (VCD), Raman optical activity (ROA), and electronic circular dichroism (CD, ECD), provide enhanced information about the amide nonplanarity, because planar amide is not optically active (chiral). For VCD, however, the inherently chiral contribution in most peptides and proteins most probably provides very weak signal in comparison with other contributions, such as the dipolar coupling. For the electronic CD, the nonplanarity contribution is relatively big and causes a strong CD couplet in the n-pi* absorption region accompanied by a red frequency shift. The pi-pi* CD region is relatively unaffected. The ROA spectroscopy appears most promising for the nonplanarity detection and the inherent chiral signal may dominate entire spectral parts. The amide I and III vibrational ROA bands are most challenging experimentally because of their relatively weak coupling to other peptide vibrations.     

Membrane interactions and biological activity of antimicrobial peptides from Australian scorpion

UyCT peptides are antimicrobial peptides isolated from the venom of the Australian scorpion. The activity of the UyCT peptides against Gram positive and Gram negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release (DR) of the fluorophore calcein from liposomes and isothermal titration calorimetry (ITC); and their secondary structure was determined by circular dichroism (CD). Three different lipid systems were used to mimic red blood cells, Escherichia coli and Staphylococcus aureus membranes. UyCT peptides exhibited broad spectrum antimicrobial activity with low MIC for S. aureus and multi-drug resistant Gram negative strains. Peptide combinations showed some synergy enhancing their potency but not hemolytic activity. The UyCT peptides adopted a helical structure in lipid environments and DR results confirmed that the mechanism of action is by disrupting the membrane. ITC data indicated that UyCT peptides preferred prokaryotic rather than eukaryotic membranes. The overall results suggest that UyCT peptides could be pharmaceutical leads for the treatment of Gram negative multiresistant bacterial infections, especially against Acinetobacter baumanni, and candidates for peptidomimetics to enhance their potency and minimize hemolysis. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-π interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular α-helices and β-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

Effect of proline position on the antimicrobial mechanism of buforin II

Buforin II (BF2) is a histone-derived antimicrobial peptide that causes cell death by translocating across membranes and interacting with nucleic acids. It contains one proline residue critical for its function. Previous research found that mutations replacing proline lead to decreased membrane translocation and antimicrobial activity as well as increased membrane permeabilization. This study further investigates the role of proline in BF2's antimicrobial mechanism by considering the effect of changing proline position on membrane translocation, membrane permeabilization, and antimicrobial activity. For this purpose, four mutants were made with proline substitution (P11A) or relocation (P11A/G7P, P11A/V12P, P11A/V15P). These mutations altered the amount of helical content. Although antimicrobial activity correlated with the α-helical content for the peptides containing proline, membrane translocation did not. This observation suggests that factors in BF2's bactericidal mechanism other than translocation must be altered by these mutations. To better explain these trends we also measured the nucleic acid binding and membrane permeabilization of the mutant peptides. A comparison of mutant and wild type BF2 activity revealed that BF2 relies principally on membrane translocation and nucleic acid binding for antimicrobial activity, although membrane permeabilization may play a secondary role for some BF2 variants. A better understanding of the role of proline in the BF2 antimicrobial mechanism will contribute to the further design and development of BF2 analogs. Moreover, since proline residues are prevalent among other antimicrobial peptides, this systematic characterization of BF2 provides general insights that can promote our understanding of other systems.     

Leonidas Zervas award lecture: Abiotic ligands for new quaternary architectures of peptides and proteins

This review is the result of a kind invitation to provide a report on the basis of the Leonidas Zervas award lecture at the 32nd European Peptide Society in Athens, Greece, September 2012. The lecture covered several topics including contributions toward new methods for the assembly of peptides and chemical modification of proteins. The present review will focus on another topic from the Zervas lecture, namely, our recent efforts at creating new, artificial architectures for the organization of the quaternary structure of proteins. Crucially, this is achieved with abiotic ligands and not protein surface engineering. This concept was demonstrated for the important biopharmaceutical peptide insulin, by using both abiotic metal ion binding ligands and perfluoroalkyl chains. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The ascaphins: a family of antimicrobial peptides from the skin secretions of the most primitive extant frog, Ascaphus truei

The tailed frog Ascaphus truei occupies a unique position in phylogeny as the most primitive extant anuran and is regarded as the sister taxon to the clade of all other living frogs. Eight structurally related peptides, termed ascaphins 1-8, were isolated from norepinephrine-stimulated skin secretions of A. truei and were shown to possess differential growth inhibitory activity against Escherichia coli and Staphylococcus aureus. Ascaphins 2-7 may be represented by the consensus amino acid sequence GX2DX2KGAAKX3KTVAX2IANX.COOH whereas ascaphin-1 (GFRDVLKGAAKAFVKTVAGHIAN.NH2) and ascaphin-8 (GFKDLLKGAAKALVKTVLF.NH2) contain a C-terminally alpha-amidated residue. The ascaphins show no appreciable structural similarity with other families of antimicrobial peptides from frog skin but display limited sequence identity with the cationic, amphipathic alpha-helical peptides pandinin 1 and opistoporin 1, isolated from the venoms of African scorpions. Ascaphin-8 shows the highest potency against a range of pathogenic microorganisms but has the greatest haemolytic activity. The data indicate that the host defence strategy of using antimicrobial peptides in skin secretions arose early in the evolution of anurans.     

The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): Structure drives preference for simple neutral glycosphingolipids

Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds. Experimental evidence for a GLTP-fold includes: i) far-UV circular dichroism (CD) showing secondary structure with high alpha-helix content and a low thermally-induced unfolding transition (~ 41 °C); ii) near-UV-CD indicating only subtle tertiary conformational change before/after interaction with membranes containing/lacking glycolipid; iii) Red-shifted tryptophan (Trp) emission wavelength maximum (λ_max ~ 352 nm) for apo-FAPP2-C-terminal domain consistent with surface exposed intrinsic Trp residues; iv) ‘signature’ GLTP-fold Trp fluorescence response, i.e., intensity decrease (~ 30%) accompanied by strongly blue-shifted λ_max (~ 14 nm) upon interaction with membranes containing glycolipid, supporting direct involvement of Trp in glycolipid binding and enabling estimation of partitioning affinities. A structurally-based preference for other simple uncharged GSLs, in addition to glucosylceramide, makes human FAPP2-GLTP more similar to fungal HET-C2 than to plant AtGLTP1 (glucosylceramide-specific) or to broadly GSL-selective human GLTP. These findings along with the distinct mRNA exon/intron organizations originating from single-copy genes on separate human chromosomes suggest adaptive evolutionary divergence by these two GLTP-folds.     

Influence of hydrocarbon-stapling on membrane interactions of synthetic antimicrobial peptides

Cyclization has been recognized as a valuable technique for increasing the efficacy of small molecule and peptide therapeutics. Here we report the application of a hydrocarbon staple to a rationally-designed cationic antimicrobial peptide (CAP) that acquires increased membrane targeting and interaction vs. its linear counterpart. The previously-described CAP, 6K-F17 (KKKKKK-AAFAAWAAFAA-NH₂) was used as the backbone for incorporation of an i to i?+?4 helical hydrocarbon staple through olefin ring closing metathesis. Stapled versions of 6K-F17 showed an increase in non-selective membrane interaction, where the staple itself enhances the degree of membrane interaction and rate of cell death while maintaining high potency against bacterial membranes. However, the higher averaged hydrophobicity imparted by the staple also significantly increases toxicity to mammalian cells. This deleterious effect is countered through stepwise reduction of the stapled 6K-F17’s backbone hydrophobicity through polar amino acid substitutions. Circular dichroism assessment of secondary structure in various bacterial membrane mimetics reveals that a helical structure may improve – but is not an absolute requirement for – antimicrobial activity of 6K-F17. Further, phosphorus-31 static solid state NMR spectra revealed that both non-toxic stapled and linear peptides bind bacterial membranes in a similar manner that does not involve a detergent-like mechanism of lipid removal. The overall results suggest that the technique of hydrocarbon stapling can be readily applied to membrane-interactive CAPs to modulate how they interact and target biological membranes.