Hydrophobic Residues Research Articles

Debittering and antioxidant improvement of soy protein hydrolysates using curcumin as hydrophobic core.

Protein hydrolysates possess various bioactive functions (e.g. antioxidant), but their bitter taste is unacceptable to most consumers. In the present study, a novel approach for debittering was introduced, which involved the utilization of a hydrophobic compound, curcumin (Cur). Soy protein hydrolysates (SPH), prepared through alcalase hydrolysis, served as the research model for this investigation. It was found that bitter intensity of SPH was dominated by the hydrophobic amino acid residues, and the addition of Cur could remarkably reduce bitterness. The debittering mechanism is attributed to the direct binding of Cur to the exposed hydrophobic amino acid residues of SPH via hydrophobic interaction, thereby shielding the hydrophobic bitter groups and hindering their interaction with the bitter taste receptors. Moreover, this debittering strategy leads to the generation of stable nanoparticles with a Cur-core/SPH-shell architecture, which can significantly improve the antioxidant capacity of SPH compared to those using biomacromolecules for encapsulation. Using curcumin as a hydrophobic core is a facile and feasible strategy with bifunction of debittering as well as improving bioactive effect of SPH, which may extend its application in foods. © 2024 Society of Chemical Industry.

Anchorage of bacterial effector at plasma membrane via selective phosphatidic acid binding to modulate host cell signaling.

Binding phospholipid is a simple, yet flexible, strategy for anchorage of bacterial effectors at cell membrane to manipulate host signaling responses. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate are the only two phospholipid species known to direct bacterial effectors to establish inner leaflet localization at the plasma membrane. Here, selectivity of phosphatidic acid (PA) by bacterial effectors for the plasma membrane anchorage and its molecular entity was identified. C-terminal BID domain of Bartonella T4SS effectors (Beps) directed the plasma membrane localization of Beps in host cells through binding with PA. A hydrophobic segment of the 'HOOK' subdomain from BID is inserted into the bilayer to enhance the interaction of positively charged residues with the lipid headgroups. Mutations of a conserved arginine facilitating the electrostatic interaction, a conserved glycine maintaining the stability of the PA binding groove, and hydrophobic residues determining membrane insertion, prevented the anchorage of Beps at the plasma membrane. Disassociation from plasma membrane to cytosol attenuated the BepC capacity to induce stress fiber formation and cell fragmentation in host cells. The substitution of alanine with aspartic acid at the -1 position preceding the conserved arginine residue hindered BepD anchoring at the plasma membrane, a vital prerequisite for its ability to elicit IL-10 secretion in host macrophages. In conclusion, our findings reveal the PA-binding properties of bacterial effectors to establish plasma membrane localization and will shed light on the intricate mechanisms employed by bacterial effectors within host cells.

P14ARF forms meso-scale assemblies upon phase separation with NPM1.

NPM1 is an abundant nucleolar chaperone that, in addition to facilitating ribosome biogenesis, contributes to nucleolar stress responses and tumor suppression through its regulation of the p14 Alternative Reading Frame tumor suppressor protein (p14ARF). Oncogenic stress induces p14ARF to inhibit MDM2, stabilize p53 and arrest the cell cycle. Under non-stress conditions, NPM1 stabilizes p14ARF in nucleoli, preventing its degradation and blocking p53 activation. However, the mechanisms underlying the regulation of p14ARF by NPM1 are unclear because the structural features of the p14ARF-NPM1 complex were elusive. Here we show that p14ARF assembles into a gel-like meso-scale network upon phase separation with NPM1. This assembly is mediated by intermolecular contacts formed by hydrophobic residues in an α-helix and β-strands within a partially folded N-terminal portion of p14ARF. These hydrophobic interactions promote phase separation with NPM1, enhance p14ARF nucleolar partitioning, restrict NPM1 diffusion within condensates and nucleoli, and reduce cellular proliferation. Our structural analysis provides insights into the multifaceted chaperone function of NPM1 in nucleoli by mechanistically linking the nucleolar localization of p14ARF to its partial folding and meso-scale assembly upon phase separation with NPM1.

Detectability of cytokine and chemokine using ELISA, following sample-inactivation using Triton X-100 or heat

Clinical samples are routinely inactivated before molecular assays to prevent pathogen transmission. Antibody-based assays are sensitive to changes in analyte conformation, but the impact of inactivation on the analyte detectability has been overlooked. This study assessed the effects of commonly used inactivation-methods, Triton X-100 (0.5%) and heat (60 °C, 1 h), on cytokine/chemokine detection in plasma, lung aspirates, and nasopharyngeal samples. Heat significantly reduced analyte detectability in plasma (IL-12p40, IL-15, IL-16, VEGF, IL-7, TNF-β) by 33–99% (p ≤ 0.02), while Triton X-100 minimally affected analytes in plasma and nasopharyngeal samples (11–37%, p ≤ 0.04) and had no significant impact on lung aspirates. Structural analysis revealed that cytokines affected by heat had more hydrophobic residues and higher instability-indices. As the protein-detectability was affected differently in different sample types, the sample environment could also influence protein stability. This underscores the importance of selecting the most suitable inactivation methods for clinical samples to ensure accurate cytokine/chemokine analysis in both clinical and research settings.

Exploring structural and biological insights of TEAD through rational design and synthesis of niflumic acid derivatives

Transcriptional enhanced associate domain (TEAD) transcription factors undergo auto-palmitoylation, which is critical to mediate their function and maintain stability. Targeting the palmitate binding pocket of TEAD holds considerable promise for drug discovery, and it can be characterised into three components: a conserved cysteine, a hydrophobic main pocket, and a hydrophilic side pocket. Endogenous palmitate and several known TEAD inhibitors interact with the cysteine and hydrophobic residues in the deep hydrophobic pocket. We anticipate that precise targeting of the polar side pocket could facilitate the discovery of inhibitors with enhanced potencies and properties. Herein, we selected niflumic acid as the core scaffold suitable for targeting the three characteristic components of TEAD palmitate pocket. Reversible and irreversible compounds with substituents capable of directing each part of the palmitate pocket were designed. The newly synthesised compounds inhibited the palmitoylation and transcriptional activity of TEAD and elicited growth-inhibitory effects against several carcinomas, including mesothelioma.

Computational Analysis of Interactions Between Drugs and Human Serum Albumin.

Drug molecules exist as complexed with serum proteins such as human serum albumin (HSA) and/or unbound free form in the blood circulation. Drugs can be effective only when they are free. Thus, it is important to understand aspects that are important for interaction between drugs and interacting proteins. In this study, interactions among 2990 FDA approved drugs and HSA were computational analyzed to unravel principles that are critical for drug-HSA interactions. Docking results showed that drugs have higher affinity toward cavity-1 (C1) than cavity-2 (C2). A total of 1131 drug molecules have docking score greater than 60 while 768 molecules have docking score greater than 60 when they are docked in C2. In addition, three solvent channels have potential to direct solvent to C1 cavity while C2 does not have any effective channel. The post MD analyses demonstrated that drugs are making polar interactions with basic amino acids in the binding cavities. Verbscoside and ceftazidime both have stable low RMSD values throughout MD simulation with 2 Å on average in C1 cavity. The ligand RMSD shows less stability for verbscoside, which is around 4 Å when it is in complex with HSA in C1. The individual contribution of the residues K192, K196, R215, and R254 to ceftazidime are -1.92 ± 0.18, -3.09 ± 0.09, -2.17 ± 0.17, and - 2.32 ± 0.098, respectively. These residues contribute the binding energy of the verbscoside by -6.06 ± 0.08, -2.10 ± 0.06, and - 1.57 ± 0.03 kcal/mol individually in C1 cavity. C2 is making polar interactions with drug via R469, K472, and K488 residues and their contribution to the two drugs are -3.13 ± 0.21 kcal/mol for R469, -1.94 ± 0.18 kcal/mol for K472, and -1.96 ± 0.11 kcal/mol for K488 to total binding energy of ceftazidime. The binding energy of verbscoside is 57.17 ± 7.00 kcal/mol and Arg-407 has the highest contribution this bind energy individually with -4.29 ± 0.12 kcal/mol. Drugs with hydrogen bond donor/acceptor chemical adducts such as verbscoside involve higher hydrogen bond formation in C1 pocket. Ceftazidime makes interaction with HSA toward hydrophobic residues, L384, L404, L487, and L488 in the C2 cavity.

Screening and computational characterization of novel antimicrobial cathelicidins from amphibian transcriptomic data

As cold-blooded organisms living in damp and dark environments, amphibians have evolved robust defense mechanisms to protect themselves from predators and infections. Among the wide repertoire of bioactive compounds they produce are antimicrobial peptides (AMPs), which are required as part of innate immunity. One important class of AMPs is cathelicidins, known for their broad-spectrum activity against pathogens and their immunoregulatory roles. However, despite their promising biomedical potential and the increasing availability of omics data, few cathelicidins have been studied in amphibians, mostly through conventional experimental techniques. Here, we present 210 novel cathelicidin sequences from amphibian transcriptomes, identified through a comprehensive computational pipeline, which employed HMMER and BLAST tools to screen cathelicidin domains. These sequences reveal a typical tripartite domain architecture that was confirmed by SignalP and InterProScan analysis. Phylogenetic inference with IQ-TREE classified the sequences into six categories based on evolutionary relationships. Compared to cathelicidins from other vertebrates, amphibian mature peptides exhibit longer average lengths (around 50 amino acids), fewer aromatic and hydrophobic residues, and reduced thermal stability. Furthermore, these amphibian cathelicidins were characterized for their physicochemical and biological properties, revealing significant antimicrobial potential with lower hemolytic capability, especially in anurans, which suggests a balance between their antimicrobial and hemolytic activities predicted through AMPlify, ampir, AmpGram, and HemoPI. Secondary structure estimations, including three-dimensional modeling using AlphaFold2, indicate that amphibian cathelicidins predominantly feature α-helices and coils. Some representative models also display a high α-helix composition with amphipathic topology, facilitating interactions with simulated bacterial membranes as assessed by the PPM approach. Thus, these findings highlight the functional role of cathelicidins in amphibian immunity and their promising biomedical applicability, emphasizing the importance of applying computational methods to expand the scope and reveal the diverse landscape of cathelicidins across vertebrates.

Impact of missense mutations on the structure–function relationship of human succinyl-CoA synthetase using in silico analysis

The encephalomyopathic mtDNA depletion syndrome with methylmalonic aciduria is associated with succinyl-CoA synthetase (SCS) deficiency caused by pathogenic variants in genes encoding its two subunits. SCS is a mitochondrial enzyme involved in several metabolic pathways and acts as a heterodimer composed of α and β subunits encoded by SUCLG1 and SUCLA2 genes, respectively. The purpose of this study was to analyze the effects of the most pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) by applying, using different prediction tools, a filtering strategy, on the 343 and 365 nsSNPs found in SUCLG1 and SUCLA2 genes, respectively, retrieved from the databases, then to evaluate their structural and functional effects using homology modeling and molecular docking. Results showed that most deleterious mutations selected for structural analysis were located in loop regions critical for protein stability and function, especially, variants altering glycine and proline residues in these regions supporting their importance. We also showed that variants leading to hydrophobic and hydrophilic residues can destabilize the folding and binding of the protein. Molecular docking has also been used to identify the most important regions of ligand binding site (CoA binding site, ADP-Mg2+ binding site and phosphate ion binding site) and between the two subunits themselves, which mainly involving the ligase CoA domain. Our structural analysis, performed on selected nsSNP, are in accordance with experimental studies reported in the literature and predicted that they would responsible to either nonfunctional protein, subunit instability resulting in reduced amounts of misassembled protein, or in a protein unable to phosphorylate ADP.

Biophysical characterization of blood coagulation factor VIII binding to lipid nanodiscs that mimic activated platelet surfaces.

Following proteolytic activation, activated blood coagulation factor VIII (FVIIIa) binds to activated platelet membranes, forming the intrinsic tenase complex with activated factor IX (FIXa). Previous studies have identified the C1 and C2 domains as the membrane binding domains of FVIII through conserved arginine residues. A membrane binding model for the FVIII C domains proposes that surface exposed hydrophobic and positively charged residues at each C domain interact with the membrane, yet a comprehensive thermodynamic and structural description of this interaction is lacking. To determine residues of interaction, thermodynamics, and membrane binding preference for FVIII membrane association. Binding of FVIII constructs to lipid nanodiscs were characterized with nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), bio-layer interferometry (BLI), and X-ray crystallography. The thermodynamics of FVIII membrane binding indicate that the C1 domain associates through an enthalpically driven process while the C2 domain is entropically driven. Alanine mutations to surface-exposed hydrophobic residues in the C2 domain reveal differential effects on membrane binding, highlighting important determinants at the residue-level. The structure of a C2 double mutant, L2251A/L2252A, demonstrates that its decreased affinity is likely due to decreasing the surface area hydrophobicity. NMR studies with the C2 domain identified residues of interaction with soluble O-phospho-L-serine (OPLS) as well as lipid nanodiscs. Lastly, increasing phosphatidylethanolamine (PE) and decreasing PS content decreases overall FVIII affinity for membrane surfaces. This study provides further insight into the molecular basis for how FVIII interacts with platelets to form the intrinsic tenase complex.

The CTR hydrophobic residues of Nem1 catalytic subunit are required to form a protein phosphatase complex with Spo7 to activate yeast Pah1 PA phosphatase

The CTR hydrophobic residues of Nem1 catalytic subunit are required to form a protein phosphatase complex with Spo7 to activate yeast Pah1 PA phosphatase

Novel structural determinants and bacterial death-related regulatory effects of the scorpion defensin BmKDfsin4 against gram-positive bacteria

Numerous defensins constitute a family of cationic antimicrobial peptides with high degrees of sequence variability, and in-depth characterization of their structural basis and antibacterial mechanisms remains limited. Here, a representative scorpion defensin, BmKDfsin4, with two distinct hydrophobic and basic residue clusters, was extensively investigated. The hydrophobic residue cluster, formed by Phe2, Pro5, Phe6, Phe28 and Leu29 residues, strongly influences the antibacterial activity of BmKDfsin4 against Gram-positive bacteria. Compared with the three scattered Lys13, Lys30 and Arg32 residues, the basic residue cluster, consisting of the Arg19, Arg20, Arg21 and Arg37 residues, played a less important role. The synergistic interaction between the basic residue cluster and Arg32 significantly affected BmKDfsin4 function. The bacterial growth inhibition by BmKDfsin4 was associated with regulating expression levels of cell division-related genes, cell wall synthesis-related genes and bacterial autolysis-related genes rather than destroying the bacterial cell membrane. The coincubation of BmKDfsin4 with the bacterial strains induced gradual changes in the bacterial surface from a rough and thin surface to a noticeably wrinkled surface together with abundant white spots and even complete cavities within the bacteria. These findings revealed novel structural determinants and bacterial death-related regulatory effects of the defensin BmKDfsin4 and highlighted diverse antibacterial mechanisms of defensins.

Potential effects of pH changes in the micro-environment during desalination on infant powder: The perspective of pH shift affecting whey protein molecular structure and function

In order to simulate the effect of pH environment change caused by different desalting methods on whey protein (WP) in actual production, the WP was treated under different acidic pH (2.0–6.0) and pH shift (pH recovery). The molecular structure, properties, and functions of the proteins were further analyzed. The results showed that the spatial structure of WP was unfolded under acidic conditions, α-helix and β-sheet were transformed into random coils, hydrophobic residues and free sulfhydryl groups were exposed. The particle size of WP was increased, and the emulsification, foaming and thermal stability were improved significantly. Especially after pH2 treatment, WP has the largest expansion degree, emulsification and foaming properties increased to 232.96 m2/g and 158.83 %. After pH recovery, the partially unfolded structure refolded, reducing the surface hydrophobicity, forming a smaller particle size of soluble protein particles, solubility has increased to 83.95 % compared to the natural. However, the emulsification, foamability, and thermal stability after pH shift are slightly reduced compared to acidic conditions, making it easier to form a gel after heating.

Amyloid fibrils for β-carotene delivery – Influence of self-assembled structures on binding and in vitro release behavior

Two whey protein isolate amyloid fibrils (WPIF) with different structure were prepared, and the effects of these structures on binding of β-carotene (BC) and in vitro digestibility were evaluated. Whey protein isolate (WPI) in water (80 °C, pH 2.0) self-assembled into elongated WPIF (E-WPIF), whereas WPI formed to worm-like WPIF (W-WPIF) in trifluoroethanol. Compared to E-WPIF, W-WPIF showed higher surface hydrophobicity, indicating exposure of more hydrophobic residues. The encapsulation efficiency and loading capacity of BC in W-WPIF were higher than that of E-WPIF. The hydrophobic interaction were the main driving forces of WPIF/BC. During gastric digestion, WPIF lost intact fibrils structures, resulting in unordered small aggregates and most BC still bound to them. Then they were destroyed in the following intestinal digestion, leading to the release of BC. Compared with W-WPIF/BC, E-WPIF/BC had higher release of BC in gastrointestinal digestion due to weaker binding of BC and better digestibility of E-WPIF.

Effect of pH-Shift Treatment on IgE-Binding Capacity and Conformational Structures of Peanut Protein.

Hypoallergenic processing is an area worthy of continued exploration. In the treatment of the peanut protein (PP), pH shift was applied by acidic (pH 1.0-4.0) and alkaline (pH 9.0-12.0) treatment, after which the pH was adjusted to 7.0. Following pH-shift treatment, PP showed a larger particle size than in neutral solutions. SDS-PAGE, CD analysis, intrinsic fluorescence, UV spectra, and surface hydrophobicity indicated the protein conformation was unfolded with the exposure of more buried hydrophobic residues. Additionally, the IgE-binding capacity of PP decreased after pH-shift treatment on both sides. Label-free LC-MS/MS results demonstrated that the pH-shift treatment induced the structural changes on allergens, which altered the abundance of peptides after tryptic digestion. Less linear IgE-binding epitopes were detected in PP with pH-shift treatment. Our results suggested the pH-shift treatment is a promising alternative approach in the peanut hypoallergenic processing. This study also provides a theoretical basis for the development of hypoallergenic food processing.

Controlled Translocation of Proteins through a Biological Nanopore for Single-Protein Fingerprint Identification.

After the successful sequencing of nucleic acids, nanopore technology has now been applied to proteins. Recently, it has been demonstrated that an electro-osmotic flow can be used to induce the transport of unraveled polypeptides across nanopores. Polypeptide translocation, however, is too fast for accurate reading its amino acid compositions. Here, we show that the introduction of hydrophobic residues into the lumen of the nanopore reduces the protein translocation speed. Importantly, the introduction of a tyrosine at the entry of the nanopore and an isoleucine at the entry of the β-barrel of the nanopore reduced the speed of translocation to ∼10 amino acids/millisecond while keeping a relatively large ionic current, a crucial component for protein identification. These nanopores showed unique features within their current signatures, which may pave the way toward protein fingerprinting using nanopores.

Protein folding: Funnel model revised

The spatial structure of proteins, largely determined by their amino acid sequences, is also dependent on the environmental conditions under which the folding process takes place. In aqueous environments, exposure of polar amino acids is the driving factor, whereas protein stabilization in amphipathic membranes requires exposure to hydrophobic residues. This observation can be extended to all other environmental conditions under which proteins exhibit biological activity and, most importantly, to the folding process. The fuzzy oil drop (FOD) model assumes a centric location of hydrophobic residues (hydrophobic core) with exposure of polar residues towards the aqueous environment, as the influence of the aqueous environment is extended to include the contribution of other non-aqueous factors, enabling the assessment of their influence on protein structuring. The application of the modified FOD model (FOD-M) we have developed allows the environment to be represented as an external force field in the form of a continuum. The role of environmental conditions allows modification of the funnel model expressing the localization of the energy minimum as dependent on external conditions expressed by the K scale, where K measures the degree of other than polar water factors participating in folding process.

A machine learning approach uncovers principles and determinants of eukaryotic ribosome pausing.

Nonuniform local translation speed dictates diverse protein biogenesis outcomes. To unify known and uncover unknown principles governing eukaryotic elongation rate, we developed a machine learning pipeline to analyze RiboSeq datasets. We find that the chemical nature of the incoming amino acid determines how codon optimality influences elongation rate, with hydrophobic residues more dependent on transfer RNA (tRNA) levels than charged residues. Unexpectedly, we find that wobble interactions exert a widespread effect on elongation pausing, with wobble-mediated decoding being slower than Watson-Crick decoding, irrespective of tRNA levels. Applying our ribosome pausing principles to ribosome collisions reveals that disomes arise upon apposition of fast-decoding and slow-decoding signatures. We conclude that codon choice and tRNA pools are evolutionarily constrained to harmonize elongation rate with cotranslational folding while minimizing wobble pairing and deleterious stalling.

Interactive Structural Analysis of KH3-4 Didomains of IGF2BPs with Preferred RNA Motif Having m6A Through Dynamics Simulation Studies.

m6A modification is the most common internal modification of messenger RNA in eukaryotes, and the disorder of m6A can trigger cancer progression. The GGACU is considered the most frequent consensus sequence of target transcripts which have a GGAC m6A core motif. Newly identified m6A 'readers' insulin-like growth factor 2 mRNA-binding proteins modulate gene expression by binding to the m6A binding sites of target mRNAs, thereby affecting various cancer-related processes. The dynamic impact of the methylation at m6A within the GGAC motif on human IGF2BPs has not been investigated at the structural level. In this study, through in silico analysis, we mapped IGF2BPs binding sites for the GGm6AC RNA core motif of target mRNAs. Subsequent molecular dynamics simulation analysis at 400 ns revealed that only the KH4 domain of IGF2BP1, containing the 503GKGG506 motif and its periphery residues, was involved in the interaction with the GGm6AC backbone. Meanwhile, the methyl group of m6A is accommodated by a shallow hydrophobic cradle formed by hydrophobic residues. Interestingly, in IGF2BP2 and IGF2BP3 complexes, the RNA was observed to shift from the KH4 domain to the KH3 domain in the simulation at 400 ns, indicating a distinct dynamic behavior. This suggests a conformational stabilization upon binding, likely essential for the functional interactions involving the KH3-4 domains. These findings highlight the potential of targeting IGF2BPs' interactions with m6A modifications for the development of novel oncological therapies.

A Nonlinear Peptide Topology for Synthetic Virions.

a nonlinear de novo peptide topology for the assembly of synthetic virions is reported. The topology is a backbone cyclized amino-acid sequence in which polar l- and hydrophobic d-amino acid residues of the same-type alternate. This arrangement introduces pseudo C4 symmetries of side chains within the same cyclopeptide ring, allowing for the lateral propagation of cyclopeptides into networks with a [3/6, 4]-fold rotational symmetry closing into virus-like shells. A combination of computational and experimental approaches was used to establish that the topology forms morphologically uniform, nonaggregating and nontoxic nanoscale shells. These effectively encapsulate genetic cargo and promote its intracellular delivery and a target genetic response. The design introduces a nanotechnology inspired solution for engineering virus-like systems thereby expanding traditional molecular biology approaches used to create artificial biology to chemical space.

Comparative Properties of Helical and Linear Amphipathicity of Peptides Composed of Arginine, Tryptophan, and Valine.

The persistence of antibiotic resistance has incited a strong interest in the discovery of agents with novel antimicrobial mechanisms. The direct killing of multidrug-resistant bacteria by cationic antimicrobial peptides (AMPs) underscores their importance in the fight against infections associated with antibiotic resistance. Despite a vast body of AMP literature demonstrating a plurality in structural classes, AMP engineering has been largely skewed toward peptides with idealized amphipathic helices (H-amphipathic). In contrast to helical amphipathicity, we designed a series of peptides that display the amphipathic motifs in the primary structure. We previously developed a rational framework for designing AMP libraries of H-amphipathic peptides consisting of Arg, Trp, and Val (H-RWV, with a confirmed helicity up to 88% in the presence of membrane lipids) tested against the most common MDR organisms. In this study, we re-engineered one of the series of the H-RWV peptides (8, 10, 12, 14, and 16 residues in length) to display the amphipathicity in the primary structure by side-by-side (linear) alignment of the cationic and hydrophobic residues into the 2 separate linear amphipathic (L-amphipathic) motifs. We compared the 2 series of peptides for antibacterial activity, red blood cell (RBC) lysis, killing and membrane-perturbation properties. The L-RWV peptides achieved the highest antibacterial activity at a minimum length of 12 residues (L-RWV12, minimum optimal length or MOL) with the lowest mean MIC of 3-4 µM, whereas the MOL for the H-RWV series was reached at 16 residues (H-RWV16). Overall, H-RWV16 displayed the lowest mean MIC at 2 µM but higher levels of RBC lysis (25-30%), while the L-RWV series displayed minor RBC lytic effects at the test concentrations. Interestingly, when the S. aureus strain SA719 was chosen because of its susceptibility to most of the peptides, none of the L-RWV peptides demonstrated a high level of membrane perturbation determined by propidium iodide incorporation measured by flow cytometry, with <50% PI incorporation for the L-RWV peptides. By contrast, most H-RWV peptides displayed almost up to 100% PI incorporation. The results suggest that membrane perturbation is not the primary killing mechanism of the L-amphipathic RWV peptides, in contrast to the H-RWV peptides. Taken together, the data indicate that both types of amphipathicity may provide different ideal pharmacological properties that deserve further investigation.