Peptide-membrane Interactions Research Articles

Rationally designed PMAP-23 derivatives with enhanced bactericidal and anticancer activity based on the molecular mechanism of peptide-membrane interactions.

Antimicrobial peptides (AMPs) are a crucial component of the natural defense system that the host employs to protect itself against invading pathogens. PMAP-23, a cathelicidin-derived AMP, has potent and broad-spectrum antimicrobial activity. Our earlier studies led us to hypothesize that PMAP-23 adopts a dynamic helix-hinge-helix structure, initially attaching to membrane surfaces through the N-helix and subsequently inserting the C-helix into the lipid bilayer. Here, we rationally designed PMAP-NC with increased amphipathicity and hydrophobicity in the N- and C-helix, respectively, based on the hypothesis of the interaction of PMAP-23 with membranes. Compared to the parental PMAP-23, PMAP-NC showed two-eightfold improved bactericidal activity against both Gram-positive and Gram-negative strains with fast killing kinetics. Fluorescence studies demonstrated that PMAP-NC largely disrupted membrane integrity, indicating that efficiency and kinetics of bacterial killing are associated with the membrane permeabilization. Interestingly, PMAP-NC exhibited much better anticancer activity against tumor cells than PMAP-23 but displayed low hemolytic activity against human erythrocytes. Collectively, our findings suggest that PMAP-NC, with the structural arrangement of an amphipathic helix-hinge-hydrophobic helix that plays a critical role in rapid and efficient membrane permeabilization, can be an attractive candidate for novel antimicrobial and/or anticancer drugs.

The Significance of Hypervariability and Conserved Motifs in Antimicrobial Peptides from Tree Frogs.

Amphibians' skin is a rich source of natural antimicrobial peptides (AMPs). These AMPs exhibit marked inter- and intraspecific sequence divergence linked to the arms race between host and pathogens. Here, we combine peptidomics, molecular modeling, and phylogenetic analyses to understand the evolution of AMPs in Cophomantini, a diverse clade of neotropical tree frogs, and to investigate their interaction with bacterial membranes. Consistent with results in other amphibians, all species of Cophomantini secrete a mixture of peptides. We selected the hylin peptide family to survey sequence variability and the presence of common amino acid motifs. We found that most species secrete a unique set of hylins that, though variable, share the conserved motif Gly-X-X-X-Pro-Ala-X-X-Gly, with Gly and Pro colocalizing with charged or polar residues. Our modeling revealed that Pro curves the peptide through a hinge, facilitating its insertion into the bacterial membrane and, once inserted, contributes to stabilizing the pore structure. The phylogenetic inference using hylid prepro-peptides showed the need to classify new AMPs using the full-length sequence of the prepro-peptide region and highlighted the complex relationships between peptide families. Our findings revealed that conserved motifs occurred independently in distinct AMP families, suggesting a convergent evolution and a significant role in peptide-membrane interactions.

Transition between Different Diffusion Modes of Individual Lipids during the Membrane-Specific Action of As-CATH4 Peptides.

The cell membrane permeabilization ability of immune defense antimicrobial peptides (AMPs) is widely applied in biomedicine. Although the mechanisms of peptide-membrane interactions have been widely investigated, analyses at the molecular level are still lacking. Herein, the membrane-specific action of a native AMP, As-CATH4, is investigated using a single-lipid tracking method in combination with live cell and model membrane assays conducted at different scales. The peptide-membrane interaction process is characterized by analyzing single-lipid diffusion behaviors. As-CATH4 exhibits potent antimicrobial activity through bacterial membrane permeabilization, with moderate cytotoxicity against mammalian cells. In-plane diffusion analyses of individual lipids show that the lipid molecules exhibit non-Gaussian and heterogeneous diffusion behaviors in both pristine and peptide-treated membranes, which can be decomposed into two Gaussian subgroups corresponding to normal- and slow-diffusive lipids. Assessment of the temporal evolution of these two diffusion modes of lipids reveal that the peptide action states of As-CATH4 include surface binding, transmembrane defect formation, and dynamic equilibrium. The action mechanisms of As-CATH4 at varying concentrations and against different membranes are distinguished. This work resolves the simultaneous mixed diffusion mechanisms of single lipids in biomimetic cell membranes, especially during dynamic membrane permeabilization by AMPs.

The Dynamic Interactions of a Multitargeting Domain in Ameloblastin Protein with Amelogenin and Membrane.

The enamel matrix protein Ameloblastin (Ambn) has critical physiological functions, including regulation of mineral formation, cell differentiation, and cell-matrix adhesion. We investigated localized structural changes in Ambn during its interactions with its targets. We performed biophysical assays and used liposomes as a cell membrane model. The xAB2N and AB2 peptides were rationally designed to encompass regions of Ambn that contained self-assembly and helix-containing membrane-binding motifs. Electron paramagnetic resonance (EPR) on spin-labeled peptides showed localized structural gains in the presence of liposomes, amelogenin (Amel), and Ambn. Vesicle clearance and leakage assays indicated that peptide-membrane interactions were independent from peptide self-association. Tryptophan fluorescence and EPR showed competition between Ambn-Amel and Ambn-membrane interactions. We demonstrate localized structural changes in Ambn upon interaction with different targets via a multitargeting domain, spanning residues 57 to 90 of mouse Ambn. Structural changes of Ambn following its interaction with different targets have relevant implications for the multifunctionality of Ambn in enamel formation.

Unravelling the atomistic details of peptide induced membrane alterations.

Membrane active peptides (MAPs) directly target the cell membrane to disrupt or translocate through it. This membrane modulating capacity of MAPs has unravelled their potential in various theranostic applications. Broad spectrum activity, low cytotoxicity and slow rate of emergence of antibiotic resistance make them an ideal alternative for the current therapeutics regime. However, despite significant progress in the field, the number of peptides reaching clinical trials is very low. The major hurdle is the poor understanding of their mechanism of action, which is not well elucidated to date; however, specific models have been proposed. Multidisciplinary approaches involving simulations and biophysics provide valuable insights into understanding the interaction of the peptide with the membrane. Herein, we have focused on two major classes of membrane-active peptides involving a cell-penetrating peptide (CPP) and an antimicrobial peptide (AMP). The cytotoxicity of the peptides was evaluated using a colourimetric assay. Morphological alterations of the cell membrane induced by peptides were examined using scanning electron microscopy (SEM) studies. With atomic force microscopy (AFM), cell surface roughness and height profiles were evaluated before and after peptide treatment. Significant changes in the roughness were observed. Zeta potential measurement was employed to observe the change in cell surface charge on peptide membrane interaction. Binding affinities of the peptide with membrane were assessed calorimetrically. Furthermore, molecular dynamic simulation studies were employed to provide atomic level resolution and detailed biophysical characterization of the membrane interaction process.

Untangling the rivalry between cholesterol and antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising candidates for next generation antimicrobial therapeutics. In mammalian cell membranes cholesterol plays a key regulatory function in antibiotic drug resistance and the immune response. However, the mechanism of action and the selectivity of AMPs towards bacterial membranes are not yet well understood. We hypothesized that differences in biophysical properties of mammalian versus bacterial membranes give rise to differences in peptide-membrane interactions which underlie the AMP mechanism and selectivity. Solid-state 2H NMR spectroscopy was employed to study di-monounsaturated phosphatidylcholine and phosphatidylethanolaminelipids (DOPC versus DOPE) bilayers in the presence of the antimicrobial peptide LL37 (2−4 mol%) and cholesterol (20−30% mol) as model systems mimicking mammalian and bacterial membranes. The lipid systems were probed with POPC-d31 (10 mol%) where the residual quadrupolar couplings yielded segmental order parameters (SCD) for the individual acyl segments. The LL37-peptide decreased the quadrupolar splittings indicating it increased the area per lipid, while cholesterol showed the opposite. The mean-torque model was used to calculate the key bilayer properties of area per lipid and bilayer thickness [2]. The model-free functional dependence of spin-lattice relaxation rates on SCD (square-law plots) indicated greater bending rigidity with cholesterol for both DOPC and DOPE membranes. By contrast LL37 decreased the bending rigidity of the membranes. Additionally all-atom molecular dynamics simulations for bilayers comprising POPE/DMPG/cardiolipin (90:5:5) and DMPC/cholesterol (90:10) were conducted with LL37 peptides on the bilayer surface to capture the molecular-level interactions. We observed partial insertion of the LL37-peptides in the POPE system, while the peptides were repelled from the membrane in the DMPC/cholesterol system. These observations reveal the surprising rivalry between cholesterol and AMPs which underlies AMP mechanism and selectivity.

Fluorescence Resonance Energy Transfer to Detect Plasma Membrane Perturbations in Giant Plasma Membrane Vesicles.

Disruptions and perturbations of the cellular plasma membrane by peptides have garnered significant interest in the elucidation of biological phenomena. Typically, these complex processes are studied using liposomes as model membranes-either by encapsulating a fluorescent dye or by other spectroscopic approaches, such as nuclear magnetic resonance. Despite incorporating physiologically relevant lipids, no synthetic model truly recapitulates the full complexity and molecular diversity of the plasma membrane. Here, biologically representative membrane models, giant plasma membrane vesicles (GPMVs), are prepared from eukaryotic cells by inducing a budding event with a chemical stressor. The GPMVs are then isolated, and bilayers are labelled with fluorescent lipophilic tracers and incubated in a microplate with a membrane-active peptide. As the membranes become damaged and/or aggregate, the resulting fluorescence resonance energy transfer (FRET) between the two tracers increases and is measured periodically in a microplate. This approach offers a particularly useful way to detect perturbations when the membrane complexity is an important variable to consider. Additionally, it provides a way to kinetically detect damage to the plasma membrane, which can be correlated with the kinetics of peptide self-assembly or structural rearrangements. Key features • Allows testing of various peptide-membrane interaction conditions (peptide:phospholipid ratio, ionic strength, buffer, etc.) at once. • Uses intact plasma membrane vesicles that can be prepared from a variety of cell lines. • Can offer comparable throughput as with traditional synthetic lipid models (e.g., dye-encapsulated liposomes).

Lipid oxidation controls peptide self-assembly near membranes through a surface attraction mechanism.

The self-assembly of peptides into supramolecular structures has been linked to neurodegenerative diseases but has also been observed in functional roles. Peptides are physiologically exposed to crowded environments of biomacromolecules, and particularly cellular membrane lipids. Previous research has shown that membranes can both accelerate and inhibit peptide self-assembly. Here, we studied the impact of membrane models that mimic cellular oxidative stress and compared this to mammalian and bacterial membranes. Using molecular dynamics simulations and experiments, we propose a model that explains how changes in peptide-membrane binding, electrostatics, and peptide secondary structure stabilization determine the nature of peptide self-assembly. We explored the influence of zwitterionic (POPC), anionic (POPG) and oxidized (PazePC) phospholipids, as well as cholesterol, and mixtures thereof, on the self-assembly kinetics of the amyloid β (1-40) peptide (Aβ40), linked to Alzheimer's disease, and the amyloid-forming antimicrobial peptide uperin 3.5 (U3.5). We show that the presence of an oxidized lipid had similar effects on peptide self-assembly as the bacterial mimetic membrane. While Aβ40 fibril formation was accelerated, U3.5 aggregation was inhibited by the same lipids at the same peptide-to-lipid ratio. We attribute these findings and peptide-specific effects to differences in peptide-membrane adsorption with U3.5 being more strongly bound to the membrane surface and stabilized in an α-helical conformation compared to Aβ40. Different peptide-to-lipid ratios resulted in different effects. We found that electrostatic interactions are a primary driving force for peptide-membrane interaction, enabling us to propose a model for predicting how cellular changes might impact peptide self-assembly in vivo.

Recent Advances of Studies on Cell-Penetrating Peptides Based on Molecular Dynamics Simulations

With the ability to transport cargo molecules across cell membranes with low toxicity, cell-penetrating peptides (CPPs) have become promising candidates for next generation peptide-based drug delivery vectors. Over the past three decades since the first CPP was discovered, a great deal of work has been done on the cellular uptake mechanisms and the applications for the delivery of therapeutic molecules, and significant advances have been made. But so far, we still do not have a precise and unified understanding of the structure-activity relationship of the CPPs. Molecular dynamics (MD) simulations provide a method to reveal peptide-membrane interactions at the atomistic level and have become an effective complement to experiments. In this paper, we review the progress of the MD simulations on CPP-membrane interactions, including the computational methods and technical improvements in the MD simulations, the research achievements in the CPP internalization mechanism, CPP decoration and coupling, and the peptide-induced membrane reactions during the penetration process, as well as the comparison of simulated and experimental results.

Bending Elasticity of Phospholipid Bilayers Containing an Amphipathic Peptide with Low Mammalian Cytotoxicity


 
 
 Peptide mimetics imitate natural peptides’ structure but they could be specifically designed to be more selective concerning their toxicity to mammalian cells. In most cases this specificity is due to their ability to form α-helix in amphipathic environment. In addition, the specific activity depends on the ability of final structure to penetrate cell membrane. Being responsible for the cell integrity and compartmentalization, biomembranes also play a major role in cellular processes, in which the membrane deformations are important. In the present study we probe peptide-membrane interactions for a shortened amino acid sequence KLAKLAK-NH2 of an antimicrobial peptide with apoptotic effect. The bending rigidity of model lipid bilayers is measured by flicker spectroscopy of quasispherical unilamellar vesicles monitored and analyzed in phase contrast light microscopy. At high peptide concentrations ∼ 30 μmol/L and peptide-to-lipid total molar ratios ∼ 0.90 bilayer stacking formation is observed. A reduction of the bending constant is reported at peptide-to-lipid total molar ratio ∼ 0.80. The membrane softening indicates peripheral peptide orientation at the lipid bilayer, which is considered a prerequisite for channel formation. Based on KLAKLAK-NH2 effect on the membrane bending elasticity we provide an evaluation of the peptide partition coefficient characterizing its affinity to POPC bilayers. The acquired results might be helpful in efforts to further tailor the pharmacokinetic properties of antimicrobial peptides in combination with strengthened stability towards enzymatic degradation.
 
 

Probing human proteins for short encrypted antimicrobial peptides reveals Hs10, a peptide with selective activity for gram-negative bacteria

BackgroundSome cationic and amphiphilic α-helical segments of proteins adsorb to prokaryotic membranes when synthesized as individual polypeptide sequences, resulting in broad and potent antimicrobial activity. However, amphiphilicity, a determinant physicochemical property for peptide-membrane interactions, can also be observed in some β-sheets. MethodsThe software Kamal was used to scan the human reference proteome for short (7–11 amino acid residues) cationic and amphiphilic protein segments with the characteristic periodicity of β-sheets. Some of the uncovered peptides were chemically synthesized, and antimicrobial assays were conducted. Biophysical techniques were used to probe the molecular interaction of one peptide with phospholipid vesicles, lipopolysaccharides (LPS) and the bacterium Escherichia coli. ResultsThousands of compatible segments were found in human proteins, five were synthesized, and three presented antimicrobial activity in the micromolar range. Hs10, a nonapeptide fragment of the Complement C3 protein, could inhibit only the growth of tested Gram-negative microorganisms, presenting also little cytotoxicity to human fibroblasts. Hs10 interacted with LPS while transitioning from an unstructured segment to a β-sheet and increased the hydrodynamic radius of LPS particles. This peptide also promoted morphological alterations in E. coli cells. Conclusions: Data presented herein introduce yet another molecular template to probe proteins in search for encrypted membrane-active segments and demonstrates that, using this approach, short peptides with low cytotoxicity and high selectivity to prokaryotic cells might be obtained. General SignificanceThis work widens the biotechnological potential of the human proteome as a source of antimicrobial peptides with application in human health.

The Antimicrobial Peptide 1018-K6 Interacts Distinctly with Eukaryotic and Bacterial Membranes, the Basis of Its Specificity and Bactericidal Activity.

Since penicillin was discovered, antibiotics have been critical in the fight against infections. However, antibiotic misuse has led to drug resistance, which now constitutes a serious health problem. In this context, antimicrobial peptides (AMPs) constitute a natural group of short proteins, varying in structure and length, that act against certain types of bacterial pathogens. The antimicrobial peptide 1018-K6 (VRLIVKVRIWRR- NH2) has significant bactericidal and antibiofilm activity against Listeria monocytogenes isolates, and against different strains and serotypes of Salmonella. Here, the mechanism of action of 1018-K6 was explored further to understand the peptide–membrane interactions relevant to its activity, and to define their determinants. We combined studies with model synthetic membranes (liposomes) and model biological membranes, assessing the absorption maximum and the quenching of 1018-K6 fluorescence in aqueous and lipid environments, the self-quenching of carboxyfluorescein, as well as performing lipid sedimentation assays. The data obtained reflect the differential interactions of the 1018-K6 peptide with eukaryotic and prokaryotic membranes, and the specific interactions and mechanisms of action in the three prokaryotic species studied: Salmonella Typhimurium2GN, Escherichia coli3GN, and Staphylococcus aureus3GP. The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics.

PEGylation of the antimicrobial peptide LyeTx I-b maintains structure-related biological properties and improves selectivity.

The biological activity of antimicrobial peptides and proteins is closely related to their structural aspects and is sensitive to certain post-translational modifications such as glycosylation, lipidation and PEGylation. However, PEGylation of protein and peptide drugs has expanded in recent years due to the reduction of their toxicity. Due to their size, the PEGylation process can either preserve or compromise the overall structure of these biopolymers and their biological properties. The antimicrobial peptide LyeTx I-bcys was synthesized by Fmoc strategy and coupled to polyethylene glycol 2.0 kDa. The conjugates were purified by HPLC and characterized by MALDI-ToF-MS analysis. Microbiological assays with LyeTx I-bcys and LyeTx I-bPEG were performed against Staphylococcus aureus (ATCC 33591) and Escherichia coli (ATCC 25922) in liquid medium. MIC values of 2.0 and 1.0 µM for LyeTx I-bcys and 8.0 and 4.0 µM for LyeTx I-bPEG were observed against S. aureus and E. coli, respectively. PEGylation of LyeTx I-bcys (LyeTx I-bPEG) decreased the cytotoxicity determined by MTT method for VERO cells compared to the non-PEGylated peptide. In addition, structural and biophysical studies were performed to evaluate the effects of PEGylation on the nature of peptide-membrane interactions. Surface Plasmon Resonance experiments showed that LyeTx I-b binds to anionic membranes with an association constant twice higher than the PEGylated form. The three-dimensional NMR structures of LyeTx I-bcys and LyeTx I-bPEG were determined and compared with the LyeTx I-b structure, and the hydrodynamic diameter and zeta potential of POPC:POPG vesicles were similar upon the addition of both peptides. The mPEG-MAL conjugation of LyeTx I-bcys gave epimers, and it, together with LyeTx I-bPEG, showed clear α-helical profiles. While LyeTx I-bcys showed no significant change in amphipathicity compared to LyeTx I-b, LyeTx I-bPEG was found to have a slightly less clear separation between hydrophilic and hydrophobic faces. However, the similar conformational freedom of LyeTx I-b and LyeTx I-bPEG suggests that PEGylation does not cause significant structural changes. Overall, our structural and biophysical studies indicate that the PEGylation does not alter the mode of peptide interaction and maintains antimicrobial activity while minimizing tissue toxicity, which confirmed previous results obtained in vivo. Interestingly, significantly improved proteolytic resistance to trypsin and proteinase K was observed after PEGylation.

Biophysical Characterization of LTX-315 Anticancer Peptide Interactions with Model Membrane Platforms: Effect of Membrane Surface Charge.

LTX-315 is a clinical-stage, anticancer peptide therapeutic that disrupts cancer cell membranes. Existing mechanistic knowledge about LTX-315 has been obtained from cell-based biological assays, and there is an outstanding need to directly characterize the corresponding membrane-peptide interactions from a biophysical perspective. Herein, we investigated the membrane-disruptive properties of the LTX-315 peptide using three cell-membrane-mimicking membrane platforms on solid supports, namely the supported lipid bilayer, intact vesicle adlayer, and tethered lipid bilayer, in combination with quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) measurements. The results showed that the cationic LTX-315 peptide selectively disrupted negatively charged phospholipid membranes to a greater extent than zwitterionic or positively charged phospholipid membranes, whereby electrostatic interactions were the main factor to influence peptide attachment and membrane curvature was a secondary factor. Of note, the EIS measurements showed that the LTX-315 peptide extensively and irreversibly permeabilized negatively charged, tethered lipid bilayers that contained high phosphatidylserine lipid levels representative of the outer leaflet of cancer cell membranes, while circular dichroism (CD) spectroscopy experiments indicated that the LTX-315 peptide was structureless and the corresponding membrane-disruptive interactions did not involve peptide conformational changes. Dynamic light scattering (DLS) measurements further verified that the LTX-315 peptide selectively caused irreversible disruption of negatively charged lipid vesicles. Together, our findings demonstrate that the LTX-315 peptide preferentially disrupts negatively charged phospholipid membranes in an irreversible manner, which reinforces its potential as an emerging cancer immunotherapy and offers a biophysical framework to guide future peptide engineering efforts.

WITHDRAWN: Role of lipopolysaccharide in antimicrobial and cell penetrating peptide membrane interactions probed by deuterium NMR of whole cells

Understanding how non-lipid components of bacteria affect antimicrobial peptide (AMP)-induced membrane disruption is important for a comprehensive understanding of AMP mechanisms and informing AMP-based drug development. This study investigates how lipopolysaccharide (LPS) affects membrane disruption by the AMP MSI-78 and compares the results to the effect of TP2, a cell-penetrating peptide that crosses membrane bilayers without permeabilizing them. We destabilize the LPS layer of Escherichia coli (E. coli) cells via chelation of the stabilizing divalent cations. 2H NMR spectra of E. coli demonstrate that EDTA concentrations of 2.5 mM and 9.0 mM alone have very minor effects on lipid acyl chain order. Interestingly, we find that E. coli pre-treated with 9.0 mM EDTA before treatment with MSI-78 are more sensitive to AMP-induced acyl chain disruption, indicating that intact LPS reduces MSI-78-induced membrane disruption in E. coli. Surprisingly, we also found that at the level of 2H NMR, the peptide-induced acyl chain disruption is similar for MSI-78 and TP2, although MSI-78 permeabilizes the bilayer and TP2 does not. Furthermore, LPS disruption appears to protect the bacteria from TP2, although it sensitizes them to MSI-78.

Binding Models of Aβ42 Peptide with Membranes Explored by Molecular Simulations.

One of the factors contributing to the toxicity of amyloid-β (Aβ) peptides is the destruction of membrane integrity through Aβ peptide-membrane interactions. The binding of Aβ peptides to membranes has been studied by experiments and theoretical simulations extensively. The exact binding mechanism, however, still remains elusive. In the present study, the molecular basis of the peptide-bilayer binding mechanism of the full-length Aβ42 monomer with POPC/POPS/CHOL bilayers is investigated by all-atom (AA) simulations. Three main binding models in coil, bend, and turn structures are obtained. Model 1 of the three models with the central hydrophobic core (CHC) buried inside the membrane is the dominant binding model. The structural features of the peptide, the peptide-bilayer interacting regions, the intrapeptide interactions, and peptide-water interactions are studied. The binding of the Aβ42 monomer to the POPC/POPS/CHOL bilayer is also explored by coarse-grained (CG) simulations as a complement. Both the AA and CG simulations show that residues in CHC prefer forming interactions with the bilayer, indicating the crucial role of CHC in peptide-bilayer binding. Our results can provide new insights for the investigation of the peptide-bilayer binding mechanism of the Aβ peptide.

Confocal Laser Scanning Microscopy and Model Membranes to Study Translocation Mechanisms of Membrane Active Peptides.

Membrane active peptides hold great potential for targeted drug delivery systems and understanding their mechanism of uptake is a key step in the development of peptide based therapeutics and clinical use. Giant unilamellar vesicles are cell-sized model membranes that can be individually observed under the microscope. The lipid composition of these membranes can be controlled, and interaction with peptides and changes induced by the peptides can be directly followed. Relevant information on the specific steps of peptides uptake can be obtained using membranes of different lipid composition. The present work provides a selection of dynamics and kinetics of peptides at interaction with model membranes of different lipid composition. The systematic peptide-membrane interaction was investigated by laser scanning confocal microscopy. The peptides used in this study neither internalized nor induced pore formation in neutral membranes composed of phosphatidylcholine and cholesterol. In membranes with anionic phosphatidylserine or cone-shaped phosphatidylethanolamine, all peptides internalized but only two of them were able to form pores, showing that the length of the peptide, the numbers of the arginine amino acid or the length of the α–helix are also relevant for the penetration efficiency of peptides.

Transmembrane peptide effects on bacterial membrane integrity and organization

As the bacterial multidrug resistance crisis continues, membrane-active antimicrobial peptides are being explored as an alternate treatment to conventional antibiotics. In contrast to antimicrobial peptides, which function by a nonspecific membrane disruption mechanism, here we describe a series of transmembrane (TM) peptides that are designed to act as drug efflux inhibitors by aligning with and out-competing a conserved TM4-TM4 homodimerization motif within bacterial small multidrug resistance proteins. The peptides contain two terminal tags: a C-terminal lysine tag to direct the peptides toward the negatively charged bacterial membrane, and an uncharged N-terminal sarcosine (N-methyl-glycine) tag to promote membrane insertion. While effective at inhibiting efflux activity, ostensibly through their designed mechanism of action, the impact of the peptides on the bacterial inner membrane remains undetermined. To evaluate the extant peptide-membrane interactions, we performed a series of biophysical measurements. Circular dichroism spectroscopy and Trp fluorescence showed that the peptides insert into the membrane generally in helical form. Interestingly, differential scanning calorimetry of the peptides added to bacterial-like membranes (POPE:POPG 3:1) revealed the peptides’ ability to demix the POPE and POPG lipids, creating two pools, one of which is likely a peptide-POPG conglomerate, and the other a POPE-rich component where the native POPG content has been depleted. However, dye leakage assays confirmed that these events occur without causing significant membrane disruption both in vitro and in vivo, indicating that the peptides can target the small multidrug resistance TM4-TM4 motif without nonspecific membrane disruption. In related studies, DiOC2(3) fluorescence indicated moderate peptide-mediated reduction of the proton motive force for all peptides, including control peptides that did not display inhibitory activity. The overall findings suggest that peptides designed with suitable tags, sequence hydrophobicity, and charge distribution can be directed more generally to impact proteins whose function involves membrane-embedded protein-protein interactions.

Amyloids on Membrane Interfaces: Implications for Neurodegeneration.

Membrane interfaces are vital for various cellular processes, and their involvement in neurodegenerative disorders such as Alzheimer's and Parkinson's disease has taken precedence in recent years. The amyloidogenic proteins associated with neurodegenerative diseases interact with the neuronal membrane through various means, which has implications for both the onset and progression of the disease. The parameters that regulate the interaction between the membrane and the amyloids remain poorly understood. The review focuses on the various aspects of membrane interactions of amyloids, particularly amyloid-β (Aβ) peptides and Tau involved in Alzheimer's and α-synuclein involved in Parkinson's disease. The genetic, cell biological, biochemical, and biophysical studies that form the basis for our current understanding of the membrane interactions of Aβ peptides, Tau, and α-synuclein are discussed.

Circularized fluorescent nanodiscs for probing protein–lipid interactions

Protein–lipid interactions are vital for numerous transmembrane signaling pathways. However, simple tools to characterize these interactions remain scarce and are much needed to advance our understanding of signal transduction across lipid bilayers. To tackle this challenge, we herein engineer nanodisc as a robust fluorescent sensor for reporting membrane biochemical reactions. We circularize nanodiscs via split GFP and thereby create an intensity-based fluorescent sensor (isenND) for detecting membrane binding and remodeling events. We show that isenND responds robustly and specifically to the action of a diverse array of membrane-interacting proteins and peptides, ranging from synaptotagmin and synuclein involved in neurotransmission to viral fusion peptides of HIV-1 and SARS-CoV-2. Together, isenND can serve as a versatile biochemical reagent useful for basic and translational research of membrane biology.