Membrane-associated Peptides Research Articles

Lighting up Mycobacteria with membrane-targeting peptides.

We report a series of fluorescent probes based on mycobacteria membrane-associated disruption peptide, containing either L- or D-amino acids which were originally designed to kill Mycobacterium tuberculosis via membrane disruption. These peptides were decorated with "always on" and environmentally sensitive fluorophores and showed the rapid and efficient labelling of Mycobacterium smegmatis, with labelling of Mycobacterium tuberculosis demonstrated by two of the probes.

Improving Cell-Free Expression of Model Membrane Proteins by Tuning Ribosome Cotranslational Membrane Association and Nascent Chain Aggregation.

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than that for soluble proteins. In this paper, we study the CFE of membrane proteins and develop a quantitative kinetic model. We identify that ribosome stalling during the translation of membrane proteins is a strong predictor of membrane protein synthesis due to aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes, which incorporate protein nascent chains and, therefore, kinetically compete with aggregation. We show that the balance between peptide-membrane association and peptide aggregation rates determines the yield of the synthesized membrane protein. We define a membrane protein expression score that can be used to rationalize the engineering of lipid composition and the N-terminal domain of a native and computationally designed membrane proteins produced through CFE.

Mechanism of action of sprG1-encoded type I toxins in Staphylococcus aureus: from membrane alterations to mesosome-like structures formation and bacterial lysis.

sprG1/SprF1 is a type I toxin-antitoxin system located on Staphylococcus aureus prophage. It has previously been shown that the two toxins, SprG131 and SprG144, encoded by the sprG1 gene, are two membrane-associated peptides structured in a single α-helix. Overexpression of these two peptides leads to growth inhibition and even S. aureus death. In this study, we investigated the involvement of each peptide in this toxicity, the sequence requirements necessary for SprG131 toxicity, and the mechanism of action of these two peptides. Our findings show that both peptides, when expressed individually, are able to stop growth, with higher toxicity observed for SprG131. The combination of a hydrophobic domain and a charged domain located only at the C-terminus is necessary for this toxicity, likely to retain the orientation of the transmembrane domain. A net cationic charge for SprG131 is not essential to induce a growth defect in S. aureus. Furthermore, we established a chronology of toxic events following overexpression to gain insights into the mode of action of SprG144 and SprG131. We demonstrated that mesosome-like structures are already formed when membrane is depolarized, about 20 min after peptides induction. This membrane depolarization occurs concomitantly with a depletion of intracellular ATP, leading to S. aureus growth arrest. Moreover, we hypothesized that SprG144 and SprG131 do not form large pores in the S. aureus membrane, as ATP is not excreted into the extracellular medium, and membrane permeabilization is delayed relative to membrane depolarization. The next challenge is to identify the conditions under which SprG144 and SprG131 are naturally expressed, and to uncover their potential roles during staphylococcal growth, colonization, and infection.

Potent dual block to HIV-1 infection using lentiviral vectors expressing fusion inhibitor peptide mC46- and Vif-resistant APOBEC3G

Gene therapy strategies that effectively inhibit HIV-1 replication are needed to reduce the requirement for lifelong antiviral therapy and potentially achieve a functional cure. We previously designed self-activating lentiviral vectors that efficiently delivered and expressed a Vif-resistant mutant of APOBEC3G (A3G-D128K) to Tcells, which potently inhibited HIV-1 replication and spread with no detectable virus. Here, we developed vectors that express A3G-D128K, membrane-associated fusion inhibitor peptide mC46, and O6-methylguanine-DNA-methyltransferase (MGMT) selectable marker for invivo selection of transduced CD34+ hematopoietic stem and progenitor cells. MGMT-selected Tcell lines MT4, CEM, and PM1 expressing A3G-D128K (with or without mC46) potently inhibited NL4-3 infection up to 45days post infection with no detectable viral replication. Expression of mC46 was sufficient to block infection >80% in a single-cycle assay. Importantly, expression of mC46 provided a selective advantage to the A3G-D128K-modified Tcells in the presence of replication competent virus. This combinational approach to first block HIV-1 entry with mC46, and then block any breakthrough infection with A3G-D128K, could provide an effective gene therapy treatment and a potential functional cure for HIV-1 infection.

Detergent-Free Isolation of Membrane Proteins and Strategies to Study Them in a Near-Native Membrane Environment.

Atomic-resolution structural studies of membrane-associated proteins and peptides in a membrane environment are important to fully understand their biological function and the roles played by them in the pathology of many diseases. However, the complexity of the cell membrane has severely limited the application of commonly used biophysical and biochemical techniques. Recent advancements in NMR spectroscopy and cryoEM approaches and the development of novel membrane mimetics have overcome some of the major challenges in this area. For example, the development of a variety of lipid-nanodiscs has enabled stable reconstitution and structural and functional studies of membrane proteins. In particular, the ability of synthetic amphipathic polymers to isolate membrane proteins directly from the cell membrane, along with the associated membrane components such as lipids, without the use of a detergent, has opened new avenues to study the structure and function of membrane proteins using a variety of biophysical and biological approaches. This review article is focused on covering the various polymers and approaches developed and their applications for the functional reconstitution and structural investigation of membrane proteins. The unique advantages and limitations of the use of synthetic polymers are also discussed.

Engineering Polyhistidine Tags on Surface Proteins of Acidithiobacillus ferrooxidans: Impact of Localization on the Binding and Recovery of Divalent Metal Cations.

Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies.Acidithiobacillus ferrooxidansis an iron- and sulfur-oxidizing chemolithoautotroph that is known to participate in metal bioleaching, and its metabolic capabilities have been exploited for industrial-scale copper and gold biomining. In addition to bioleaching, microorganisms could also be engineered for selective metal binding, enabling new opportunities for metal bioseparation and recovery. Here, we explored the ability of polyhistidine (polyHis) tags appended to two recombinantly expressed endogenous proteins to enhance the metal binding capacity of A. ferrooxidans. The genetically engineered cells achieved enhanced cobalt and copper binding capacities, and the Langmuir isotherm captures their interaction behavior with these divalent metals. Additionally, the cellular localization of the recombinant proteins correlated with kinetic modeling of the binding interactions, where the outer membrane-associated polyHis-tagged licanantase peptide bound the metals faster than the periplasmically expressed polyHis-tagged rusticyanin protein. The selectivity of the polyHis sequences for cobalt over copper from mixed metal solutions suggests potential utility in practical applications, and further engineering could be used to create metal-selective bioleaching microorganisms.

Comparative molecular dynamics simulation studies of realistic eukaryotic, prokaryotic, and archaeal membranes

The OPM (Orientation of Proteins in Membranes) database currently holds, classifies, and annotates around 14,000 three-dimensional structures of transmembrane and peripheral membrane proteins and peptides positioned with respect to membrane boundaries. Developing uniform templates of biological membranes with realistic native-like lipid composition would provide a convenient and accurate platform for researchers who simulate membrane-associated proteins and peptides in natural environments. In this study, for the first time, we performed the standardized molecular dynamic simulations and comparative analysis of structures and properties of 18 native-like membrane models.

Degradation of Diatom Protein in Seawater: A Peptide-Level View

Peptides and proteins were identified during a controlled laboratory degradation of the marine diatom Thalassiosira weissflogii by a surface seawater microbiome. Samples from each time point were processed both with and without the protease trypsin, allowing a partial differentiation between peptides produced naturally by microbial enzymatic degradation and peptides produced from the laboratory digestion of intact protein. Over the 12-day degradation experiment, 31% of the particulate organic carbon was depleted, and there was no preferential degradation of the overall protein pool. However, there was distinct differentiation in the cellular location, secondary structure and modifications between peptides produced by microbial vs. laboratory breakdown. During the initial period of rapid algal decay and bacterial growth, intracellular components from the cytoplasm were consumed first, resulting in the accumulation of membrane-associated proteins and peptides in the detrital pool. Accompanying the enrichment of membrane protein material was an increase in the importance of ɑ-helix motifs. Methylated arginine, a post-translational modification common in cell senescence, was found in high amounts within the microbially produced detrital peptide pool, suggesting a link between in-cell modification and resistance to immediate degradation. Another modification—asparagine deamidation—accumulated within the detrital peptides. Protein taxonomies showed the bacterial community decomposing the algal material was rich in Proteobacteria, and protein annotations showed abundant transportation of solubilized carbohydrates and small peptides across membranes. At this early stage of diagenesis, no changes in bulk amino acids (THAA) were observed, yet a proteomic approach allowed us to observe selective changes in diatom protein preservation by using amino acid sequences to infer subcellular location, secondary structures, and post-translational modifications (PTMs).

High-yield production and purification of the fusion pH-responsive peptide GST-pHLIP in <i>Escherichia coli</i> BL21

<abstract> <p>The pH Low Insertion Peptide (pHLIP) has versatile applications in several diseases due to its differential behavior at slightly different pH values. pHLIP is an unstructured and peripheral membrane-associated peptide at neutral pH and an α-helical transmembrane peptide at acidic values. Similar to what happened to insulin and growth hormone, pHLIP´s expanding applications require high-yield production to further scale-up its usefulness. To date, synthesis of the pHLIP has not been reported in a prokaryotic platform, mainly relying on solid-phase synthesis. Bacterial production arises as an option for high-amount peptide generation and larger pHLIP fusion protein-synthesis; however, cell-based pH-responsive peptide production could be challenging due to intracellular peptide interactions or degradation due to unstructured conformations. An <italic>Escherichia coli</italic> (E. coli)-BL21 cell culture was induced with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) in order to produce a Glutathione S-transferase-pHLIP (GST-pHLIP) fusion construct. Purification was done with Glutathione (GSH)-decorated magnetic beads using 4 ml of the induced cell culture. The production was quantified with Bradford reagent and characterized with SDS-PAGE and Western blot, contrasting Bradford results with densitometry analysis to obtain production approximate absolute values. A purified approximate total yield of ~26 µg with an apparent GSH-bead saturation and a total production of ~82 µg was obtained. Our Western Blot assay confirmed the presence of the GST-pHLIP construct in all the IPTG-induced fractions. Conclusion: A high-yield pHLIP production irrespective of its membrane affinity in acidic environments or its unstructured nature was achieved. Our study could be useful to scale up pHLIP synthesis for future applications.</p> </abstract>

Leaflet Asymmetry Modeling in the Lipid Composition of Escherichia coli Cytoplasmic Membranes.

Lipid composition asymmetry between leaflets is important to cell function and plays a key role in the "positive inside" rule in transmembrane proteins. In this work, Escherichia coli inner plasma membrane models reflecting this asymmetry have been investigated at the early-log and stationary stages during the bacterial lifecycle using all-atom molecular dynamics simulations. The CHARMM36 lipid force field is used, and selected membrane properties are tested for variations between two leaflets and whole membranes. Our models include bacterial lipids with a cyclopropane moiety on the sn-2 acyl chain in the stationary membrane model. The PE/PG ratio for two leaflets reflects the "positive inside" rule of membrane proteins, set to 6.8 and 2.8 for the inner and outer leaflets of the two models, respectively. We are the first to model leaflet asymmetry in the lipid composition of E. coli cytoplasmic membranes and observe the effect on membrane properties in leaflets and whole membranes. Specifically, our results show that for the stationary phase bilayer, the surface area per lipid (SA/lipid) is larger, the thickness (2DC and DB) is smaller, the tilt angle is larger, the tilt modulus is smaller, and the deuterium order parameters (SCD) of sn-1 and sn-2 tails are lower, compared to the early-log stage. Moreover, the stationary stage bilayer has a positive spontaneous curvature, while the early-log stage has a near flat spontaneous curvature. For leaflet asymmetry, the inner leaflet has a larger SA/lipid, a smaller thickness, a smaller elastic tilt modulus (a larger tilt angle), and lower SCD, compared to the outer leaflet in both stages. Moreover, an asymmetric membrane involves a lipid tilt and a lateral extension, varying from a reference state of a pre-equilibrium membrane. This work encourages a more profound exploration of leaflet asymmetry in various other membrane models and how this might affect the structure and function of membrane-associated peptides and proteins.

Thermodynamics-Based Molecular Modeling of α-Helices in Membranes and Micelles.

The Folding of Membrane-Associated Peptides (FMAP) method was developed for modeling α-helix formation by linear peptides in micelles and lipid bilayers. FMAP 2.0 identifies locations of α-helices in the amino acid sequence, generates their three-dimensional models in planar bilayers or spherical micelles, and estimates their thermodynamic stabilities and tilt angles, depending on temperature and pH. The method was tested for 723 peptides (926 data points) experimentally studied in different environments and for 170 single-pass transmembrane (TM) proteins with available crystal structures. FMAP 2.0 detected more than 95% of experimentally observed α-helices with an average error in helix end determination of around 2, 3, 4, and 5 residues per helix for peptides in water, micelles, bilayers, and TM proteins, respectively. Helical and nonhelical residue states were predicted with an accuracy from 0.86 to 0.96, and the Matthews correlation coefficient was from 0.64 to 0.88 depending on the environment. Experimental micelle- and membrane-binding energies and tilt angles of peptides were reproduced with a root-mean-square deviation of around 2 kcal/mol and 7°, respectively. The TM and non-TM states of hydrophobic and pH-triggered α-helical peptides in various lipid bilayers were reproduced in more than 95% of cases. The FMAP 2.0 web server (https://membranome.org/fmap) is publicly available to explore the structural polymorphism of antimicrobial, cell-penetrating, fusion, and other membrane-binding peptides, which is important for understanding the mechanisms of their biological activities.

Coordination of Redox Ions within a Membrane-Binding Peptide: A Tale of Aromatic Rings.

The amino-terminal-copper-and-nickel-binding (ATCUN) motif, a tripeptide sequence ending with a histidine, confers important functions to proteins and peptides. Few high-resolution studies have been performed on the ATCUN motifs of membrane-associated proteins and peptides, limiting our understanding of how they stabilize Cu2+/Ni2+ in membranes. Here, we leverage solid-state NMR to investigate metal-binding to piscidin-1 (P1), a host-defense peptide featuring F1F2H3 as its ATCUN motif. Bound to redox ions, P1 chemically and physically damages pathogenic cell membranes. We design 13C/15N correlation experiments to detect and assign the deprotonated nitrogens produced and/or shifted by Ni2+-binding. Occupying multiple chemical states in P1-apo, H3 and the neighboring H4 respond to metalation by populating only the τ-tautomer. H3, as a proximal histidine, directly coordinates the metal, compared to the distal H4. Density functional theory calculations reflect this noncanonical arrangement and point toward cation-π interactions between the F1/F2/H4 aromatic rings and metal. These structural findings, which are relevant to other ATCUN-containing membrane peptides, could help design new therapeutics and materials for use in the areas of drug-resistant bacteria, neurological disorders, and biomedical imaging.

Native Chemical Ligation of Highly Hydrophobic Peptides in Ionic Liquid-Containing Media.

The chemical synthesis of a highly hydrophobic membrane-associated peptide by native chemical ligation (NCL) in an ionic liquid (IL) [C2mim][OAc]/buffer mixture was achieved by employing peptide concentrations up to 11 mM. NCL was studied at different pH and water content and compared to several “gold-standard” ligation protocols. The optimized reaction protocol for the NCL in IL required the addition of 40% water and pH adjustment to 7.0–7.5, resulting in ligation yields of up to 80–95% within 1 to 4 h. This new ligation protocol is generally applicable and outperforms current “gold-standard” NCL methods.

Peptide lipidation in lysophospholipid micelles and lysophospholipid-enriched membranes.

Acyl transfer from lipids to membrane-associated peptides is a well-documented process, leading to the generation of a lipidated peptide and a lysolipid. In this article, we demonstrate that acyl transfer from lysophosphatidylcholines (lysoPCs) to the peptide melittin also occurs, both in micelles of pure lysolipid and in lipid/lysolipid mixtures. In the case of bilayers containing lysolipids, acyl transfer from the lysolipid is marginally favoured over transfer from the lipid. In pure bilayers of saturated lipids, the introduction of even small amounts of lysolipid appears to significantly increase the reactivity towards lipidation.

New Evidence for P-gp-Mediated Export of Amyloid-β PEPTIDES in Molecular, Blood-Brain Barrier and Neuronal Models.

Defective clearance mechanisms lead to the accumulation of amyloid-beta (Aβ) peptides in the Alzheimer’s brain. Though predominantly generated in neurons, little is known about how these hydrophobic, aggregation-prone, and tightly membrane-associated peptides exit into the extracellular space where they deposit and propagate neurotoxicity. The ability for P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, to export Aβ across the blood-brain barrier (BBB) has previously been reported. However, controversies surrounding the P-gp–Aβ interaction persist. Here, molecular data affirm that both Aβ40 and Aβ42 peptide isoforms directly interact with and are substrates of P-gp. This was reinforced ex vivo by the inhibition of Aβ42 transport in brain capillaries from P-gp-knockout mice. Moreover, we explored whether P-gp could exert the same role in neurons. Comparison between non-neuronal CHO-APP and human neuroblastoma SK-N-SH cells revealed that P-gp is expressed and active in both cell types. Inhibiting P-gp activity using verapamil and nicardipine impaired Aβ40 and Aβ42 secretion from both cell types, as determined by ELISA. Collectively, these findings implicate P-gp in Aβ export from neurons, as well as across the BBB endothelium, and suggest that restoring or enhancing P-gp function could be a viable therapeutic approach for removing excess Aβ out of the brain in Alzheimer’s disease.

Structural Plasticity of EAK-16 Peptide Inducing Vesicle Membrane Leakage.

Ionic complementary peptide EAK-16 has been studies for anticancer drug delivery application. This is a 16 residues, short sequence peptide has ability to trosnform into micro/nanoparticle via self-assembly. However, it is still not clear that how this can bind with cell membrane to induce membrane leakage or delivering their cargo inside cell membrane. The main objective of this work was to understand behaviour of secondary structure conformation of peptide in solution and at lipid membrane interfaces and membrane permeability of synthetic ionic complementary peptide EAK-16. The corresponding secondary structure conformation was evaluated. We performed biophysical investigation to probe the interaction of synthesised ionic complementary peptide (EAK-16) with dimyristoylphospholcholine (DMPC) and dimyristoylphosphoserine (DMPS) membrane interfaces. The folding behaviours of EAK-16 were studied with Circular Dichroism (CD) spectroscopy. Membrane leakage with peptide was confirmed with calcein leakage assay. Our finding of this study showed that in aqueous phase EAK-16 was predominantly folded into β-sheets. The temperature could alter the β-sheets. However, in DMPC and DMPS membrane interfaces, EAK-16 adopted helical conformation. EAK-16 has preference in perturbing anionic compared Zwitterionic lipid vesicles. This study proposed that hydrophobic grooves of EAK-16 might be a key in the association with lipid bilayers. Secondly, a charge distribution of ionic residues would also support the orientation at lipid bilayers. This peptide membrane association would facilitate the membrane destabilisation. This study demonstrated the supporting evidence that EAK-16 could interact with lipid membranes and conforming to helical structure, while the helical conformation induced the lipid membrane leakage. Overall, this study provides a physical rationale that ionic complementary peptide can be a useful tool for designing and development of novel antibiotics and anticancer agents along its previous drug delivery applications.

Targeting Acidic Diseased Tissues by pH-Triggered Membrane-Associated Peptide Folding.

The advantages of targeted therapy have motivated many efforts to find distinguishing features between the molecular cell surface landscapes of diseased and normal cells. Typically, the features have been proteins, lipids or carbohydrates, but other approaches are emerging. In this discussion, we examine the use of cell surface acidity as a feature that can be exploited by using pH-sensitive peptide folding to target agents to diseased cell surfaces or cytoplasms.

Optimized aqueous Kinugasa reactions for bioorthogonal chemistry applications.

Kinugasa reactions hold potential for bioorthogonal chemistry in that the reagents can be biocompatible. Unlike other bioorthogonal reaction products, β-lactams are potentially reactive, which can be useful for synthesizing new biomaterials. A limiting factor for applications consists of slow reaction rates. Herein, we report an optimized aqueous copper(i)-catalyzed alkyne-nitrone cycloaddition involving rearrangement (CuANCR) with rate accelerations made possible by the use of surfactant micelles. We have investigated the factors that accelerate the aqueous CuANCR reaction and demonstrate enhanced modification of a model membrane-associated peptide. We discovered that lipids/surfactants and alkyne structure have a significant impact on the reaction rate, with biological lipids and electron-poor alkynes showing greater reactivity. These new findings have implications for the use of CuANCR for modifying integral membrane proteins as well as live cell labelling and other bioorthogonal applications.

CXCR2 specific endocytosis of immunomodulatory peptide LL-37 in human monocytes and formation of LL-37 positive large vesicles in differentiated monoosteophils

Immunomodulatory peptide cathelicidin/LL-37 induces human monocyte differentiation into a novel bone repair cell, the monoosteophil. We now demonstrate that LL-37 is endocytosed by monocytes over a period of 6 days producing large (10 × 2 μm), specialized LL-37 and integrin α3 positive vesicles. CXCR2, a membrane receptor previously associated with the binding of LL-37 to neutrophils, was co-endocytosed with LL-37 where both markers remained within the cytosol over a 16 h observation period. Endocytosis of LL-37 was mediated by a clathrin- and cavoelin/lipid raft-dependent pathway into early Rab5+ endosomes expressing APPL1 and EEA1. From 4 to 16 h, LL-37 vesicles co-localized with the Golgi, mitochondria, and to a lesser extent lysosomes and ER. By day 6, LL-37 was associated with large (>10 μm) vesicles, adjacent to Golgi, mitochondria, ER and lysosomes. LL-37 co-stained with integrin α3, tetraspanin CD9, GPI-linked CD59 and costimulatory molecule CD276 (B7-H3) in these vesicles. Continuous tracking of LL-37 with its associated vesicles over 6 days indicates that LL-37 is an extremely stable, membrane-associated peptide that plays a critical role in the differentiation of monocytes into monoosteophils.

Retention of Native Quaternary Structure in Racemic Melittin Crystals.

Racemic crystallography has been used to elucidate the secondary and tertiary structures of peptides and small proteins that are recalcitrant to conventional crystallization. It is unclear, however, whether racemic crystallography can capture native quaternary structure, which could be disrupted by heterochiral associations. We are exploring the use of racemic crystallography to characterize the self-assembly behavior of membrane-associated peptides, very few of which have been crystallized. We report a racemic crystal structure of the membrane-active peptide melittin; the new structure allows comparison with a previously reported crystal structure of L-melittin. The tetrameric assembly observed in crystalline L-melittin has been proposed to represent the tetrameric state detected in solution for this peptide. This tetrameric assembly is precisely reproduced in the racemic crystal, which strengthens the conclusion that the tetramer is biologically relevant. More broadly, these findings suggest that racemic crystallography can provide insight on native quaternary structure.