Alpha-helical Conformation Research Articles

Characterization of the Arabidopsis thaliana chromatin remodeler DEK3 for its interaction with histones and DNA

Chromatin structure and dynamics regulate all DNA-templated processes, such as transcription, replication, and repair. Chromatin binding factors, chromatin architectural proteins, and nucleosome remodelers modulate chromatin structure and dynamics and, thereby, the various DNA-dependent processes. Arabidopsis thaliana DEK3, a member of the evolutionarily conserved DEK domain-containing chromatin architectural proteins, is an important factor for chromatin structure and function, involved in transcriptional programming to regulate flowering time and abiotic stress tolerance. AtDEK3 contains an uncharacterized N-terminal domain, a middle SAF domain (winged helix-like domain), and a C-terminal DEK domain, but their role in the interaction of AtDEK3 with histones and DNA remained poorly understood. Using biochemical and biophysical analyses, we provide a comprehensive invitro characterization of the different AtDEK3 domains for their interaction with histone H3/H4 and DNA. AtDEK3 directly interacts with histone H3/H4 tetramers through its N-terminal domain and the C-terminal DEK domain in a 1:1 stoichiometry. Upon interaction with H3/H4, the unstructured N-terminal domain of AtDEK3 undergoes a conformational change and adopts an alpha-helical conformation. In addition, the in-solution envelope structures of the AtDEK3 domains and their complex with H3/H4 have been characterized. The SAF and DEK domains associate with double-stranded and four-way junction DNA. As DEK3 possesses a histone-interacting domain at the N- and the C-terminus and a DNA-binding domain in the middle and at the C-terminus, the protein might play a complex role as a chromatin remodeler.

The spatial separation of basic amino acids is similar in RHAMM and hyaluronan binding peptide P15-1 despite different sequences and conformations.

Peptides that increase pro-reparative responses to injury and disease by modulating the functional organization of hyaluronan (HA) with its cell surface binding proteins (e.g., soluble receptor for hyaluronan-mediated motility [RHAMM] and integral membrane CD44) have potential therapeutic value. The binding of RHAMM to HA is an attractive target, since RHAMM is normally absent or expressed at low levels in homeostatic conditions, but its expression is significantly elevated in the extracellular matrix during tissue stress, response-to-injury, and in cancers and inflammation-based diseases. The HA-binding site in RHAMM contains two closely spaced sequences of clustered basic amino acids, in an alpha-helical conformation. In the present communication, we test whether an alpha-helical conformation is required for effective peptide binding to HA, and competitive disruption of HA-RHAMM interaction. The HA-binding RHAMM-competitive peptide P15-1, identified using the unbiased approach of phage display, was examined using circular dichroism spectroscopy and the conformation-predictive AI-based AlphaFold2 algorithm. Unlike the HA-binding site in RHAMM, peptide P15-1 was found to adopt irregular conformations in solution rather than alpha helices. Instead, our structural analysis suggests that the primary determinant of peptide-HA binding is associated with a specific clustering and spacing pattern of basic amino acids, allowing favorable electrostatic interaction with carboxylate groups on HA.

Conformational Switch of a Peptide Provides a Novel Strategy to Design Peptide Loaded Porous Organic Polymer for Pyroptosis Pathway Mediated Cancer Therapy.

While peptide-based drug development is extensively explored, this strategy has limitations due to rapid excretion from the body (or shorter half-life in the body) and vulnerability to protease-mediated degradation. To overcome these limitations, a novel strategy for the development of a peptide-based anticancer agent is introduced, utilizing the conformation switch property of a chameleon sequence stretch (PEP1) derived from a mycobacterium secretory protein, MPT63. The selected peptide is then loaded into a new porous organic polymer (PG-DFC-POP) synthesized using phloroglucinol and a cresol derivative via a condensation reaction to deliver the peptide selectively to cancer cells. Utilizing ensemble and single-molecule approaches, this peptide undergoes a transition from a disordered to an alpha-helical conformation, triggered by the acidic environment within cancer cells that is demonstrated. This adopted alpha-helical conformation resulted in the formation of proteolysis-resistant oligomers, which showed efficient membrane pore-forming activity selectively for negatively charged phospholipids accumulated in cancer cell membranes. The experimental results demonstrated that the peptide-loaded PG-DFC-POP-PEP1 exhibited significant cytotoxicity in cancer cells, leading to cell death through the Pyroptosis pathway, which is established by monitoring numerous associated events starting from lysosome membrane damage to GSDMD-induced cell membrane demolition. This novel conformational switch-based drug design strategy is believed to have great potential in endogenous environment-responsive cancer therapy and the development of future drug candidates to mitigate cancers.

Therapeutic stapled peptides: Efficacy and molecular targets

Peptide stapling, by employing a stable, preformed alpha-helical conformation, results in the production of peptides with improved membrane permeability and enhanced proteolytic stability, compared to the original peptides, and provides an effective solution to accelerate the rapid development of peptide drugs. Various reviews present peptide stapling chemistries, anchoring residues and one- or two-component cyclization, however, therapeutic stapled peptides have not been systematically summarized, especially focusing on various disease-related targets. This review highlights the latest advances in therapeutic peptide drug development facilitated by the application of stapling technology, including different stapling techniques, synthetic accessibility, applicability to biological targets, potential for solving biological problems, as well as the current status of development. Stapled peptides as therapeutic drug candidates have been classified and analysed mainly by receptor- and ligand-based stapled peptide design against various diseases, including cancer, infectious diseases, inflammation, and diabetes. This review is expected to provide a comprehensive reference for the rational design of stapled peptides for different diseases and targets to facilitate the development of therapeutic peptides with enhanced pharmacokinetic and biological properties.

Structural Shifts of the Parvovirus B19 Capsid Receptor-binding Domain: A Peptide Study.

Binding appropriate cellular receptors is a crucial step of a lifecycle for any virus. Structure of receptor-binding domain for a viral surface protein has to be determined before the start of future drug design projects. Investigation of pH-induced changes in the secondary structure for a capsid peptide with loss of function mutation can shed some light on the mechanism of entrance. Spectroscopic methods were accompanied by electrophoresis, ultrafiltration, and computational biochemistry. In this study, we showed that a peptide from the receptor-binding domain of Parvovirus B19 VP1 capsid (residues 13-31) is beta-structural at pH=7.4 in 0.01 M phosphate buffer, but alpha- helical at pH=5.0, according to the circular dichroism (CD) spectroscopy results. Results of infra- red (IR) spectroscopy showed that the same peptide exists in both alpha-helical and beta-structural conformations in partial dehydration conditions both at pH=7.4 and pH=5.0. In contrast, the peptide with Y20W mutation, which is known to block the internalization of the virus, forms mostly alpha-helical conformation in partial dehydration conditions at pH=7.4. According to our hypothesis, an intermolecular antiparallel beta structure formed by the wild-type peptide in its tetramers at pH=7.4 is the prototype of the similar intermolecular antiparallel beta structure formed by the corresponding part of Parvovirus B19 receptor-binding domain with its cellular receptor (AXL). Loss of function Y20W substitution in VP1 capsid protein prevents the shift into the beta-structural state by the way of alpha helix stabilization and the decrease of its ability to turn into the disordered state.

Screening and Functional Analyses of Novel Cecropins from Insect Transcriptome.

Antibiotic resistance is a significant and growing threat to global public health. However, antimicrobial peptides (AMPs) have shown promise as they exhibit a broad spectrum of antibacterial activities with low potential for resistance development. Insects, which inhabit a wide range of environments and are incredibly diverse, remain largely unexplored as a source of novel AMPs. To address this, we conducted a screening of the representative transcriptomes from the 1000 Insect Transcriptome Evolution (1KITE) dataset, focusing on the homologous reference genes of Cecropins, the first identified AMPs in insects known for its high efficiency. Our analysis identified 108 Cecropin genes from 105 insect transcriptomes, covering all major hexapod lineages. We validated the gene sequences and synthesized mature peptides for three identified Cecropin genes. Through minimal inhibition concentration and agar diffusion assays, we confirmed that these peptides exhibited antimicrobial activities against Gram-negative bacteria. Similar to the known Cecropin, the three Cecropins adopted an alpha-helical conformation in membrane-like environments, efficiently disrupting bacterial membranes through permeabilization. Importantly, none of the three Cecropins demonstrated cytotoxicity in erythrocyte hemolysis tests, suggesting their safety in real-world applications. Overall, this newly developed methodology provides a high-throughput bioinformatic pipeline for the discovery of AMP, taking advantage of the expanding genomic resources available for diverse organisms.

Competition co-immunoprecipitation reveals the interactors of the chloroplast CPN60 chaperonin machinery.

The functionality of all metabolic processes in chloroplasts depends on a balanced integration of nuclear- and chloroplast-encoded polypeptides into the plastid's proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding under ambient and stressful conditions, with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin of Escherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of very limited knowledge about the interactors. We employed the competition immunoprecipitation method for the identification of the chaperonin's interactors in Chlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL and the expected complex partners, we revealed numerous new interactors with high confidence. Proteins that qualify as putative substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical conformation and increased aggregation propensity. Immunoprecipitations targeted against a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.

Epitope-directed anti-SARS-CoV-2 scFv engineered against the key spike protein region could block membrane fusion.

The newly emerged SARS-CoV-2 causing coronavirus disease (COVID-19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2' site, by the TMPRSS2 protease. S2' cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2' site to be conserved among all the SARS-CoV-2 variants, we chose an S2' epitope encompassing the S2' cleavage site and generated single-chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2' epitope was determined. Incidentally, S2' epitope in the scFv bound structure adopts an alpha-helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody-S2' epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2' cleavage competitively.

Transforming Cross-Linked Cyclic Dimers of KR-12 into Stable and Potent Antimicrobial Drug Leads.

Is it possible to enhance structural stability and biological activity of KR-12, a truncated antimicrobial peptide derived from the human host defense peptide LL-37? Based on the mapping of essential residues in KR-12, we have designed backbone-cyclized dimers, cross-linked via a disulfide bond to improve peptide stability, while at the same time improving on-target activity. Circular dichroism showed that each of the dimers adopts a primarily alpha-helical conformation (55% helical content) when bound to lyso-phosphatidylglycerol micelles, indicating that the helical propensity of the parent peptide is maintained in the new cross-linked cyclic form. Compared to KR-12, one of the cross-linked dimers showed 16-fold more potent antimicrobial activity against human pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans and 8-fold increased activity against Escherichia coli. Furthermore, these peptides retained antimicrobial activity at physiologically relevant conditions, including in the presence of salts and in human serum, and with selective Gram-negative antibacterial activity in rich growth media. In addition to giving further insight into the structure-activity relationship of KR-12, the current work demonstrates that by combining peptide stabilization strategies (dimerization, backbone cyclization, and cross-linking via a disulfide bond), KR-12 can be engineered into a potent antimicrobial peptide drug lead with potential utility in a therapeutic context.

The dynein adapter BicD2 recognizes its cargo through short cargo-recognition alpha-helices.

Nup358, a protein of the nuclear pore complex, facilitates a nuclear positioning pathway that is essential for brain development. Nup358 interacts with the dynein adaptor Bicaudal D2 (BicD2), which in turn recruits the dynein machinery to position the nucleus. However, the molecular mechanisms of the Nup358/BicD2 interaction and the activation of transport remain poorly understood. Using nuclear magnetic resonance, mutagenesis and circular dichroism spectroscopy, a short Nup358 α-helix was identified, which transitioned from a random coil to an α-helical conformation upon BicD2-binding and formed the core of the Nup358-BicD2 interface. Mutations in this region of Nup358 decreased the Nup358/BicD2 interaction, resulting in decreased dynein recruitment and impaired motility. BicD2 thus recognizes Nup358 though a “cargo recognition α-helix”, a structural feature that may stabilize BicD2 in its activated state and promote processive dynein motility. To test the hypothesis that the other BicD2 cargoes are recognized by similar α-helical motifs, we identified the BicD2-binding site on Rab6GTP, which recruits BicD2 to Golgi-derived vesicles for transport. The BicD2 binding site is located in a region of Rab6 that undergoes conformational changes in the GTP- compared to the GDP-bound state, explaining why GTP-bound Rab6 has a higher affinity. This region is also intrinsically disordered, and we propose that it transitions to an alpha-helical conformation upon binding to BicD2, similar as observed for Nup358. Our results provide insights into the molecular mechanism of cargo selection of BicD2, which facilitates transport pathways that are important for brain development and the transport of Golgi-derived vesicles. Our results also pave the way for studies that establish the function of BicD2/dynein-dependent transport pathways and their regulatory mechanisms in the developing brain and their contributions to neuromuscular diseases.

GxxxG Motif Stabilize Ion-Channel like Pores through Cα-H···O Interaction in Aβ (1-40).

Aβ (1-40) can transfer from the aqueous phase to the bilayer and thus form stable ion-channel-like pores where the protein has alpha-helical conformation. The stability of the pores is due to the presence of the GXXXG motif. It has been reported that these ion-channel-like pores are stabilized by a Cα-H···O hydrogen bond that is established between a glycine of the GXXXG sequence of an alpha-helix and another amino acid of a vicinal alpha-helix. However, conflicting data are reported in the literature. Some authors have suggested that hydrogen bonding does not have a stabilizing function. Here we synthesized pentapeptides having a GXXXG motif to explore its role in pore stability. We used molecular dynamics simulations, quantum mechanics, and experimental biophysical techniques to determine whether hydrogen bonding was formed and had a stabilizing function in ion-channel-like structures. Starting from our previous molecular dynamics data, molecular quantum mechanics simulations, and ATR data showed that a stable ion-channel-like pore formed and a band centered at 2910 cm-1 was attributed to the interaction between Gly 7 of an alpha-helix and Asp 23 of a vicinal alpha-helix.

Uncovering the Contributions of Charge Regulation to the Stability of Single Alpha Helices.

The single alpha helix (SAH) is a recurring motif in biology. The consensus sequence has a di-block architecture that includes repeats of four consecutive glutamate residues followed by four consecutive lysine residues. Measurements show that the overall helicity of sequences with consensus E4 K4 repeats is insensitive to a wide range of pH values. Here, we use the recently introduced q-canonical ensemble, which allows us to decouple measurements of charge state and conformation, to explain the observed insensitivity of SAH helicity to pH. We couple the outputs from separate measurements of charge and conformation with atomistic simulations to derive residue-specific quantifications of preferences for being in an alpha helix and for the ionizable residues to be charged vs. uncharged. We find a clear preference for accommodating uncharged Glu residues within internal positions of SAH-forming sequences. The stabilities of alpha helical conformations increase with the number of E4 K4 repeats and so do the numbers of accessible charge states that are compatible with forming conformations of high helical content. There is conformational buffering whereby charge state heterogeneity buffers against large-scale conformational changes thus making the overall helicity insensitive to large changes in pH. Further, the results clearly argue against a single, rod-like alpha helical conformation being the only or even dominant conformation in the ensembles of so-called SAH sequences.

Ligand recognition and biased agonism of the D1 dopamine receptor

Dopamine receptors are widely distributed in the central nervous system and are important therapeutic targets for treatment of various psychiatric and neurological diseases. Here, we report three cryo-electron microscopy structures of the D1 dopamine receptor (D1R)-Gs complex bound to two agonists, fenoldopam and tavapadon, and a positive allosteric modulator LY3154207. The structure reveals unusual binding of two fenoldopam molecules, one to the orthosteric binding pocket (OBP) and the other to the extended binding pocket (EBP). In contrast, one elongated tavapadon molecule binds to D1R, extending from OBP to EBP. Moreover, LY3154207 stabilizes the second intracellular loop of D1R in an alpha helical conformation to efficiently engage the G protein. Through a combination of biochemical, biophysical and cellular assays, we further show that the broad conformation stabilized by two fenoldopam molecules and interaction between TM5 and the agonist are important for biased signaling of D1R.

Elucidation of Bacterial Peptidoglycan Signaling and Recognition in the Human Commensal Candida. albicans through the LRR domain of the peripheral Membrane Protein Cyr1p

The human commensal Candida. albicans (C. albicans) is the most common fungal resident of the human microbiome. In healthy individuals this fungus occupies a budding morphology, but during immune incompetence morphologically transitions to a pathogenic hyphal form. The long filamentous form of hyphae readily invades human epithelial cells and if not successfully treated, cause lethal systemic infections. One of the key molecular integrators in this morphological transition is the binding of bacterial peptidoglycan (PG) fragments to the Leucine Rich Repeat (LRR) domain of the adenyl cyclase Cyr1p. PG is a highly complex polymer consisting of repeating units of the disaccharide N‐acetyl glucosamine‐N acetyl muramic acid, which are crosslinked by pentapeptide side chains. Considerable diversity exists in this polymer within and among Gram (‐) and Gram (+) species of bacteria, rendering a plethora of potential ligands for Cyr1p. Identification of specific PG fragments that are able to induce hyphal formation in C. albicans, and stabilize Cyr1p through the LRR domain can elude to small molecule inhibitors to combat these lethal fungal infections that are continuously becoming resistant to current antifungal therapies. To approach a congruency of PG fragments from both Gram (‐) and Gram (+) bacterium that can serve as ligands for the LRR domain, we began by characterizing recombinant LRR and in doing so have determined this protein is a peripheral membrane protein that adopts an alpha helical conformation. Furthermore, to assess Cyr1p's ability to interact with a repertoire of PG fragments, we conducted Cellular Thermal Shift Assays (CETSA) with various PG fragments and have observed a temperature induced stabilization in comparison to apo‐protein, suggesting that there is a positive interaction with the LRR domain and the fragments. We further investigated the ability of these PG fragments to induce hyphal formation in WT C. albicans cells via microscopy assays and evaluated upregulation of hyphae specific genes at various timepoints via RT‐PCR. We observed differential hyphal gene regulation at various timepoints, suggesting that some fragments although able to initiate hyphal growth, failed to maintain growth and subsequent elongation. In addition, we observed that despite a positive association of the LRR with some fragments through CETSA assays, these fragments failed to illicit hyphal formation in WT cells. We infer that stabilization of the LRR is not linearly attributed to hyphal elongation, although hyphal initiation may be present. Collectively, these data help to identify new mechanisms to treat pathogenic fungal infections through the elucidation of promiscuity of the Cyr1p LRR domain, and steers us towards the identification of PG decoys and/or small molecule inhibitors for the treatment of these infections.

Probing the competitive inhibitor efficacy of frog-skin alpha helical AMPs identified against ACE2 binding to SARS-CoV-2 S1 spike protein as therapeutic scaffold to prevent COVID-19.

In COVID-19 infection, the SARS-CoV-2 spike protein S1 interacts to the ACE2 receptor of human host, instigating the viral infection. To examine the competitive inhibitor efficacy of broad spectrum alpha helical AMPs extracted from frog skin, a comparative study of intermolecular interactions between viral S1 and AMPs was performed relative to S1-ACE2p interactions. The ACE2 binding region with S1 was extracted as ACE2p from the complex for ease of computation. Surprisingly, the Spike-Dermaseptin-S9 complex had more intermolecular interactions than the other peptide complexes and importantly, the S1-ACE2p complex. We observed how atomic displacements in docked complexes impacted structural integrity of a receptor-binding domain in S1 through conformational sampling analysis. Notably, this geometry-based sampling approach confers the robust interactions that endure in S1-Dermaseptin-S9 complex, demonstrating its conformational transition. Additionally, QM calculations revealed that the global hardness to resist chemical perturbations was found more in Dermaseptin-S9 compared to ACE2p. Moreover, the conventional MD through PCA and the torsional angle analyses indicated that Dermaseptin-S9 altered the conformations of S1 considerably. Our analysis further revealed the high structural stability of S1-Dermaseptin-S9 complex and particularly, the trajectory analysis of the secondary structural elements established the alpha helical conformations to be retained in S1-Dermaseptin-S9 complex, as substantiated by SMD results. In conclusion, the functional dynamics proved to be significant for viral Spike S1 and Dermaseptin-S9 peptide when compared to ACE2p complex. Hence, Dermaseptin-S9 peptide inhibitor could be a strong candidate for therapeutic scaffold to prevent infection of SARS-CoV-2.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00894-022-05117-8.

Do membranes or SNARE proteins mediate the effects of alcohol on vesicle fusion?

In neurons, neurotransmitter release is highly regulated and sensitive to alcohol. Release of neurotransmitters occurs when synaptic vesicles undergo membrane fusion with the plasma membrane. The SNARE proteins catalyze this reaction by zippering together moving from a more disordered conformation to a more ordered alpha helical conformation. The SNARE proteins Syntaxin-1A and SNAP-25 are associated with the plasma membrane and Synaptobrevin-2 is associated with the vesicle. Alcohol may act directly on the fusion machinery or on the fusing membranes, separately from any upstream or downstream process.

Isolation and Chemical Characterization of an Alpha-Helical Peptide, Dendrocin-ZM1, Derived from Zataria multiflora Boiss with Potent Antibacterial Activity.

Today, resistance of microorganisms to antibiotics has become a major challenge. To overcome this problem, development of new drugs, besides research on their antibacterial activity, is essential. Among chemical components, antimicrobial peptides (AMPs) exhibit antibacterial activity and can be selected as suitable antimicrobial candidates. In this study, a novel antimicrobial peptide, called dendrocin-ZM1, with a molecular weight of ~3716.48Da, was isolated from Zataria multiflora Boiss (ZM) and purified via precipitation with ammonium sulfate and reverse-phase HPLC chromatography; it was then sequenced via Edman degradation. The in silico method was used to examine the physicochemical properties of dendrocin-ZM1. In this study, four reference strains (gram-positive and gram-negative) and one clinical vancomycin-resistant Staphylococcus aureus strain were used to survey the antimicrobial activities. Moreover, to examine cytotoxicity and hemolytic activity, a HEK-293 cell line and human red blood cells (RBCs) were used, respectively. Evaluation of the physicochemical properties of dendrocin-ZM1, as an AMP, indicated a net charge of + 7 and a hydrophobicity percentage of 54%. This peptide had an amphipathic alpha-helical conformation. It exhibited broad-spectrum antibacterial activities against the tested strains at minimum inhibitory concentrations (MICs) of 4-16μg/mL. Besides, this peptide showed negligible hemolysis and cytotoxicity in the MIC range. It also exhibited heat stability at temperatures of 20 to 80°C and was active in a broad pH range (from 6.0 to 10.0). Overall, the present results suggested dendrocin-ZM1 as a remarkable antimicrobial candidate.

The impact of phosphatidylserine exposure on cancer cell membranes on the activity of the anticancer peptide HB43.

HB43 (FAKLLAKLAKKLL) is a synthetic peptide active against cell lines derived from breast, colon, melanoma, lung, prostate, and cervical cancers. Despite its remarkable spectrum of activity, the mechanism of action at the molecular level has never been investigated, preventing further optimization of its selectivity. The alternation of charged and hydrophobic residues suggests amphipathicity, but the formation of alpha-helical structure seems discouraged by its short length and the large number of positively charged residues. Using different biophysical and in silico approaches we show that HB43 is completely unstructured in solution but assumes alpha-helical conformation in the presence of DPC micelles and liposomes exposing phosphatidylserine (PS) used as mimics of cancer cell membranes. Membrane permeabilization assays demonstrate that the interaction leads to the preferential destabilization of PS-containing vesicles with respect to PC-containing ones, here used as noncancerous cell mimics. ssNMR reveals that HB43 is able to fluidify the internal structure of cancer-cell mimicking liposomes while MD simulations show its internalization in such bilayers. This is achieved by the formation of specific interactions between the lysine side chains and the carboxylate group of phosphatidylserine and/or the phosphate oxygen atoms of targeted phospholipids, which could catalyze the formation of the alpha helix required for internalization. With the aim of better understanding the peptide biocompatibility and the additional antibacterial activity, the interaction with noncancerous cell mimicking liposomes exposing phosphatidylcholine (PC) and bacterial mimicking bilayers exposing phosphatidylglycerol (PG) is also described.

Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca

Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defence peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-d_{3} mixture. The structure is defined by an alpha-helix that spans between Ile^{6}-Ala^{26}, and a cyclic disulfide-bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide’s interactions with sodium dodecyl sulfate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its alpha-helical conformation between residues Ile^{6}-Ala^{26}, while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.

Structural and functional stability of the sulfur-free surfactant protein B peptide mimic B-YL in synthetic surfactant lipids

BackgroundOptimal functionality of synthetic lung surfactant for treatment of respiratory distress syndrome in preterm infants largely depends on the quality and quantity of the surfactant protein B (SP-B) peptide mimic and the lipid mixture. B-YL peptide is a 41-residue sulfur-free SP-B mimic with its cysteine and methionine residues replaced by tyrosine and leucine, respectively, to enhance its oxidation resistance.AimTesting the structural and functional stability of the B-YL peptide in synthetic surfactant lipids after long-term storage.MethodsThe structural and functional properties of B-YL peptide in surfactant lipids were studied using three production runs of B-YL peptides in synthetic surfactant lipids. Each run was held at 5 °C ambient temperature for three years and analyzed with structural and computational techniques, i.e., MALDI-TOF mass spectrometry, ATR-Fourier Transform Infrared Spectroscopy (ATR-FTIR), secondary homology modeling of a preliminary B-YL structure, and tertiary Molecular Dynamic simulations of B-YL in surfactant lipids, and with functional methods, i.e., captive bubble surfactometry (CBS) and retesting in vivo surface activity in surfactant-deficient young adult rabbits.ResultsMALDI-TOF mass spectrometry showed no degradation of the B-YL peptide as a function of stored time. ATR-FTIR studies demonstrated that the B-YL peptide still assumed stable alpha-helical conformations in synthetic surfactant lipids. These structural findings correlated with excellent in vitro surface activity during both quasi-static and dynamic cycling on CBS after three years of cold storage and in vivo surface activity of the aged formulations with improvements in oxygenation and dynamic lung compliance approaching those of the positive control surfactant Curosurf®.ConclusionsThe structure of the B-YL peptide and the in vitro and in vivo functions of the B-YL surfactant were each maintained after three years of refrigeration storage.