Anionic Lipid Bilayers Research Articles

Polarized ATR-FTIR studies of DPPC/DPPG lipid bilayers doped with PLL

The structural characterization of lipid bilayers is crucial for understanding various biological processes and designing effective systems of drugs. Attenuated Total Reflection Fourier Transformed Infrared (ATR-FTIR) spectroscopy is a powerful technique for probing lipid membrane properties, but conventional methods face challenges in discerning molecular orientation and intermolecular interactions. The polarized ATR-FTIR spectroscopy combined with Principal Component Analysis (PCA) was used to investigate the interactions between positively charged peptide (poly-L-lysine (PLL)) and anionic lipid bilayers (dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol, DPPC/DPPG). This integrated approach offers deeper insight into mutual interactions between proteins and lipids of membrane, providing a comprehensive understanding of structure of both protein and lipid bilayers components. The findings of this study shed light on the orientational order of lipid molecules in DPPC/DPPG membrane structure as well as secondary strictures of PPL peptides. It was shown that the presence of PLL peptide disrupts lipid molecule ordering within the lipid bilayer, primarily through electrostatic and H-bond interactions. The utility of polarized ATR-FTIR spectroscopy supported by PCA calculations in elucidating molecular organizations within complex membrane systems is emphasized.

Bilayer lipids modulate ligand binding to atypical chemokine receptor 3

Chemokine receptors belong to the large class of G protein-coupled receptors (GPCRs) and are involved in a number of (patho)physiological processes. Previous studies highlighted the importance of membrane lipids for modulating GPCR structure and function. However, the underlying mechanisms of how lipids regulate GPCRs are often poorly understood. Here, we report that anionic lipid bilayers increase the binding affinity of the chemokine CXCL12 for the atypical chemokine receptor 3 (ACKR3) by modulating the CXCL12 binding kinetics. Notably, the anionic bilayer favors CXCL12 over the more positively charged chemokine CXCL11, which we explained by bilayer interactions orienting CXCL12 but not CXCL11 for productive ACKR3 binding. Furthermore, our data suggest a stabilization of active ACKR3 conformations in anionic bilayers. Taken together, the described regulation of chemokine selectivity of ACKR3 by the lipid bilayer proposes an extended version of the classical model of chemokine binding including the lipid environment of the receptor.

Binding and dimerization of PGLa peptides in anionic lipid bilayer studied by replica exchange molecular dynamics

The 21-residue PGLa peptide is well known for antimicrobial activity attributed to its ability to compromize bacterial membranes. Using all-atom explicit solvent replica exchange molecular dynamics with solute tempering, we studied PGLa binding to a model anionic DMPC/DMPG bilayer at the high peptide:lipid ratio that promotes PGLa dimerization (a two peptides per leaflet system). As a reference we used our previous simulations at the low peptide:lipid ratio (a one peptide per leaflet system). We found that the increase in the peptide:lipid ratio suppresses PGLa helical propensity, tilts the bound peptide toward the bilayer hydrophobic core, and forces it deeper into the bilayer. Surprisingly, at the high peptide:lipid ratio PGLa binding induces weaker bilayer thinning, but deeper water permeation. We explain these effects by the cross-correlations between lipid shells surrounding PGLa that leads to a much diminished efflux of DMPC lipids from the peptide proximity at the high peptide:lipid ratio. Consistent with the experimental data the propensity for PGLa dimerization was found to be weak resulting in coexistence of monomers and dimers with distinctive properties. PGLa dimers assemble via apolar criss-cross interface and become partially expelled from the bilayer residing at the bilayer-water boundary. We rationalize their properties by the dimer tendency to preserve favorable electrostatic interactions between lysine and phosphate lipid groups as well as to avoid electrostatic repulsion between lysines in the low dielectric environment of the bilayer core. PGLa homedimer interface is predicted to be distinct from that involved in PGLa-magainin heterodimers.

Design, Synthesis, and Anticancer Activity of N‐1 Terminal Segment of α‐(2, 5,7‐Tri‐tert‐butylindol‐3‐yl)alanine‐Incorporated Short Peptides

AbstractUsing a minimallised approach,we designed and synthesized N‐terminal modified peptides W‐KRGNKWLA‐CONH2 (P1) and W‐ARGDGGNGRGA‐CONH2 (P2) using Fmoc‐Chemistry solid phase peptide synthesis protocol. High‐performance liquid chromatograms and mass spectrometry (LCMS and analytical HPLC) were employed to determine the purity and molecular weight of the peptide. Surface tension measurements are used to evaluate peptide surface activity. The primary structure of the peptideis anlaysed by NMR spectroscopy. Circular dichroism studies demonstrate that the secondary structure of the peptide changes minimally when it attached to anionic lipid bilayers (POPC : POPG). According to a steady‐state fluorescence experiment, peptides P1 and P2 preferentially interact with anionic (POPC : POPG) lipid bilayers rather than zwitterionic (POPC) lipid bilayers. The peptide P2 interacts preferentially with anionic lipid membranes and has an anticancer effect due to RGD and NGR binding motifs. The cytotoxicity of the MTT experiment demonstrated that both normal and cancerous cells could lower peptide concentrations below their IC50 levels. Molecular docking also supports in‐vitro anticancer research. Both peptides P1 and P2 were non‐toxic to vero cells and appeared to be more promising as it demonstrated wide spectrum anticancer activity.

Probing Mastoparan-like Antimicrobial Peptides Interaction with Model Membrane Through Energy Landscape Analysis.

Antimicrobial Peptides (AMPs) have emerged as promising alternatives to conventional antibiotics due to their capacity to disrupt the lipid packing of bacterial cell membranes. This mechanism of action may prevent the development of resistance by bacteria. Understanding their role in lipid packing disruption and their structural properties upon interaction with bacterial membranes is highly desirable. In this study, we employed Molecular Dynamics simulations and the Energy Landscape Visualization Method (ELViM) to characterize and compare the conformational ensembles of mastoparan-like Polybia-MP1 and its analogous H-MP1, in which histidines replace lysine residues. Two situations were analyzed: (i) the peptides in their free state in an aqueous solution containing water and ions and (ii) the peptides spontaneously adsorbing onto an anionic lipid bilayer, used as a bacteria membrane mimetic. ELViM was used to project a single effective conformational phase space for both peptides, providing a comparative analysis. This projection enabled us to map the conformational ensembles of each peptide in an aqueous solution and assess the structural effects of substituting lysines with histidines in H-MP1. Furthermore, a single conformational phase space analysis was employed to describe structural changes during the adsorption process using the same framework. We show that ELViM provides a comprehensive analysis, able to identify discrepancies in the conformational ensembles of these peptides that may affect their affinity to the membrane and adsorption kinetics.

Lateral Transport of Domains in Anionic Lipid Bilayer Membranes under DC Electric Fields: A Coarse-Grained Molecular Dynamics Study.

Dynamic lateral transport of lipids, proteins, and self-assembled structures in biomembranes plays a crucial role in diverse cellular processes. In this study, we perform coarse-grained molecular dynamics simulations on a vesicle composed of a binary mixture of neutral and anionic lipids to investigate the lateral transport of individual lipid molecules and the self-assembled lipid domains upon an applied direct current (DC) electric field. Under the potential force of the electric field, a phase-separated domain rich in anionic lipids is trapped in the opposite direction of the electric field. The subsequent reversal of the electric field induces unidirectional domain motion. During the domain motion, the domain size remains constant, but a considerable amount of the anionic lipids is exchanged between the anionic-lipid-rich domain and the surrounding bulk. While the speed of the domain motion (collective lipid motion) shows a significant positive correlation with the electric field strength, the exchange of anionic lipids between the domain and bulk (individual lipid motion) exhibits no clear correlation with the field strength. The mean velocity field of the lipids surrounding the domain displays a two-dimensional (2D) source dipole. We revealed that the balance between the potential force of the applied electric field and the quasi-2D hydrodynamic frictional force well explains the dependence of the domain motions on the electric field strengths. The present results provide insight into the hierarchical dynamic responses of self-assembled lipid domains to the applied electric field and contribute to controlling the lateral transportation of lipids and membrane inclusions.

Abstract 3845: Structural mechanisms of how PTEN mutations degrade function at the membrane and life expectancy of carriers of mutations in the human brain

Abstract PTEN negatively regulates PI3K/AKT/mTOR signaling through dephosphorylation of PIP3 to PIP2. PTEN dysfunction triggers disease phenotypes, ranging from cancer and PHTS to NDDs such as ASD. The most common PTEN mutations, nonsense, frameshift, and deletion/insertion, may cause premature termination of translation. PTEN has a considerable number of missense mutations that may result in loss of protein function through reduced catalytic activity and protein stability. To decipher mechanistic details of the mutations and structural features of the mutant proteins, comprehensive computational studies were performed for PTEN mutants at an anionic lipid bilayer containing PC, PS, PIP2, and PIP3. Six PTP mutations (Y68H, H93R, A126T, R130Q, G132D, R173C) and two C2 mutations (F241S, D252G) were considered. The PTP mutations are known to associate with cancer, PHTS, macrocephaly, and ASD, while the C2 mutations are exclusively related to macrocephaly and ASD. Our studies indicate that the PTEN mutants can effectively absorb the anionic lipid bilayer, similar to wild-type PTEN. However, Y68H significantly disrupts the core of PTP domain, reducing protein stability. H93R hijacks the substate PIP3, interrupting the catalytic site residues recruitment of the substate for catalysis. A126T, R130Q, and G132D in the P loop directly influence the dynamics of the loop, yielding a collapsed loop conformation. The collapsed P loop loses the interaction with the WPD loop, leading to dehydration in the catalytic site. R173C and D252G at the interface between two major domains disrupt the domain-domain interaction, allosterically biasing the P loop dynamics. However, F241S in C2 exhibits less effective allosteric connection to the catalytic site than that observed in the wild type. PCA of the sampled conformations found that the macrocephaly and ASD-related H93R and F241S are likely to sample the conformations present in the wild type. In contrast, the sampled conformations for the cancer and PHTS-related Y68H, A126T, and G132D are different from those for the wild type. These distinct structural features seem to correlate with mutation strength and timing of the expression of the genes that determine the cancer and NDD outcomes. The isoform expression data extracted from the BrainSpan indicate that exon 5 is impacted by NDD or non-NDD mutations. Interestingly, PTEN splicing isoforms that do not carry exon 5 are exclusively impacted by the NDD mutations, F241S and D252G. The increased life expectancy of PTEN variants carriers carrying exons 5 and 7 is highly correlated to increased lifetime risk for certain cancers. Our results underscore the detailed structural and functional mechanisms of PTEN with mutations at the membrane, and suggest that tissue-specific splicing isoform expression should be taken into consideration while predicting the effect of mutations in various diseases. Citation Format: Hyunbum Jang, Jiaye Chen, Lilia M. Iakoucheva, Ruth Nussinov. Structural mechanisms of how PTEN mutations degrade function at the membrane and life expectancy of carriers of mutations in the human brain. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3845.

Design and synthesis of novel N-terminal peptides of integrin and aminopeptidase are new finding for anticancer activity

The short peptides, containing the amino acid sequence asparagine-glycine-arginine (NGR) and arginine-glycine-aspartic acid (RGD), possess the strong binding ability to N (APN/CD13) aminopeptidase receptor and integrin proteins involved in antitumor properties are overexpressed. A novel short N-terminal modified hexapeptides P1 and P2 was designed and synthesized using the Fmoc-chemistry solid phase peptide synthesis protocol. Notably, the cytotoxicity of the MTT assay demonstrated the viability of normal and cancer cells up to lower peptide concentrations. Interestingly, both peptides show good anticancer activities against the four cancer cells and normal cells namely, Hep-2, HepG2, MCF-7, A375, and Vero and compared with standard drugs, doxorubicin and paclitaxel. Additionally, in silico studies were applied to predict the binding sites and binding orientation of the peptides for potential anticancer targets. Steady-state fluorescence measurements showed that peptide P1 exhibits preferential interactions with POPC/POPG anionic bilayers rather than the zwitterionic POPC lipid bilayers and peptide P2, did not show any preferential interaction with lipids bilayers. But impressively, peptide P2 shows anticancer activity due to the NGR/RGD motif. Circular dichroism studies demonstrated that the peptide's secondary structure changes only minimally upon binding to the anionic lipid bilayers.

The interaction of tryptophan enantiomers with model membranes is modulated by polar head type and physical state of phospholipids

The mutual influence of chiral bioactive molecules and supramolecular assemblies is currently being studied in many research fields, including medical-pharmaceutical applications. Model membranes of phospholipids, such as the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the anionic dipalmitoylphosphatidylglycerol (DPPG), interact with a variety of chiral compounds that include amino acids. In this work, the interaction of tryptophan enantiomers, L-Trp and D-Trp, on DPPC and DPPG bilayers was investigated by using differential scanning calorimetry, attenuated total reflectance-Fourier transform infrared and spin-label electron spin resonance spectroscopies as well as molecular docking simulations. The results show that Trp enantiomers slightly perturb the bilayer thermotropic phase transitions. For both membranes, O atoms in the carbonyl groups have a propensity to act as acceptors of a (weak) hydrogen bond. The Trp chiral forms also promote formation of hydrogen bonds and/or hydration in the PO2– moiety of the phosphate group, especially for the DPPC bilayer. In contrast, they interact more closely with the glycerol group of DPPG polar head. Only for DPPC bilayers, both enantiomers increase the packing of the first hydrocarbon chain segments for temperatures through the gel state, whereas they do not affect the lipid chain order and mobility in the fluid state. The results are consistent with a Trp association in the upper region of the bilayers without permeation in the innermost hydrophobic region. The findings suggest that neutral and anionic lipid bilayers are differently sensitive to amino acid chirality.

Modulation of Anionic Lipid Bilayers by Specific Interplay of Protons and Calcium Ions.

Biomembranes, important building blocks of living organisms, are often exposed to large local fluctuations of pH and ionic strength. To capture changes in the membrane organization under such harsh conditions, we investigated the mobility and hydration of zwitterionic and anionic lipid bilayers upon elevated H3O+ and Ca2+ content by the time-dependent fluorescence shift (TDFS) technique. While the zwitterionic bilayers remain inert to lower pH and increased calcium concentrations, anionic membranes are responsive. Specifically, both bilayers enriched in phosphatidylserine (PS) and phosphatidylglycerol (PG) become dehydrated and rigidified at pH 4.0 compared to at pH 7.0. However, their reaction to the gradual Ca2+ increase in the acidic environment differs. While the PG bilayers exhibit strong rehydration and mild loosening of the carbonyl region, restoring membrane properties to those observed at pH 7.0, the PS bilayers remain dehydrated with minor bilayer stiffening. Molecular dynamics (MD) simulations support the strong binding of H3O+ to both PS and PG. Compared to PS, PG exhibits a weaker binding of Ca2+ also at a low pH.

α-Synuclein Interaction with Lipid Bilayer Discs.

α-Synuclein (aSyn) is a 140 residue long protein present in presynaptic termini of nerve cells. The protein is associated with Parkinson’s disease, in which case it has been found to self-assemble into long amyloid fibrils forming intracellular inclusions that are also rich in lipids. Furthermore, its synaptic function is proposed to involve interaction with lipid membranes, and hence, it is of interest to understand aSyn–lipid membrane interactions in detail. In this paper we report on the interaction of aSyn with model membranes in the form of lipid bilayer discs. Using a combination of cryogenic transmission electron microscopy and small-angle neutron scattering, we show that circular discs undergo a significant shape transition after the adsorption of aSyn. When aSyn self-assembles into fibrils, aSyn molecules desorb from the bilayer discs, allowing them to recover to their original shape. Interestingly, the desorption process has an all-or-none character, resulting in a binary coexistence of circular bilayer discs with no adsorbed aSyn and deformed bilayer discs having a maximum amount of adsorbed protein. The observed coexistence is consistent with the recent finding of cooperative aSyn adsorption to anionic lipid bilayers.

Abstract 2918: Comprehensive survey of KRas4B membrane interaction and orientation

Abstract Three RAS genes encode four Ras proteins, HRas, NRas, and KRas splice variants, KRas4A and KRas4B. In human cancers, KRAS oncogene is highly expressed (85%) as compared to other RAS oncogenes, NRAS (11%) and HRAS (4%). Oncogenic Ras leads to aberrant cell proliferation signaling, primarily through the MAPK (Raf/MEK/ERK) pathway. At the plasma membrane, nanoclustered Ras proteins promote Raf activation, mediating the downstream signaling pathway. Among the Ras isoforms, KRas4B in the membrane has been studied extensively due to its higher level of mutations in cancer. Although a number of experimental studies have focused on KRas4B subcellular localization and their nanocluster formation at the signaling platform, the mechanistic details of the membrane anchorage and the membrane orientation of KRas4B at atomic resolution are still unknown. Here, comprehensive computational studies were performed for KRas4B at the anionic lipid bilayers, composed of the phospholipids, PS and PA, and the phosphoinositides, PIP2, PI4P, and PI5P. Wild-type and two oncogenic mutants, G12D and G12V KRas4B in the GTP- and GDP-bound states were considered. The lysine rich HVR was post-translationally modified with the farnesyl and methyl groups. Previous studies showed that wild-type KRas4B-GDP interacts weakly and nonspecifically with anionic phospholipids, while oncogenic mutations alter selective anionic lipid binding toward anionic lipids, PA, PI3P, PI4P, and PI5P. Our studies indicate that the membrane interaction of KRas4B is highly sensitive depending on the types of nucleotides, mutational states, and anionic lipids enriched the lipid bilayer. While the GTP-bound spontaneously inserts its prenyl group into the hydrophobic core of the bilayer, the occurrence of spontaneous insertion is reduced in the GDP-bound form. The distance of GTP-bound catalytic domain from the bilayer surface is longer than the GDP-bound form. The HVR is sequestered by the GDP-bound catalytic domain, which causes the catalytic domain to be occluded and increases the membrane interaction by the catalytic domain. In contrast, the GTP-bound catalytic domain releases its HVR and liberates from the membrane interaction. The occlusion of catalytic domain is less prevalent in the GTP-bound state. KRas4BG12D/G12V-GTP exhibits the active-state orientation, exposing its effector binding site for recruiting the effectors. While PA and PIP2 support the active-state orientation, the PIP4 enriched bilayer prevents the spontaneous prenyl insertion resulting in the occlusion of catalytic domain. Our results underscore the importance of detailed structural mechanisms of KRas4B membrane interaction and orientation, elucidating vital mechanistic role of KRas4B, a key membrane-anchored protein in cancer. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261201500003I. Citation Format: Hyunbum Jang, Mingzhen Zhang, Vadim Gaponenko, Ruth Nussinov. Comprehensive survey of KRas4B membrane interaction and orientation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2918.

Membrane Interactions of α-Synuclein Revealed by Multiscale Molecular Dynamics Simulations, Markov State Models, and NMR.

α-Synuclein (αS) is a presynaptic protein that binds to cell membranes and is linked to Parkinson’s disease (PD). Binding of αS to membranes is a likely first step in the molecular pathophysiology of PD. The αS molecule can adopt multiple conformations, being largely disordered in water, adopting a β-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of α-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces. Multiscale molecular dynamics simulations in conjunction with nuclear magnetic resonance (NMR) and cross-linking mass spectrometry (XLMS) measurements are used to explore the interactions of αS with an anionic lipid bilayer. The simulations and NMR measurements together reveal a break in the helical structure of the central non-amyloid-β component (NAC) region of αS in the vicinity of residues 65–70, which may facilitate subsequent oligomer formation. Coarse-grained simulations of αS starting from the structure of αS when bound to a detergent micelle reveal the overall pattern of protein contacts to anionic lipid bilayers, while subsequent all-atom simulations provide details of conformational changes upon membrane binding. In particular, simulations and NMR data for liposome-bound αS indicate incipient β-strand formation in the NAC region, which is supported by intramolecular contacts seen via XLMS and simulations. Markov state models based on the all-atom simulations suggest a mechanism of conformational change of membrane-bound αS via a dynamic helix break in the region of residue 65 in the NAC region. The emergent dynamic model of membrane-interacting αS advances our understanding of the mechanism of PD, potentially aiding the design of novel therapeutic approaches.

Tuning of a Membrane-Perforating Antimicrobial Peptide to Selectively Target Membranes of Different Lipid Composition.

The use of designed antimicrobial peptides as drugs has been impeded by the absence of simple sequence-structure-function relationships and design rules. The likely cause is that many of these peptides permeabilize membranes via highly disordered, heterogeneous mechanisms, forming aggregates without well-defined tertiary or secondary structure. We suggest that the combination of high-throughput library screening with atomistic computer simulations can successfully address this challenge by tuning a previously developed general pore-forming peptide into a selective pore-former for different lipid types. A library of 2916 peptides was designed based on the LDKA template. The library peptides were synthesized and screened using a high-throughput orthogonal vesicle leakage assay. Dyes of different sizes were entrapped inside vesicles with varying lipid composition to simultaneously screen for both pore size and affinity for negatively charged and neutral lipid membranes. From this screen, nine different LDKA variants that have unique activity were selected, sequenced, synthesized, and characterized. Despite the minor sequence changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic lipid bilayers. Long-scale, unbiased atomistic molecular dynamics (MD) simulations directly reveal that rather than rigid, well-defined pores, these peptides can form a large repertoire of functional dynamic and heterogeneous aggregates, strongly affected by single mutations. Predicting the propensity to aggregate and assemble in a given environment from sequence alone holds the key to functional prediction of membrane permeabilization.

Polycation-Anionic Lipid Membrane Interactions.

Natural or synthetic polycations are used as biocides or as drug/gene carriers. Understanding the interactions between these macromolecules and cell membranes at the molecular level is therefore of great importance for the design of effective polymer biocides or biocompatible polycation-based delivery systems. Until now, details of the processes at the interface between polycations and biological systems have not been fully recognized. In this study, we consider the effect of strong polycations with quaternary ammonium groups on the properties of anionic lipid membranes that we use as a model system for protein-free cell membranes. For this purpose, we employed experimental measurements and atomic-scale molecular dynamics (MD) simulations. MD simulations reveal that the polycations are strongly hydrated in the aqueous phase and do not lose the water shell after adsorption at the bilayer surface. As a result of strong hydration, the polymer chains reside at the phospholipid headgroup and do not penetrate to the acyl chain region. The polycation adsorption involves the formation of anionic lipid-rich domains, and the density of anionic lipids in these domains depends on the length of the polycation chain. We observed the accumulation of anionic lipids only in the leaflet interacting with the polymer, which leads to the formation of compositionally asymmetric domains. Asymmetric adsorption of the polycation on only one leaflet of the anionic membrane strongly affects the membrane properties in the polycation–membrane contact areas: (i) anionic lipid accumulates in the region near the adsorbed polymer, (ii) acyl chain ordering and lipid packing are reduced, which results in a decrease in the thickness of the bilayer, and (iii) polycation–anionic membrane interactions are strongly influenced by the presence and concentration of salt. Our results provide an atomic-scale description of the interactions of polycations with anionic lipid bilayers and are fully supported by the experimental data. The outcomes are important for understanding the correlation of the structure of polycations with their activity on biomembranes.

Effect of Non-natural Hydrophobic Amino Acids on the Efficacy and Properties of the Antimicrobial Peptide C18G.

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules through evolution. The peptides are known to selectively bind to bacterial cell surfaces through electrostatic interactions, and subsequently, the peptides insert into the cell membrane and cause local disruptions of membrane integrity leading to cell death. Previous experiments showed that replacing the Leu residues in the AMP C18G with other naturally occurring hydrophobic residues resulted in side-chain-dependent activities. This work extends the investigation to non-natural hydrophobic amino acids and the effect on peptide activity. Minimal inhibitory concentration (MIC) results demonstrated that amino acid substitutions containing long flexible carbon chains maintained or increased antimicrobial activity compared to natural analogues. In solution, the peptide showed aggregation only with the most hydrophobic non-natural amino acid substitutions. Binding assays using Trp fluorescence confirm a binding preference for anionic lipids while quenching experiments demonstrated that the more hydrophobic peptides are more deeply buried in the anionic lipid bilayers compared to the zwitterionic bilayers. The most effective peptides at killing bacteria were also those which showed some level of disruption of bacterial membranes; however, one peptide sequence exhibited very strong activity and very low levels of red blood cell hemolysis, yielding a promising target for future development.

Pathogen associated molecular pattern-decorated mesoporous silica-A colloidal model for studying bacterial-host cell interactions.

Tuberculosis is the top infectious disease worldwide and the development of a vaccine and diagnostic tools to control the disease is a priority that requires a better understanding of the factors involved in the pathogenesis of Mycobacterium tuberculosis, the infectious agent. It is known that bacterial cell surface components are released, interact with immune cell receptors, and may traffic toward host cell structures. Many of these compounds are lipids that have been associated with mycobacterial virulence. However, their hydrophobic nature has frequently hampered their biological study. In this work, silica particles were coated with functional lipids to obtain a colloidal bioinspired system based on nonhydrosoluble glycolipids. Mycobacterium tuberculosis phosphatidylinositol mannosides (PIMs), known to interact with receptors of innate immune cells, were purified from the M. tuberculosis H37Rv type strain, and used to prepare large unilamellar liposomes in combination with zwitterionic phosphatidyl choline. Then, bacillary-like Santa Barbara Amorphous-15 (SBA-15) silica particles were cationized and the vesicle fusion method was used to promote the attachment of anionic PIM-containing lipid bilayers. Thermogravimetric analysis, x-ray diffraction, N2 adsorption-desorption isotherm analysis, Fourier transform infrared spectroscopy, electron microscopy, and zeta potential analyses were used to characterize the materials obtained. The as-prepared PIM-containing colloids, named PIM@SBA-15, showed biocompatibility toward human fibroblasts and were found to colocalize with Toll-like receptor (TLR)2 upon their incubation with THP1-derived macrophages. Furthermore, the particles induced the formation of pseudopods and were internalized into phagocytic cells. In all, these data suggest the usefulness of PIM@SBA-15 particles to better comprehend the interactions between immune cells and PIMs.

Design and synthesis of a novel peptide for selective detection of cancer cells.

Using a minimalist approach, an 11-residue peptide (Peptide 1) tagged with rhodamine fluorophore was designed and synthesized for selective detection of cancer cells. Peptide 1 contains RGD and NGR motifs to bind, respectively, integrins and aminopeptidase CD13, which are over expressed in cancer cells. Surface tension measurements revealed that peptide 1 possess surface-active property owing to the overall hydrophobicity and cationic nature of the peptide. Peptide 1 displays cancer cell-selective binding at ≤5.0µM concentrations, while peptide 2 (randomized sequence of 1) shows non-selective binding to normal and cancer cells. Fluorescence microscopy and FACS analysis demonstrated the intracellular localization of peptide 1 in three different cancer cell lines, confirming the role of RGD and NGR motifs. Cytotoxicity assay exhibited the viability of normal and cancer cells up to 100µM concentrations of peptide 1. Steady-state fluorescence measurements disclosed the preferential interactions of the peptide 1 with anionic POPC/POPG bilayers rather than with zwitterionic POPC lipid bilayers. Circular dichroism studies showed minimal changes in the secondary structure of peptide 1 upon binding with the anionic lipid bilayers. Peptide 1 is largely unordered, non-toxic, and useful for identification of cancer cells. Peptide 1 provides a template for designing drug-loaded peptides for targeted delivery into cancer cells.

Conformational Dynamics and Energetics of Melittin and its Diastereomer Interacting with POPC and POPG Lipid Bilayers: A Molecular Dynamics Study

Often amphipathic membrane-active peptides assume helical secondary structure when partitioning into lipid membranes. Characterizing this membrane-induced folding process at single molecule level is fundamental to understanding peptide lipid-membrane interactions. To this end, we use all-atom, long time scale (∼1µs) molecular dynamics (MD) simulations to investigate and compare the conformational dynamics and energetics of the well-studied peptide melittin (MWT) and one of its less-studied diastereomer, with four D-amino acids, (MD4) interacting respectively with a zwitterionic POPC and an anionic POPG lipid bilayer. Unlike in previous MD studies, we start from a completely folded conformation of the peptides and follow their unfolding through free MD simulations. We find that (i) both MWT and MD4 unfold in solution and remain mostly disordered; (ii) MWT binds to both POPC and POPG, remaining mostly folded (with more than 70% helical content); and (iii) MD4 binds only to POPG and it is mostly disordered (with less than 40% helical content). Furthermore, by calculating the potential of mean force, as a function of peptide-lipid bilayer separation, we have determined the relative dissociation energy of MWT and MD4 from POPG, which can be related to the membrane induced folding energy of melittin. To our knowledge, this is the first MD study of the MD4 melittin analog, and the obtained results are in good agreement with previous experimental studies. Work supported in part by a grant from the MU Research Board. The computation for this work was performed on the high HPC infrastructure provided by RCSS and in part by the NSF under grant number CNS-1429294 at the University of Missouri, Columbia MO.

Enveloped artificial viral capsids self-assembled from anionic β-annulus peptide and cationic lipid bilayer.

Anionic artificial viral capsids were self-assembled from β-annulus-EE peptide, then complexed with lipid-bilayer-containing cationic lipids via electrostatic interaction to form enveloped artificial viral capsids. The critical aggregation concentration of the enveloped artificial viral capsid was significantly lower than that of the uncomplexed artificial viral capsid, indicating that the lipid bilayer stabilised the capsid structure.