Self-immolation Assisted Morphology Transformation of Prebiotic Lipidated-cationic Amino Acids: Electro-droplet Mediated C-C Coupling Reaction to Synthesize Macromolecules.

Compartmentalization protected biomolecules from the fluctuating environments of early Earth. Although contemporary cells mostly use phospholipid-based bilayer membranes, the utility of non-bilayer compartments was not ruled out during the prebiotic and modern eras. In the present study, we demonstrated the prebiotic synthesis of lipidated cationic amino acid-based amphiphiles [lauryl ester of lysine (LysL); ornithine (OrnL); and 2,4-diamino butyric acid (DabL)] using model dry-down reaction. These amphiphiles self-assemble into micellar membranes. However, the OrnL and DabL-based micelles undergo pH-responsive transformation to lipid droplet-like morphologies, a modelcompartment in the prebiotic Earth. These cationic droplets encapsulated prebiotic molecules (isoprene) and assisted electron transfer reaction to synthesize isoprenoid derivatives at primitive Earth conditions. The self-assembly of prebiotic amphiphiles, their transformation to droplet compartments, and droplet-assisted C-C bond formation reaction might have helped the evolution to synthesize various biomolecules required for the origin of life.

Phosphate-Based Amphiphile and Lipidated Lysine Assemble into Superior Protocellular Membranes over Carboxylate and Sulfate-Based Systems: A Potential Missing Link between Prebiotic and the Modern Era?

Amphiphiles are among the most extensively studied building blocks that self-assemble into cell-like compartments. Most literature suggested that the building blocks/amphiphiles of early Earth (fatty acid-based membrane) were much simpler than today's phospholipids. To establish the bridge between the prebiotic fatty acid era and the modern phospholipid era, the investigation and characterization of alternate building blocks that form protocellular membranes are necessary. Herein, we report the potential prebiotic synthesis of alkyl phosphate, alkyl carboxylate, and alkyl sulfate amphiphiles (anionic) using dry-down reactions and demonstrate a more general role of cationic amino acid-based amphiphiles to recruit the anionic amphiphiles via ion-pair, hydrogen bonding, and hydrophobic interactions. The formation and self-assembly of the catanionic (mixed) amphiphilic system to vesicular morphology were characterized by turbidimetric, dynamic light scattering, transmission electron microscopy, fluorescence lifetime imaging microscopy, and glucose encapsulation experiments. Further experiments suggest that the phosphate-based vesicles were more stable than the alkyl sulfate and alkyl carboxylate-based systems. Moreover, the alkyl phosphate system can form vesicles at prebiotically relevant acidic pH (5.0), while alkyl carboxylate mainly forms cluster-type aggregates. An extended supramolecular polymer-type network formation via H-bonding and ion-pair interactions might order the membrane interface and stabilize the phosphate-based vesicles. The results suggest that phosphate-based amphiphiles might be a superior successor to fatty acids as early compartment building blocks. The work highlights the importance of previously unexplored building blocks that participate in protocellular membrane formation to encapsulate important precursors required for the functions of early life.

Heterogeneity and Compositional Diversities of Campylobacter jejuni Outer Membrane Vesicles (OMVs) Drive Multiple Cellular Uptake Processes.

Naturally secreted outer membrane vesicles (OMVs) from gut microbes carry diverse cargo, including proteins, nucleic acids, toxins, and many unidentified secretory factors. Bacterial OMVs can shuttle molecules across different cell types as a generalized secretion system, facilitating bacterial pathogenicity and self-survival. Numerous mucosal pathogens, including Campylobacter jejuni (C. jejuni), share a mechanism of harmonized secretion of major virulence factors. Intriguingly, as a common gut pathogen, C. jejuni lacks some classical virulence-associated secretion systems; alternatively, it often employs nanosized lipid-bound OMVs as an intensive strategy to deliver toxins, including secretory proteins, into the target cells. To better understand how the biophysical and compositional attributes of natural OMVs of C. jejuni regulate their cellular interactions to induce a biologically relevant host response, we conducted an in-depth morphological and compositional analysis of naturally secreted OMVs of C. jejuni. Next, we focused on understanding the mechanism of host cell-specific OMVs uptake from the extracellular milieu. We showed that intracellular perfusion of OMVs is mediated by cytosolic as well as multiple endocytic uptake processes due to the heterogenic nature, abundance of surface proteins, and membrane phospholipids acquired from the source bacteria. Furthermore, we used human and avian cells as two different host targets to provide evidence of target cell-specific preferential uptake of OMVs. Together, the present study provides insight into the unique functionality of natural OMVs of C. jejuni at the cellular interface, upholding their potential for multimodal use as prophylactic and therapeutic carriers.

ATP-Assisted Protocellular Membrane Formation with Ethanolamine-Based Amphiphiles.

Prebiotic membranes are one of the essential elements of the origin of life because they build compartments to keep genetic materials and metabolic machinery safe. Since modern cell membranes are made up of ethanolamine-based phospholipids, prebiotic membrane formation with ethanolamine-based amphiphiles and phosphates might act as a bridge between the prebiotic and contemporary eras. Here, we report the prebiotic synthesis of O-lauroyl ethanolamine (OLEA), O-lauroyl methyl ethanolamine (OLMEA), and O-lauroyl dimethylethanolamine (OLDMEA) under wet-dry cycles. Turbidimetric, NMR, DLS, fluorescence, microscopy, and glucose encapsulation studies highlighted that OLEA-ATP and OLMEA-ATP form protocellular membranes in a 3:1 ratio, where ATP acts as a template. OLDMEA with a dimethyl group did not form any membrane in the presence of ATP. ADP can also template OLEA to form vesicles in a 2:1 ratio, but the ADP-templated vesicles were smaller. This suggests the critical role of the phosphate backbone in controlling the curvature of supramolecular assembly. The mechanisms of hierarchical assembly and transient dissipative assembly are discussed based on templated-complex formation via electrostatic, hydrophobic, and H-bonding interactions. Our results suggest that N-methylethanolamine-based amphiphiles could be used to form prebiotic vesicles, but the superior H-bonding ability of the ethanolamine moiety likely provides an evolutionary advantage for stable protocell formation during the fluctuating environments of early earth.

Self-assembled prebiotic amphiphile-mixture exhibits tunable catalytic properties.

Protocellular surface formation via the self-assembly of amphiphiles, and catalysis by simple peptides/proto-RNA are two important pillars in the evolution of protocells. To hunt for prebiotic self-assembly-supported catalytic reactions, we thought that amino-acid-based amphiphiles might play an important role. In this paper, we investigate the formation of histidine-based and serine-based amphiphiles under mild prebiotic conditions from amino acid : fatty alcohol and amino acid : fatty acid mixtures. The histidine-based amphiphiles were able to catalyze hydrolytic reactions at the self-assembled surface (with a rate increase of ∼1000-fold), and the catalytic ability can be tuned by linkage of the fatty carbon part to histidine (N-acylated vs. O-acylated). Moreover, the presence of cationic serine-based amphiphiles on the surface enhances the catalytic efficiency by another ∼2-fold, whereas the presence of anionic aspartic acid-based amphiphiles reduces the catalytic activity. Ester partitioning into the surface, reactivity, and the accumulation of liberated fatty acid explain the substrate selectivity of the catalytic surface, where the hexyl esters were found to be more hydrolytic than other fatty acyl esters. Di-methylation of the -NH2 of OLH increases the catalytic efficacy by a further ∼2-fold, whereas trimethylation reduces the catalytic ability. The self-assembly, charge-charge repulsion, and the H-bonding to the ester carbonyl are likely to be responsible for the superior (∼2500-fold higher rate than the pre-micellar OLH) catalytic efficiency of O-lauryl dimethyl histidine (OLDMH). Thus, prebiotic amino-acid-based surfaces served as an efficient catalyst that exhibits regulation of catalytic function, substrate selectivity, and further adaptability to perform bio-catalysis.

Molecular-level insights into a tripolyphosphate and pyrophosphate templated membrane assembly.

Templated assembly of small molecules into nano-structural architectures has been used extensively by nature throughout its evolution. These systems have also been studied in artificial systems to design a phosphate templated assembly. However, it is yet to be investigated how the molecules interact among themselves at the molecular level and whether the phosphate templated assembly has any role in the formation of prebiotic protocellular membranes. Here, we report the prebiotic synthesis of choline-based cationic amphiphiles (-N+Me3) and the templated assembly of these amphiphiles with tripolyphosphate (TPP) and pyrophosphate (PPi). SEM, TEM, FLIM, DLS, fluorescence, and encapsulation studies suggest that the number of phosphate units in the phosphate backbone controls the formation and size of the protocell vesicles. Isothermal titration calorimetry, turbidimetric studies, and NMR experiments suggest that the cationic amphiphile forms a 3 : 1 catanionic complex with TPP and a 2 : 1 catanionic complex with PPi. The templated catanionic complex further self-assembles into vesicles, and the structure of the complex guides the size of the assembly. The size-controlling ability of the phosphate backbone might have been utilized in the prebiotic era to support the dynamics and tunability of protocellular membrane compartments.

C-Terminal Lipidation of SARS-CoV-2 Fusion Peptide Reinstates Superior Membrane Fusion Catalytic Ability.

The spike (S) protein of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) mediates a critical stage in infection, the fusion between viral and host membranes. The protein is categorized as a class I viral fusion protein and has two distinct cleavage sites that can be activated by proteases. The activation deploys the fusion peptide (FP) for insertion into the target cell membranes. Recent studies including our experiments showed that the FP was unable to modulate the kinetics of fusion at a low peptide-to-lipid ratio akin to the spike density at the viral surface. Therefore, we modified the C terminus of FP and attached a myristoyl chain (C-myr-FP) to restrict the C terminus near to the interface, bridge both membranes, and increase the effective local concentration. The lipidated FP (C-myr-FP) of SARS-CoV-2 greatly accelerates membrane fusion at a low peptide-to-lipid ratio as compared to the FP with no lipidation. Biophysical experiments suggest that C-myr-FP adopts a helical structure, perturbs the membrane interface, and increases water penetration to catalyze fusion. Scrambled peptide (C-myr-sFP) and truncated peptide (C-myr-8FP) could not significantly catalyze the fusion, thus suggesting the important role of myristoylation and the N terminus. C-myr-FP enhances murine coronavirus infection by promoting syncytia formation in L2 cells. The C-terminal lipidation of the FP might be a useful strategy to induce artificial fusion in biomedical applications.

Lipidated Lysine and Fatty Acids Assemble into Protocellular Membranes to Assist Regioselective Peptide Formation: Correlation to the Natural Selection of Lysine over Nonproteinogenic Lower Analogues.

The self-assembly of prebiotically plausible amphiphiles (fatty acids) to form a bilayer membrane for compartmentalization is an important factor during protocellular evolution. Such fatty acid-based membranes assemble at relatively high concentrations, and they lack robust stability. We have demonstrated that a mixture of lipidated lysine (cationic) and prebiotic fatty acids (decanoic acid, anionic) can form protocellular membranes (amino acid-based membranes) at low concentrations via electrostatic, hydrogen bonding, and hydrophobic interactions. The formation of vesicular membranes was characterized by dynamic light scattering (DLS), pyrene and Nile Red partitioning, cryo-transmission electron microscopy (TEM) images, and glucose encapsulation studies. The lipidated nonproteinogenic analogues of lysine (Lys), such as ornithine (Orn) and 2,4-diaminobutyric acid (Dab), also form membranes with decanoate (DA). Time-dependent turbidimetric and 1H NMR studies suggested that the Lys-based membrane is more stable than the membranes prepared from nonproteinogenic lower analogues. The Lys-based membrane embeds a model acylating agent (aminoacyl-tRNA mimic) and facilitates the colocalization of substrates to support regioselective peptide formation via the α-amine of Lys. These membranes thereby assist peptide formation and control the positioning of the reactants (model acylating agent and -NH2 of amino acids) to initiate biologically relevant reactions during early evolution.

Lipid and Lipidation in Membrane Fusion.

Membrane fusion plays a lead role in the transport of vesicles, neurotransmission, mitochondrial dynamics, and viral infection. There are fusion proteins that catalyze and regulate the fusion. Interestingly, various types of fusion proteins are present in nature and they possess diverse mechanisms of action. We have highlighted the importance of the functional domains of intracellular heterotypic fusion, homotypic endoplasmic reticulum (ER), homotypic mitochondrial, and type-I viral fusion. During intracellular heterotypic fusion, the SNAREs and four-helix bundle formation are prevalent. Type-I viral fusion is controlled by the membrane destabilizing properties of fusion peptide and six-helix bundle formation. The ER/mitochondrial homotypic fusion is controlled by GTPase activity and the membrane destabilization properties of the amphipathic helix(s). Although the mechanism of action of these fusion proteins is diverse, they have some similarities. In all cases, the lipid composition of the membrane greatly affects membrane fusion. Next, examples of lipidation of the fusion proteins were discussed. We suggest that the fatty acyl hydrophobic tail not only acts as an anchor but may also modulate the energetics of membrane fusion intermediates. Lipidation is also important to design more effective peptide-based fusion inhibitors. Together, we have shown that membrane lipid composition and lipidation are important to modulate membrane fusion.

Membrane transport inspired hydrolysis of non-activated esters at near physiological pH.

A positively charged micelle loaded with substrates was transported selectively to the reaction site (cathode) to promote the proximity and localization of the reactants (ester and hydroxide). The guided vehicular delivery coupled with electrolysis allows the hydrolysis of non-activated esters at near physiological pH with significant yields along with recyclability.

The microenvironment and pKa perturbation of aminoacyl-tRNA guided the selection of cationic amino acids.

The proteinogenic lysine (Lys) and arginine (Arg) have multiple methylene groups between α-carbon and the terminal charged centre. Why nature did not select ornithine (Orn), 2,4-diamino butyric acid (Dab) and 2,3-diamino propionic acid (Dpr) with fewer methylene groups in the side chain remains an important question! The propensity of aminoacyl-tRNA (aa-tRNA) model substrates towards self-degradation via intramolecular lactamization was studied using UV spectroscopy and 1H-NMR titration, which showed that Lys and Arg remain stable, and Orn and Dab cyclize to lactam. Hydrophobicity-assisted surface mediated model peptide formation highlighted that the microenvironment and pKa perturbation led to poor regioselectivity (α-amine vs. terminal amine) in Dpr and other non-proteinogenic analogues. The α-selectivity became even poorer in the presence of phosphate, making them ill-suited for peptide synthesis. Superior regioselectivity of the Lys aa-tRNA model substrate suggests that the extra methylene bridge helped nature to separate the microenvironments of the α-amine and ε-amine to synthesize the peptide backbone.

Hydroxy-Porphyrin as an Effective, Endogenous Molecular Clamp during Early Stages of Amyloid Fibrillization.

Amyloid fibril formation of proteins is of great concern in neurodegenerative disease and can be detrimental to the storage and stability of biologics. Recent evidence suggests that insulin fibril formation reduces the efficacy of type II diabetes management and may lead to several complications. To develop anti-amyloidogenic compounds of endogenous origin, we have utilized the hydrogen bond anchoring, π stacking ability of porphyrin, and investigated its role on the inhibition of insulin amyloid formation. We report that hydroxylation and metal removal from the heme moiety yields an excellent inhibitor of insulin fibril formation. Thioflavin T, tyrosine fluorescence, Circular Dichorism (CD) spectroscopy, Field emission scanning electron microscopy (FESEM) and molecular dynamics (MD) simulation studies suggest that hematoporphyrin (HP) having hydrogen bonding ability on both sides is a superior inhibitor compared to hemin and protoporphyrin (PP). Experiments with hen egg white lysozyme (HEWL) amyloid fibril formation also validated the efficacy of endogenous porphyrin based small molecules. Our results will help to decipher a general therapeutic strategy to counter amyloidogenesis.

Translation of Mycobacterium Survival Strategy to Develop a Lipo-peptide based Fusion Inhibitor*.

The entry of enveloped virus requires the fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such membrane fusion may emerge as a broad-spectrum antiviral agent against a wide range of viral infections. Mycobacterium survives inside the phagosome by inhibiting phagosome-lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40-repeat protein suggest that the trp-asp (WD) sequence is placed at distorted ß-meander motif (more exposed) in coronin 1. The unique structural feature of coronin 1 was explored to identify a simple lipo-peptide sequence (myr-WD), which effectively inhibits membrane fusion by modulating the interfacial order, water penetration, and surface potential. The mycobacterium inspired lipo-dipeptide was successfully tested to combat type 1 influenza virus (H1N1) and murine coronavirus infections as a potential broad-spectrum antiviral agent.

Structure-activity relationship of heme and its analogues in membrane damage and inhibition of fusion.

Under pathological conditions, such as sickle cell disease and malaria, heme concentration increases considerably, and it induces membrane damage. As sickled and normal erythrocytes contain high cholesterol: phospholipid ratio, we investigated the role of lipid composition, chain length, and unsaturation on the partitioning and leakage of hemin in phospholipid vesicles. To establish structure-activity relationship in membrane damage, experiments with two other analogues, protoporphyrin-IX and hematoporphyrin (HP) were also carried out. Hemin and its analogues localize differently in membranes and exhibit distinct roles in partitioning, leakage and fusion. Hemin and HP trigger more leakage in the presence of aminophospholipids, whereas cholesterol buffers the destabilizing effect remarkably. Inhibition of fusion by hemin further suggests its unexplored and important role in membrane trafficking, particularly under diseased conditions.

A headgroup linker perturbs pKavia acyl chain migration: designing base-labile supramolecular assemblies.

Acyl chain transfer, which perturbs the protonation equilibrium of amine and reduces the apparent pKa by 2.0-2.5 units, is used to develop a liposome-based drug delivery system.

Phosphatidylserine-Dependent Catalysis of Stalk and Pore Formation by Synaptobrevin JMR-TMD Peptide.

Although the importance of a SNARE complex in neurotransmitter release is widely accepted, there exist different views on how the complex promotes fusion. One hypothesis is that the SNARE complex's ability to bring membranes into contact is sufficient for fusion, another points to possible roles of juxtamembrane regions (JMRs) and transmembrane domains (TMDs) in catalyzing lipid rearrangement, and another notes the complex's presumed ability to bend membranes near the point of contact. Here, we performed experiments with highly curved vesicles brought into contact using low concentrations of polyethylene glycol (PEG) to investigate the influence of the synaptobrevin (SB) TMD with an attached JMR (SB-JMR-TMD) on the rates of stalk and pore formation during vesicle fusion. SB-JMR-TMD enhanced the rates of stalk and fusion pore (FP) formation in a sharply sigmoidal fashion. We observed an optimal influence at an average of three peptides per vesicle, but only with phosphatidylserine (PS)-containing vesicles. Approximately three SB-JMR-TMDs per vesicle optimally ordered the bilayer interior and excluded water in a similar sigmoidal fashion. The catalytic influences of hexadecane and SB-JMR-TMD on fusion kinetics showed little in common, suggesting different mechanisms. Both kinetic and membrane structure measurements support the hypotheses that SB-JMR-TMD 1) catalyzes initial intermediate formation as a result of its basic JMR disrupting ordered interbilayer water and permitting closer interbilayer approach, and 2) catalyzes pore formation by forming a membrane-spanning complex that increases curvature stress at the circumference of the hemifused diaphragm of the prepore intermediate state.

Oligomerization, conformational stability and thermal unfolding of Harpin, HrpZPss and its hypersensitive response-inducing c-terminal fragment, C-214-HrpZPss.

HrpZ-a harpin from Pseudomonas syringae-is a highly thermostable protein that exhibits multifunctional abilities e.g., it elicits hypersensitive response (HR), enhances plant growth, acts as a virulence factor, and forms pores in plant plasma membranes as well as artificial membranes. However, the molecular mechanism of its biological activity and high thermal stability remained poorly understood. HR inducing abilities of non-overlapping short deletion mutants of harpins put further constraints on the ability to establish structure-activity relationships. We characterized HrpZPss from Pseudomonas syringae pv. syringae and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZPss) using calorimetric, spectroscopic and microscopic approaches. Both C-214-HrpZPss and HrpZPss were found to form oligomers. We propose that leucine-zipper-like motifs may take part in the formation of oligomeric aggregates, and oligomerization could be related to HR elicitation. CD, DSC and fluorescence studies showed that the thermal unfolding of these proteins is complex and involves multiple steps. The comparable conformational stability at 25°C (∼10.0 kcal/mol) of HrpZPss and C-214-HrpZPss further suggest that their structures are flexible, and the flexibility allows them to adopt proper conformation for multifunctional abilities.

Wild-type and mutant hemagglutinin fusion peptides alter bilayer structure as well as kinetics and activation thermodynamics of stalk and pore formation differently: mechanistic implications.

Viral fusion peptides are short N-terminal regions of type-1 viral fusion proteins that are critical for virus entry. Although the importance of viral fusion peptides in virus-cell membrane fusion is established, little is known about how they function. We report the effects of wild-type (WT) hemagglutinin (HA) fusion peptide and its G1S, G1V, and W14A mutants on the kinetics of poly(ethylene glycol)(PEG)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, sphingomyelin, and cholesterol (molar ratio of 35:30:15:20). Time courses of lipid mixing, content mixing, and content leakage were obtained using fluorescence assays at multiple temperatures and analyzed globally using either a two-step or three-step sequential ensemble model of the fusion process to obtain the rate constant and activation thermodynamics of each step. We also monitored the influence of peptides on bilayer interfacial order, acyl chain order, bilayer free volume, and water penetration. All these data were considered in terms of a recently published mechanistic model for the thermodynamic transition states for each step of the fusion process. We propose that WT peptide catalyzes Step 1 by occupying bilayer regions vacated by acyl chains that protrude into interbilayer space to form the Step 1 transition state. It also uniquely contributes a positive intrinsic curvature to hemi-fused leaflets to eliminate Step 2 and catalyzes Step 3 by destabilizing the highly stressed edges of the hemi-fused microstructures that dominate the ensemble of the intermediate state directly preceding fusion pore formation. Similar arguments explain the catalytic and inhibitory properties of the mutant peptides and support the hypothesis that the membrane-contacting fusion peptide of HA fusion protein is key to its catalytic activity.

A novel assay for detecting fusion pore formation: implications for the fusion mechanism.

Membrane fusion is broadly envisioned as a two- or three-step process proceeding from contacting bilayers through one or two semistable, nonlamellar lipidic intermediate structures to a fusion pore. A true fusion event requires mixing of contents between compartments and is monitored by the movement of soluble molecules between trapped compartments. We have used poly(ethylene glycol) (PEG) to rapidly generate an ensemble aggregated state A that proceeds sequentially through intermediates (I1 and/or I2) to a final fusion pore state (FP) with rate constants k1, k2, and k3. Movement of moderately sized solutes (e.g., Tb³âº/dipicolinic acid) has been used to detect pores assigned to intermediate states as well as to the final state (FP). Analysis of ensemble kinetic data has required that mixing of contents occurs with defined probabilities (αi) in each ensemble state, although it is unclear whether pores that form in different states are different. We introduce here a simple new assay that employs fluorescence resonance energy transfer (FRET) between a 6-carboxyfluorescein (donor) and tetramethylrhodamine (acceptor), which are covalently attached to complementary sequences of 10 bp oligonucleotides. Complementary sequences of fluorophore-labeled oligonucleotides were incorporated in vesicles separately, and the level of FRET increased in a simple exponential fashion during PEG-mediated fusion. The resulting rate constant corresponded closely to the slow rate constant of FP formation (k3) derived from small molecule assays. Additionally, the total extent of oligonucleotide mixing corresponded to the fraction of content mixing that occurred in state FP in the small molecule assay. The results show that both large "final pores" and small (presumably transient) pores can form between vesicles throughout the fusion process. The implications of this result for the mechanism of membrane fusion are discussed.