Anionic Phospholipid Vesicles Research Articles

Thermodynamics of oligomerization and Helix-to-sheet structural transition of amyloid β-protein on anionic phospholipid vesicles

Understanding oligomerization and aggregation of the amyloid-β protein is important to elucidate the pathological mechanisms of Alzheimer's disease, and lipid membranes play critical roles in this process. In addition to studies reported by other groups, our group has also reported that the negatively-charged lipid bilayers with a high positive curvature induced α-helix-to-β-sheet conformational transitions of amyloid-β-(1–40) upon increase in protein density on the membrane surface and promoted amyloid fibril formation of the protein. Herein, we investigated detailed mechanisms of the conformational transition and oligomer formation of the amyloid-β protein on the membrane surface. Changes in the fractions of the three protein conformers (free monomer, membrane-bound α-helix-rich conformation, and β-sheet-rich conformation) were determined from the fluorescent spectral changes of the tryptophan probe in the protein. The helix-to-sheet structural transition on the surface was described by a thermodynamic model of octamer formation driven by entropic forces including hydrophobic interactions. These findings provide useful information for understanding the self-assembly of amyloidogenic proteins on lipid membrane surfaces.

One-pot functionalization of carbon dots with ecPis-4s antimicrobial peptide

The synthesis of nanobiostructures (CD@ecPis-4s) composed of carbon dots (CD) covalently bound to the antimicrobial peptide ecPis-4s is reported. Nanoparticle functionalization was achieved by pre-activating the oxygenated functional groups using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and ethyl (hydroxyimino)cyanoacetate in an aqueous medium. The obtained nanobiostructures were characterized by several techniques, including FTIR and 1H NMR spectroscopies. The functionalization does not compromise the optical properties of CD. The polypeptide moiety of the peptide-decorated nanoparticles retains the active α-helical conformation and lytic activity in the presence of anionic phospholipid vesicles as observed in the circular dichroism and calcein release experiments. Consequently, the CD@ecPis-4s nanobiostructures present similar activity against Gram-positive (S. aureus and S. agalactiae) and Gram-negative (E. coli and P. aeruginosa) bacteria strains when compared to the free peptide. To our knowledge, this is the first report on carbon dots containing a covalently bound antimicrobial peptide. Thereby, in an era of increasing antimicrobial resistance, the synthesis and investigations performed in this work led to materials with potential for monitoring, in loco, bacterial contamination via exposure to UV radiation.

Functional and structural properties of cardiotoxin isomers produced by blocking negatively charged groups

In this study, we investigated the functional roles of Asp40, Asp57, and C-terminal Asn60 in Naja atra cardiotoxin 3 (CTX3) structure and function by modifying these three carboxyl groups with semicarbazide. The conjugation of the carboxyl groups with semicarbazide produced two conformational isomers whose gross and fine structures were different from those of CTX3. The blocking of the carboxyl groups increased the structural flexibility of CTX3 in response to trifluoroethanol-induced effect. Despite presenting modest to no effect on decreasing the induction of permeability in zwitterionic phospholipid vesicles, the carboxyl group-modified CTX3 showed a marked reduction in its permeabilizing effect on anionic phospholipid vesicles in comparison to that of the native protein. Compared with native CTX3, carboxyl group-modified CTX3 exhibited lower activity in inducing membrane leakage in U937 cells. The CD spectra of lipid-bound toxins and the color transition of polydiacetylene/lipid assay showed that the membrane interaction mode of CTX3 was distinctly changed by the modification in the carboxyl groups. Given that the selective modification of Asp40 does not cause the conformational isomerization of CTX3, our data indicate that the carboxyl groups in Asp57 and Asn60 are essential in maintaining the structural topology of CTX3. Furthermore, modification of carboxyl groups changes the interdependence between the infrastructure and the global conformation of CTX3 in modulating membrane permeabilizing activity.

Metal‐Supported Phospholipid Bilayers Formed by Redox Command

AbstractA surface‐confined redox reaction triggers the formation of single bilayer membranes from small unilamellar vesicles of anionic or zwitterionic phospholipids onto gold surfaces modified with a ferrocene‐terminated self‐assembled monolayer (SAM). The ion pairing association of the charged lipid headgroups with the electrogenerated cationic ferroceniums drives the assembly of the phospholipids on the SAM surface to produce solid‐supported bilayers of high surface coverage (≳90%) from gel‐ or fluid‐phase forming phospholipids within minutes at room temperature. The redox‐mediated strategy reported in this work is a conceptual advance in the preparation of solid‐supported lipid bilayers. It is fast, compatible with negatively‐charged and zwitterionic phospholipids, insensitive to the phase state of the phospholipid in the vesicle precursor, and not limited to hydrophilic surfaces.

Membrane interactions of the anuran antimicrobial peptide HSP1-NH2: Different aspects of the association to anionic and zwitterionic biomimetic systems

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH2 (HSP1-NH2), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH2 with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH2 presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. 2H exchange experiments of HSP1-NH2 indicated the insertion of the most N-terminal residues (1–3) in the DPC-d38 micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH2 (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH2 peptide depends on the membrane composition and peptide concentration.

Kinetoplastid Membrane Protein-11 Induces Pores in Anionic Phospholipid Membranes: Effect of Cholesterol

Kinetoplastid membrane protein-11 (KMP-11), expressed in all stages of leishmanial life cycle, is considered a potential candidate for leishmaniasis vaccine. KMP-11 is found on the membrane surface of the parasite. Although the biological function of KMP-11 is unknown, we hypothesize from its sequence analysis that it may interact with the macrophage membrane and may influence the entry process of the parasite into the host cell. To validate this hypothesis, we have investigated the interaction of KMP-11 with unilamellar anionic phospholipid vesicles and explored its pore-forming activity. The decrease in negative ζ-potential of the vesicles and reduction in the fluorescence intensity of membrane-bound dye DiI C-18 suggest a strong association of KMP-11 with the membrane. The fluorescence leakage experiment as well as phase contrast microscopy shows direct evidence of KMP-11-induced pore formation in an anionic membrane. Incorporation of cholesterol into the membrane has been found to inhibit pore formation induced by KMP-11, suggesting an important role of cholesterol in leishmaniasis. Interestingly, vesicles containing only neutral phospholipid do not exhibit any tendency toward pore formation.

Collisional mechanism of ligand release by Bombyxmori JHBP, a member of the TULIP / Takeout family of lipid transporters

Juvenile hormones (JHs) regulate important processes in insects, such as postembryonic development and reproduction. In the hemolymph of Lepidoptera, these lipophilic sesquiterpenic hormones are transported from their site of synthesis to target tissues by high affinity carriers, the juvenile hormone binding proteins (JHBPs). Lepidopteran JHBPs belong to a recently uncovered, yet very ancient family of proteins sharing a common lipid fold (TULIP domain) and involved in shuttling various lipid ligands. One important, but poorly understood aspect of JHs action, is the mechanism of hormone transfer to or through the plasma membranes of target cells. Since many membrane-active peptides and proteins, such as the pore-forming bacterial toxins, are activated by low pH or interaction with phospholipid membranes, we have examined the effect of these factors on JH binding by JHBPs. The affinity of Bombyx mori and Manduca sexta JHBPs for JH III was determined by the DCC assay, equilibrium dialysis, and isothermal titration calorimetry, and found to be greatly reduced at low pH, in agreement with previous observations. Loss of binding was accompanied by changes in fluorescence and near-UV CD spectra, indicating significant changes in protein structure in the environment of aromatic residues. The apparent dissociation rate constant (koff) of the JHBP-JH III complex was greater at acidic pH, suggesting that low pH favors ligand release by opening of the binding pocket. The affinity of recombinant B. mori JHBP (rBmJHBP) was also decreased in the presence of anionic phospholipid vesicles. Measurements of steady-state fluorescence anisotropy with the lipophilic probe TMA-DPH demonstrated that rBmJHBP specifically interacts with anionic membranes. These results suggest the existence of a collisional mechanism for ligand release that may be important for delivery of JHs to the target cells, and could be relevant to the function of related members of this emerging family of lipid-transport proteins.

Lipid Vesicle-Coated Complex Coacervates.

Compartmentalization by complex coacervation is important across a range of different fields including subcellular and prebiotic organization, biomedicine, food science, and personal care products. Often, lipid self-assemblies such as vesicles are also present intracellularly or in commercial formulations. A systematic understanding of how phospholipid vesicles interact with different complex coacervates could provide insight and improve control over these systems. In this manuscript, anionic phospholipid vesicles were added to a series of different complex coacervate samples in which coacervates were formed by mixing one of five polycations with one of three (poly)anions that varied in chemical structure and length. Vesicles were found to assemble at the coacervate/continuous phase interface and/or form aggregates. We report how factors such as the charge density of polyelectrolytes and the charge ratio of cationic-to-anionic moieties impact the vesicle distribution in coacervate samples. Our findings emphasize the importance of interactions between vesicles and polycations in the dilute supernatant phase for determining whether the vesicles aggregate prior to assembly at the liquid-liquid interface. The uptake of an RNA oligonucleotide (A15) was also investigated to understand the effect of these liposome coatings on diffusion into coacervate droplets. Systems in which uniform vesicle coronas assemble around coacervate droplets without restricting the entry of biomolecules such as RNAs could be of interest as bioreactors.

Abstract 396: Acrolein Modification Weakens the Antimicrobial Activity of Apolipoprotein A1

Human apolipoprotein A-I (apoA-I) has been show to exhibit antimicrobial activity by neutralizing lipopolysaccharides and destabilizing inner membranes of gram-negative bacteria. Previous studies showed that acrolein, a highly reactive αβ unsaturated aldehyde generated in cigarette smoking, modifies ε-amino side chains of lysine residues in apoA-I. The current study investigated the effect of acrolein exposure on the structure and antimicrobial activity of apoA-I. Incubation of apoA-I with acrolein using a 1:20 molar ratio, acrolein modification was evident by the appearance of apoA-I oligomers due to intermolecular crosslinking. Increase of the acrolein to protein ratio resulted in heavily cross-linked apoA-I, with protein bands appearing at 63, 98, and 126 kDa. The presence of acrolein-modified lysines in the oligomers was verified through Western blot analysis using mab5F6 antibody that specifically detects acrolein modified lysine residues in proteins. The structural changes of modified apoA-I was analyzed using circular dichroism. The α-helical content of acrolein-modified apoA-I was not significantly different from the unmodified protein. However, the midpoint of guanidine-induced denaturation increased from 0.97 to 1.50 M guanidine upon modification, indicating a significant increase in protein stability. This suggests that while modification did not alter the secondary structure, the protein fold was altered due to cross-linking. To measure the effect of acrolein modification on the interaction with bacterial membranes, binding experiments were performed with phosphatidylglycerol entrapped with calcein. This showed that the percentage of calcein released by apoA-I decreased from 87.5 ± 2.3 % to 4.7 ± 0.13 % when the protein was modified by acrolein. Thus acrolein-modified apoA-I binds phosphatidylglycerol less effectively, possibly due to loss of electrostatic interactions with anionic phospholipid vesicles. In addition, binding of apoA-I to lipopolysaccharides was significantly weaker when the protein was modified by acrolein. These results suggest that apoA-I modification by acrolein results in a protein with a decreased ability to bind to bacterial membranes and is thus less potent as an antimicrobial protein.

Inhibition of Membrane‐Induced Alpha‐Synuclein (aSyn) Aggregation: A Strategy to Interfere with aSyn Neurotoxicity in Parkinson's Disease (PD)

Oligomerization of the presynaptic protein aSyn is thought to play a key role in the pathogenesis of PD. aSyn exists in a number of conformations, including a membrane‐bound state likely involved in regulating synaptic vesicle trafficking. aSyn interacts with anionic phospholipid vesicles by forming an amphipathic α‐helix of various lengths. We hypothesize that disruption of interactions between aSyn and phospholipid membranes leads to a shift to a short‐helix, lipid‐bound form, which is more susceptible to forming toxic aSyn oligomers due to exposure of the protein's central hydrophobic region. Therefore, stabilization of the long‐helix form of lipid‐bound aSyn should prevent membrane‐induced aggregation and alleviate aSyn neurotoxicity. To address this hypothesis, endosulfine alpha (ENSA), a protein that interacts selectively with membrane‐bound aSyn, and a series of peptides identified as interactors of lipid‐bound aSyn via phage display screening were characterized in terms of their effects on membrane‐induced aSyn aggregation, aSyn‐mediated membrane permeabilization, and aSyn neurotoxicity in primary midbrain cultures. We found that A30P and G51D, two aSyn variants with disrupted membrane interactions, had an increased ability to form SDS‐resistant aggregates at the membrane surface and to elicit neurotoxicity than wild type (WT) aSyn, and both triggered the disruption of dye‐loaded vesicles under conditions identical to those that promoted membrane‐induced aggregation. WT ENSA (but not the non‐aSyn‐binding S109E variant) and a subset of phage display peptides attenuated aSyn self‐assembly at the membrane, vesicle disruption, and aSyn neurotoxicity. Intriguingly, ENSA was found to be down‐regulated in the brains of patients with dementia with Lewy bodies or PD versus non‐diseased individuals. Current efforts are focused on characterizing the aggregation of aSyn variants in biological membranes and monitoring aSyn‐mediated vesicle permeabilization in neuronal cells, with the ultimate aim of elucidating neuroprotective mechanisms of ENSA and aSyn‐binding peptides in cellular models relevant to PD. Our findings strongly support the idea that membrane‐induced aSyn aggregation plays a critical role in aSyn neurotoxicity, and they suggest that targeting this process is a viable therapeutic strategy for PD.Support or Funding InformationBranfman Family Foundation, Indiana CTSI Core Pilot Grant, Indiana CTSI Collaboration in Translational Research Grant, Richard F. Borch Research Enhancement Award

Interaction of insulin with anionic phospholipid (DPPG) vesicles.

The interaction between a protein/enzyme and a lipid is critical for pharmacological activity. Here, we study the interaction between insulin and the 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) lipid anionic vesicle by successfully entrapping the insulin molecule into DPPG vesicles, which are biocompatible liposomes. For the insulin-DPPG complex system, steady state emission spectroscopy at room temperature (300 K) shows a new broad and structured peak between 400 nm and 500 nm along with the tyrosine fluorescence peak at 303 nm. Temperature dependent and time resolved spectroscopy reveal that the peak between 400 nm and 500 nm in the insulin-DPPG system arises due to the tyrosine phosphorescence phenomenon. This phosphorescence peak is the signature of insulin entrapment into the liposome. A molecular dynamics study of the tyrosine-DPPG system shows that the rigidity of tyrosine increases in the lipid layer. Dynamic light scattering (DLS), and zeta potential studies also establish the attachment of insulin with the anionic liposome.

Probing Conformational Change of Intrinsically Disordered α-Synuclein to Helical Structures by Distinctive Regional Interactions with Lipid Membranes

α-Synuclein (α-Syn) is an intrinsically disordered protein, whose fibrillar aggregates are associated with the pathogenesis of Parkinson's disease. α-Syn associates with lipid membranes and forms helical structures upon membrane binding. In this study, we explored the helix formation of α-Syn in solution containing trifluoroethanol using small-angle X-ray scattering and electrospray ionization ion mobility mass spectrometry. We then investigated the structural transitions of α-Syn to helical structures via association with large unilamellar vesicles as model lipid membrane systems. Hydrogen-deuterium exchange combined with electrospray ionization mass spectrometry was further utilized to understand the details of the regional interaction mechanisms of α-Syn with lipid vesicles based on the polarity of the lipid head groups. The characteristics of the helical structures were observed with α-Syn by adsorption onto the anionic phospholipid vesicles via electrostatic interactions between the N-terminal region of the protein and the anionic head groups of the lipids. α-Syn also associates with zwitterionic lipid vesicles and forms helical structures via hydrophobic interactions. These experimental observations provide an improved understanding of the distinct structural change mechanisms of α-Syn that originate from different regional interactions of the protein with lipid membranes and subsequently provide implications regarding diverse protein-membrane interactions related to their fibrillation kinetics.

Structural Intermediates during α-Synuclein Fibrillogenesis on Phospholipid Vesicles

α-Synuclein (AS) fibrils are the main protein component of Lewy bodies, the pathological hallmark of Parkinson's disease and other related disorders. AS forms helices that bind phospholipid membranes with high affinity, but no atomic level data for AS aggregation in the presence of lipids is yet available. Here, we present direct evidence of a conversion from α-helical conformation to β-sheet fibrils in the presence of anionic phospholipid vesicles and direct conversion to β-sheet fibrils in their absence. We have trapped intermediate states throughout the fibril formation pathways to examine the structural changes using solid-state NMR spectroscopy and electron microscopy. The comparison between mature AS fibrils formed in aqueous buffer and those derived in the presence of anionic phospholipids demonstrates no major changes in the overall fibril fold. However, a site-specific comparison of these fibrillar states demonstrates major perturbations in the N-terminal domain with a partial disruption of the long β-strand located in the 40s and small perturbations in residues located in the "non-β amyloid component" (NAC) domain. Combining all these results, we propose a model for AS fibrillogenesis in the presence of phospholipid vesicles.

Complexation of cationic polyelectrolyte with anionic phospholipid vesicles: Concentration, molecular weight and salt effects

The influence of cationic poly(diallyldimethylammonium chloride) on the morphology and phase behavior of anionic phospholipid vesicles was investigated using differential scanning calorimetry, fluorescent microscopy and light scattering technique. A wide range of polymer concentration has been examined for the first time. The polycation can bind electrostatically to the vesicles to compensate, neutralize and reverse the vesicular charge, depending on the molar ratio of cationic to anionic group R. For R < 1, charge compensation weakened the electrostatic repulsion between the lipid molecules, leading to formation of polymer-modified vesicles, each with an increased number of bilayers. The bilayer exhibits a rising main phase transition temperature from a gel to liquid crystalline state. This behavior persisted until R ≈ 1 around the neutralization condition, where the complexes became largest and precipitate. With R > 1, charge reversal took place, the complex size reduced. Interestingly, the main phase transition temperature was found for the first time to shift back towards the original value in the absence of polymer for large enough R. Although the thermal behavior was nearly independent of the polymer molecular weight, the complex morphology could be different.

Structural Studies of a Peptide with Immune Modulating and Direct Antimicrobial Activity

The structure and function of the synthetic innate defense regulator peptide 1018 was investigated. This 12 residue synthetic peptide derived by substantial modification of the bovine cathelicidin bactenecin has enhanced innate immune regulatory and moderate direct antibacterial activities. The solution state NMR structure of 1018 in zwitterionic dodecyl phosphocholine (DPC) micelles indicated an α-helical conformation, while secondary structures, based on circular dichroism measurements, in anionic sodium dodecyl sulfate (SDS) and phospholipid vesicles (POPC/PG in a 1:1 molar ratio) and simulations revealed that 1018 can adopt a variety of folds, tailored to its different functions. The structural data are discussed in light of the ability of 1018 to potently induce chemokine responses, suppress the LPS-induced TNF-α response, and directly kill both Gram-positive and Gram-negative bacteria.

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides' lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.

Gadolinium-containing phosphatidylserine liposomes for molecular imaging of atherosclerosis

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE(-/-) mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.

Interaction of a potyviral VPg with anionic phospholipid vesicles

The viral genome-linked protein (VPg) of Potato virus A (PVA) is a multifunctional protein that belongs to a class of intrinsically disordered proteins. Typically, this type of protein gains a more stable structure upon interactions or posttranslational modifications. In a membrane lipid strip overlay binding assay, PVA VPg was found to bind phosphatidylserine (PS), but not phosphatidylcholine (PC). According to circular dichroism spectroscopy, the secondary structure of PVA VPg was stabilized upon interactions with PS and phosphatidylglycerol (PG), but not with PC vesicles. It is possible that this stabilization favored the formation of alpha-helical structures. Limited tryptic digestion showed that the interaction with anionic vesicles protected certain, otherwise accessible, trypsin cleavage sites. An electron microscopy study revealed that interaction with VPg substantially increased the vesicle diameter and caused the formation of pore or plaque-like electron dense spots on the vesicle surface, which gradually led to disruption of the vesicles.

Roles of lysine residues and N-terminal α-amino group in membrane-damaging activity of Taiwan cobra cardiotoxin 3 toward anionic and zwitterionic phospholipid vesicles

In contrast to a slight increase in activity toward phosphatidylcholine (EYPC)/dimyristoyl phosphatidic acid (DMPA) vesicles, guanidination of Naja naja atra cardiotoxin 3 (CTX3) and selective trinitrophenylation of N-terminal α-amino group enhanced notably membrane-damaging activity on EYPC/egg yolk sphingomyelin (EYSM) vesicles. Chemically modified CTX3 showed a reduction in its hemolytic activity and cytotoxicity. These reflected that membrane-damaging activity of CTX3 was affected by phospholipid compositions. Phospholipid-binding capability and oligomeric assembly upon binding with lipid vesicles did not closely correlate with membrane-damaging potency of native and modified CTX3. Moreover, different topographical contacts and distinctive modes for the binding of CTX3 and its modified derivatives with anionic phospholipid vesicles (EYPC/DMPA) and zwitterionic phospholipid vesicles (EYPC/EYSM) were observed. Compared with in the case of EYPC/DMPA, the interaction between CTX molecules and EYPC/EYSM was drastically reduced by increasing salt concentration and heparin. Taken together, our data indicate that guanidination of Lys residues and trinitrophenylation of α-amino group alter differently the interacted modes upon absorption on anionic phospholipid vesicles and zwitterionic phospholipid vesicles. The findings also suggest that positively charged residues of CTX3 play a distinctive role in damaging anionic and zwitterionic phospholipid vesicles.

Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity: INSIGHTS INTO PHOSPHATIDYLCHOLINE ACTIVATION AND SURFACE DILUTION INHIBITION

The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (tauc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased tauc. PI-PLC activity and phospholipid tauc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC tauc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.