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Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the β clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in β clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_βIB1). A structural comparison of available structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_βIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed that the i-face of α and β clade PLDs is involved in their binding to choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the β clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in β clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_βIB1). A structural comparison of available PLDs structures reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular-dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_βIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed the membrane binding site of α and β clade PLDs on choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

The reinforcement tendency of acrylonitrile butadiene styrene/single-walled carbon nanotubes hybrid composites is investigated using universal tensile testing machine. SWCNT are exfoliated in the ABS matrix at all concentrations, however, selective localization of SWCNT occurred in the SAN phase rather than the PB phase of ABS. Although, glass transition temperature of ABS remained intact by the incorporation of SWCNTs, but mechanical properties are enhanced significantly. The increase in tensile strength and Young's modulus of ABS/SWCNT hybrid composites is up to 48% and 103%, respectively, at considerable loss of strain at break inferring the restriction in the movement of the ABS chains by anchoring them in the vicinity of SWCNT. Modified rule of mixture (MRoM) model is employed to predict the rate of increase in the Young's modulus (dY/dVf) of ABS/SWCNT hybrid composites. The predicted trends are correlated well with the experimental values of dY/dVf in the lower volume fraction of SWCNT in ABS demonstrating the effective load transfer from the polymer matrix to the nanotubes. POLYM. COMPOS., 2017. © 2017 Society of Plastics Engineers

Membrane-binding interfaces of peripheral proteins are restricted to a small part of their exposed surface, so the ability to engage in strong selective interactions with membrane lipids at various depths in the interface, both below and above the phosphates, is an advantage. Driven by their hydrophobicity, aromatic amino acids preferentially partition into membrane interfaces, often below the phosphates, yet enthalpically favorable interactions with the lipid headgroups, above the phosphate plane, are likely to further stabilize high interfacial positions. Using free-energy perturbation, we calculate the energetic cost of alanine substitution for 11 interfacial aromatic amino acids from 3 peripheral proteins. We show that the involvement in cation−π interactions with the headgroups (i) increases the ΔΔGtransfer as compared with insertion at the same depth without cation−π stabilization and (ii) can contribute at least as much as deeper insertion below the phosphates, highlighting the multiple roles of aromatics in peripheral membrane protein affinity. acceptedVersion

Membrane binding interfaces of peripheral proteins are restricted to a small part of their exposed surface so the ability to engage in strong selective interactions with membrane lipids at various depths in the interface, both below and above the phosphates, is an advantage. Driven by their hydrophobicity aromatic amino acids preferentially partition into membrane interfaces often below the phosphates. Yet enthalpically favorable interactions with the lipid headgroups, above the phosphate plane, are likely to further stabilize high interfacial positions. Using Free Energy Perturbation we calculate the energetic cost of alanine substitution for 11 interfacial aromatic amino acids from 3 peripheral proteins. We show that involvement in cation-π interactions with the headgroups (i) increases the ΔΔGtransfer as compared to insertion at the same depth without cation-π stabilization and (ii) can contribute at least as much as deeper insertion below the phosphates, highlighting the multiple roles of aromatics in peripheral membrane protein affinity.

The reinforcement effects of two dimensional (2-D) few layer graphene (FLG) and one dimensional (1-D) multi-walled carbon nanotubes (MWCNT) in acrylonitrile-butadiene-styrene (ABS) were investigated using tensile testing machine. Both, FLG and MWCNT were dispersed well in ABS matrix but preferably reside in the styrene-acrylonitrile phase rather than in the polybutadiene phase. The reinforcement tendency of FLG in ABS was higher than MWCNT; tensile strength was increased up to 43% for ABS/FLG and 22% for ABS/MWCNT. While, tensile modulus of ABS/FLG and ABS/MWCNT was enhanced up to 71% and 54%, respectively. Modified rule of mixture (MRoM) and Halpin-Tsai (H-T) models were employed to predict the rate of increase in the Young’s modulus of ABS/FLG and ABS/MWCNT. H-T model correlated the reinforcement tendencies more effectively than MRoM model possibly due to the difference in the length efficiency factor. Moreover, the HV of the ABS/FLG was significantly increased as compared to ABS/MWCNT clearly indicating the shape difference for reinforcing the ABS matrix as 2-D platelet dispersion of graphene hindered the deformation more effectively owing to the effective load transfer from polymer matrix to the filler as compared to 1-D MWCNT.

The Salmon louse (Lepeophtheirus salmonis) is a marine ectoparasite of salmonid fish in the Northern Hemisphere and considered as a major challenge in aquaculture and a threat to wild populations of salmonids. Adult female lice produce a large number of lipid-rich eggs, however, the mechanism of maternal lipid transport into developing eggs during salmon louse reproduction has not been described. In the present study, a full-length L. salmonis lipophorin receptor (LsLpR) consisting of 16 exons was obtained by RACE and RT-PCR. The predicted ORF was 952 amino acids and structural analysis showed five functional domains that are similar to LpR of insects and decapods. Phylogenetic analysis placed the LsLpR together with LpRs from decapods and insects. Expression analysis revealed that the relative abundance of LsLpR transcripts was highest in the larvae and adult female lice. In adult females, the LsLpR transcripts and protein were found in the ovary and vitellogenic oocytes whereas, in larvae, the LsLpR transcripts were found in the neuronal somata of the brain and the intestine. Oil Red O stain results revealed that storage of neutral lipids was found in vitellogenic oocytes and ovaries of adult females, and in the yolk of larvae. Moreover, RNA interference (RNAi) was conducted to demonstrate the function of LsLpR in reproduction and lipid metabolism in L. salmonis. In larvae, the transcription of LsLpR was decreased by 44–54% while in an experiment LsLpR knockdown female lice produced 72% less offspring than control lice. Molecular characterization of the lipophorin receptor in the crustacean ectoparasite Lepeophtheirus salmonis

Nα-Acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) because it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N termini of the transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling, in vitro assays, and cellular localization studies to investigate the secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C terminus bind the membrane directly in a parallel position relative to the lipid bilayer via hydrophobic and electrostatic interactions. A peptide corresponding to the C terminus was unstructured in solution and only folded into an α-helical conformation in the presence of liposomes. Computational modeling and cellular mutational analysis revealed the hydrophobic face of two α-helices to be critical for membranous localization. Furthermore, we found a strong and specific binding preference of Naa60 toward membranes containing the phosphatidylinositol PI(4)P, thus possibly explaining the primary residency of Naa60 at the PI(4)P-rich Golgi. In conclusion, we have defined the mode of cytosolic Naa60 anchoring to the Golgi apparatus, most likely occurring post-translationally and specifically facilitating post-translational N-terminal acetylation of many transmembrane proteins.

Coarse-grained simulations of model membranes containing mixtures of phospholipid and cholesterol molecules at different concentrations and temperatures have been performed. A random mixing without tendencies for segregation or formation of domains was observed on spatial scales corresponding to a few thousand lipids and timescales up to several microseconds. The gel-to-liquid crystalline phase transition is successively weakened with increasing amounts of cholesterol without disappearing completely even at a concentration of cholesterol as high as 60%. The phase transition temperature increases slightly depending on the cholesterol concentration. The gel phase system undergoes a transition with increasing amounts of cholesterol from a solid-ordered phase into a liquid-ordered one. In the solid phase, the amplitude of the oscillations in the radial distribution function decays algebraically with a prefactor that goes to zero at the solid-liquid transition.

Classical simulations of simple water models reproduce many properties of the liquid and ice but overestimate the heat capacity by about 65% at ordinary temperatures and much more for low temperature ice. This is due to the fact that the atomic vibrations are quantum mechanical. The application of harmonic quantum corrections to the molecular motion results in good heat capacities for the liquid and for ice at low temperatures but a successively growing positive deviation from experimental results for ice above 200 K that reaches 15% just below melting. We suggest that this deviation is due to the lack of quantum corrections to the anharmonic motions. For the liquid, the anharmonicities are even larger but also softer and thus in less need of quantum correction. Therefore, harmonic quantum corrections to the classically calculated liquid heat capacities result in agreement with the experimental values. The classical model underestimates the heat of melting by 15%, while the application of quantum corrections produces fair agreement. On the other hand, the heat of vaporization is overestimated by 10% in the harmonically corrected classical model.

It is here shown that there is a considerable system size-dependence in the area compressibility calculated from area fluctuations in lipid bilayers. This is caused by the contributions to the area fluctuations from undulations. This is also the case in experiments. At present, such a contribution, in most cases, is subtracted from the experimental values to obtain a true area compressibility. This should also be done with the simulation values. Here, this is done by extrapolating area compressibility versus system size, down to very small (zero) system size, where undulations no longer exist. The area compressibility moduli obtained from such simulations do not agree with experimental true area compressibility moduli as well as the uncorrected ones from contemporary or earlier simulations, but tend, instead, to be approximately 50% too large. As a byproduct, the bending modulus can be calculated from the slope of the compressibility modulus versus system-size. The values obtained in this way for the bending modulus are then in good agreement with experiment.

Molecular dynamics simulations of atomistic models show the ordering and condensing effect of the cholesterol on phospholipid bilayers. Such simulations are, however, too time consuming to permit monitoring of the gel/liquid crystalline phase transition. We used therefore the simplified coarse grained MARTINI model to study the phase transition in dipalmitoylphosphatidylcholine (DPPC) cholesterol mixtures. A recent study by Marrink shows that this model is able to describe the main phase transition of pure phospholipid systems reasonably.The area per molecule was calculated separately for the phospholipids and cholesterol as a partial specific area (Edholm & Nagle). The condensation effect of cholesterol is obtained with the atomistic model as a negative partial specific area for cholesterol. The coarse grained model reproduce the condensation effect and gives a negative partial specific area of cholesterol molecule at low cholesterol concentration and low temperatures above the main phase transition. The main phase transition occurs already at about 295K for the coarse grained model. There is, however, another (second order) phase transition at 304K, where partial specific area suddenly becomes positive for all cholesterol concentrations. Such a transition was, however, not observed with the atomistic model. A similar transition was observed in the order parameter versus temperature at low cholesterol concentration, but this vanishes at high concentration. These results were used to test different suggested relations between area per lipid and the order parameter.Below, the main transition, the radial distribution functions and chain order parameters were used to monitor the difference between long range positional order and chain order as well as to search for inhomogeneities in the lipid distribution.

Coarse grained simulations of membranes containing mixtures of phospholipids and cholesterol at different concentrations and temperatures (below and above the main transition) have been performed. Random mixing without formation of domains was observed. On the contrary, we observed that phase separated fluid systems with different cholesterol concentrations mix into uniform systems in less than 200 nsec. For the gel phase the results are less conclusive due to the two orders of magnitude slower dynamics. The gel to liquid crystalline phase transition is successively weakened by cholesterol while the phase transition temperature increases slightly.The gel phase system undergoes a transition with increasing amounts of cholesterol from a solid ordered phase into a liquid ordered one. In the solid phase, the amplitude of the oscillations in the radial distribution functions decays algebraically with a pre-factor that goes to zero at the two-dimensional solid-liquid transition. The liquid ordered phase is characterized by liquid-like pair correlation functions that decay exponentially to one and have just one detectable peak. Angular correlation functions that measure how the orientation of the lattice vectors in the membrane plane decorrelates with distance were also calculated. They show an algebraic decay with exponent 0.15-0.25 in large regions of the solid ordered phase. This indicates that the liquid ordered phase has more structure than a two-dimensional liquid and may be a hexatic phase.To explore further whether phase segregation into cholesterol-rich and cholesterol-poor domains is favorable from a free energy point of view, the chemical potential for cholesterol insertion into lipid bilayers at different cholesterol concentrations was calculated from simulations. This shows a small bulk free energy of about 0.3kT per lipid that favors phase separation while a small line tension (a few pN) between cholesterol-rich and -poor regions favors mixing.

Experimental data for many important properties of lipid bilayers are scarce and uncertain. This includes for instance area per lipid, area and volume compressibilities, area expansion coefficients and heat capacity.Present simulation techniques can often give such properties more easily and sometimes with better accuracy than experiments. Simulation results depend, however, upon potential parameters that yet are not enough tested and validated. Still, the time is getting ripe for systematic calculation of membrane properties using simulation techniques. This is useful both since it will provide better values for a number of membranes properties but also since a more serious test of the membrane force field parameters will push the refinement and development on this side.Initially, the study has concentrated on area per lipid, area compressibility and bending modulus. We have shown that the inverse apparent area compressibility modulus obtained from the area fluctuations of the system shows a linear variation with system size. From this, the the true area compressibility modulus can be obtained by extrapolating to small areas. From the term that varies linearly with system size and is due to undulations, the bending modulus of the bilayer can easily be calculated. The method has been applied to several lipids including DPPC and DMPC.