Parity violation and the origins of biomolecular handedness

The violation of parity by the weak interactions ensures that enantiomeric chiral molecules have inequivalent energies, one being inherently stabilized with respect to the other. These parity-violating energy differences have been calculated for a number of fundamental biomolecules including a series of alpha-amino acids, polypeptide structures, and a representative of the sugar series together with its variation over a possible prebiotic reaction path leading to alpha-amino acids. In each case the natural enantiomer found in terrestrial biochemistry was shown to be intrinsically stabilized and preferred over its unnatural enantiomer. The significance of these results in accounting for the prebiotic origins of the terrestrial biomolecular homochirality is discussed and the possible consequences of parity-violating energy differences in mineral catalysts during the prebiotic era considered.     

Prospects of a Computational Origin of Life Endeavor

While the last century brought an exquisite understanding of the molecular basis of life, very little is known about the detailed chemical mechanisms that afforded the emergence of life on early earth. There is a broad agreement that the problem lies in the realm of chemistry, and likely resides in the formation and mutual interactions of carbon-based molecules in aqueous medium. Yet, present-day experimental approaches can only capture the synthesis and behavior of a few molecule types at a time. On the other hand, experimental simulations of prebiotic syntheses, as well as chemical analyses of carbonaceous meteorites, suggest that the early prebiotic hydrosphere contained many thousands of different compounds. The present paper explores the idea that given the limitations of test-tube approaches with regards to such a 'random chemistry' scenario, an alternative mode of analysis should be pursued. It is argued that as computational tools for the reconstruction of molecular interactions improve rapidly, it may soon become possible to perform adequate computer-based simulations of prebiotic evolution. We thus propose to launch a computational origin of life endeavor (http://ool.weizmann.ac.il/CORE), involving computer simulations of realistic complex prebiotic chemical networks. In the present paper we provide specific examples, based on a novel algorithmic approach, which constitutes a hybrid of molecular dynamics and stochastic chemistry. As one potential solution for the immense hardware requirements dictated by this approach, we have begun to implement an idle CPU harvesting scheme, under the title ool@home.     

Group I introns and gnra tetraloops: remnants of ‘The RNA world’?

GNRA tetraloops, found in high frequency in natural RNAs, make loop-receptor interactions, stabilizing the tertiary structure of Group I introns, a class of small RNAs. Analyzing 230 Group I introns, to study the distribution and sequence pattern of the GNRA tetraloops, we suggest that these features reflect the ancestral nature of these catalytic molecules, in a prebiotic RNA world. The adenosine rich GNRA tetraloops would have interacted with each other through long range RNA-RNA interactions to form higher order structures forming potential sites that render the propensity for the short RNAs to bind to metal ions from the prebiotic pool, aiding them to act as metalloenzymes.     

Interaction of nominally soluble proteins with phospholipid monolayers at the air-water interface

The interactions of carbonmonoxyhemoglobin (HbCO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and polyhistidine with phospholipid monolayers at the air-water interface were studied at physiological pH and ionic strength. HbCO and GAPDH both interact more strongly with monolayers containing negatively charged lipids. The interaction of HbCO and GAPDH with lipid monolayers decreases with increasing pH. Both the HbCO-monolayer and the GAPDH-monolayer interactions can be modeled as diffusion-limited processes, with kinetic data fit to a stretched exponential equation. The significance of these kinetics are discussed. Polyhistidine interacts only with monolayers containing lipids with negatively charged headgroups. In total, the results presented are consistent with an HbCO-lipid interaction with a large electrostatic component, a GAPDH-lipid interaction with comparable electrostatic and hydrophobic components, and a polyhistidine-lipid interaction that is solely electrostatic.     

Lipid-peptide interactions between fragments of the transit peptide of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast membrane lipids

The interactions of fragments of the transit peptide of ribulose-1,5-bisphosphate carboxylase/oxygenase with lipid monolayers was studied in order to investigate the possible involvement of the membrane lipids in the protein import process. The fragments are surface active and have a differential ability to insert in lipid monolayers. The fragments have a preference for the chloroplast galacto- and sulpholipids and phosphatidylglycerol and interact with envelope membrane lipid extracts. These results suggest that probably transit peptide-lipid interactions are involved in the chloroplast protein import process.     

Interactions of the antifungal mycosubtilin with ergosterol-containing interfacial monolayers

Mycosubtilin, an antimicrobial lipopeptide produced by Bacillus subtilis, is characterized by strong antifungal activities. The molecular mechanisms of its biological activities on the membranes of the sensitive yeasts or fungi have not yet been clearly elucidated. Our purpose was to mimic the mycosubtilin interactions with these membranes using various Langmuir monolayers. Since the major sterol of yeasts or fungi is ergosterol, the interactions of mycosubtilin with monolayers constituted by ergosterol, DPPC/ergosterol or DPPC/sphingomyelin/ergosterol were examined at different initial surface pressures (Πi). Plotting the mycosubtilin-induced surface pressure increases versus Πi allowed to determine that the exclusion pressures of mycosubtilin from these different monolayers is higher than the surface prevailing within the biological membranes. However, this behavior was lost when mycosubtilin was interacting with ergosteryl acetate-containing monolayers. This suggests the involvement of the sterol alcohol group in the mycosubtilin interactions within membranes. Furthermore, the behavior of mycosubtilin with stigmasterol, similar to that observed with ergosterol, differs from that previously observed with cholesterol, suggesting a role of the alkyl side chain of the sterols. The adsorption of mycosubtilin to ergosterol monolayers induced changes in the lipopeptide orientation at the air-water interface as revealed by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Moreover, imaging the air-water interface by Brewster angle microscopy (BAM) indicates that mycosubtilin induced changes in the organization and morphology of monolayers containing pure ergosterol with the appearance of small condensed dots, suggesting again that the target of mycosubtilin might be the ergosterol present in the membranes of the sensitive yeasts or fungi.     

Biased polymers in the origin of life

We delve into the study of a Markov chain formalism applied to the thermal prebiotic oligomerization of amino acids. We find for the case of only two types of electromagnetic interactions, that the steady state attainment by the Markov chain is a built in mechanism limiting the expected variability in sequences in a population of polymers. Such result may be of importance as it makes more accessible the replication of a minimal chemical machinery compatible with life.     

Probing the modes of antibacterial activity of chitosan. Effects of pH and molecular weight on chitosan interactions with membrane lipids in Langmuir films

Chitosan, a cationic biopolymer derived from chitin, has been described as having antibacterial activity. The modes of this activity, however, have not been established. One mode proposed is that chitosan perturbs bacterial cell membranes. To validate this proposal, in this study we investigated chitosan interactions with lipids in Langmuir monolayers as model membranes. The interactions were assessed by monitoring differences in the shape of the compression isotherms measured in the absence and presence of chitosan in the subphase (acetate buffer). To appraise the contribution of electrostatic interactions versus hydrogen bonding and hydrophobic interactions, three membrane lipids differing in charge were studied-anionic dipalmitoylphosphatidylglycerol (DPPG), zwitterionic dipalmitoylphosphatidylcholine (DPPC), and neutral cholesterol-and the pH of the subphase was varied between 3.5 and 6.0. In addition, the impact of the molecular weight of chitosan on the interactions was assessed at pH 3.5. It was found that while chitosan had a negligible effect on DPPC monolayers over the pH range studied, it distinctly affected DPPG and cholesterol monolayers. The effect on DPPG was found to decrease with increasing pH, that at pH 3.5 being ascribed to the charge-mediating action of chitosan on the local ionic environment and that at higher pHs to the intercalation of chitosan to the monolayers. Practically independent of pH, the effect of chitosan on cholesterol was accounted for by the formation of cholesterol-chitosan hydrogen bonds. Chitosan of lower molecular weight facilitated the interactions with all the three lipids studied. The results obtained may be helpful in identifying the mode of antibacterial activity of chitosan versus other modes that involve the disturbance of cell life cycles.     

A Langmuir film balance study of the interactions of ionic and polar solutes with glycolipid monolayers

Using a Langmuir film balance experiments have been conducted to discover if dissolved salts or carbohydrates interact with glycolipid monolayers. Two types of glycolipid were studied, simple glycosides made by ether linking monosaccharides to fatty alcohols and cerebrosides extracted from natural sources. It was found that salts or carbohydrates in the subphase expanded glycolipid monolayers. That is, a monolayer spread on a solution occupied a greater area at a given pressure than it would have spread on pure water. Of the carbohydrates galactose and glucose, galactose caused a markedly greater expansion of monolayers than glucose. However, the magnitude of the expansions measured for stearyl glucoside, mannoside and galactoside films on solutions of a particular sugar were not significantly different, demonstrating that this phenomenon is independent of the glycolipid sugar residue. As with carbohydrates, salts also have differing effects on glycolipid monolayers. Although the effect an individual ion has on a monolayer cannot be directly measured, comparisons between salts indicate that there is a correlation between the size of an ion and the extent of the monolayer expansion it causes. To explain these observations two different mechanisms are proposed. In the case of salts it is suggested that large ions which have a low charge density disrupt water structure in such a way that monolayers spread on the surface of their solutions are expanded. The ability of carbohydrates to expand monolayers is explained in terms of the carbohydrate replacing water molecules bound to the polar groups of the monolayer and in so doing increasing the effective area of the lipid molecules. It is suggested that the molecular mechanisms involved in the interactions of ions and carbohydrates with glycolipid monolayers may also operate in the interactions of glycolipids and glycoproteins with extracellular agents and surfaces.     

Interactions of chlorpromazine with phospholipid monolayers: effects of the ionization state of the drug

Molecular dynamics simulations have been performed to investigate the interactions between chlorpromazine (CPZ) and Langmuir monolayers of the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the anionic dipalmitoylphosphatidylglycerol (DPPG). Simulations for a fixed surface density and different charge states - neutral and protonated CPZ - were able to capture important features of the CPZ-phospholipid monolayer interaction. Neutral CPZ is predominantly found in the hydrophobic tail region, whereas protonated CPZ is located at the lipid-water interface. Specific interactions (hydrogen bonds) between protonated CPZ and the lipid head groups were found for both zwitterionic and anionic monolayers. We computed lipid tail order parameters and investigated the effects of the drug upon tail ordering. We also computed electrostatic surface potentials and found qualitative good agreement with experimental results.     

Penetration of the insect defensin A into phospholipid monolayers and formation of defensin A-lipid complexes.

Defensin A is an inducible cationic protein secreted in the hemolymph of fleshfly Phormia terranovae larvae in response to bacterial or septic injuries. Defensin A is known to permeabilize the bacteria cell membranes by forming voltage-dependent channels. The penetration of this small protein into lipid monolayers was studied as a function of the polar head and acyl chain length of phospholipids. The extent of penetration by defensin A is higher in monolayers made of anionic phospholipids than in monolayers made of zwitterionic phospholipids (phosphatidylcholines), because of electrostatic interactions. From the analysis of the compression isotherm parameters of mixed defensin A/phospholipid monolayers, it appears that defensin A interacts with phospholipid by forming 1:4 complexes. These complexes are not miscible in the lipid phase and induce microheterogeneity in the lipid membrane. These clusters might be related to the ion-channel structures responsible for the biological activity of defensin A.     

Kinetics and efficiency of excitation energy transfer from chlorophylls,their heavy metal-substituted derivatives,and pheophytins to singlet oxygen

The interactions of carbonmonoxyhemoglobin (HbCO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and polyhistidine with phospholipid monolayers at the air-water interface were studied at physiological pH and ionic strength. HbCO and GAPDH both interact more strongly with monolayers containing negatively charged lipids. The interaction of HbCO and GAPDH with lipid monolayers decreases with increasing pH. Both the HbCO-monolayer and the GAPDH-monolayer interactions can be modeled as diffusion-limited processes, with kinetic data fit to a stretched exponential equation. The significance of these kinetics are discussed. Polyhistidine interacts only with monolayers containing lipids with negatively charged headgroups. In total, the results presented are consistent with an HbCO-lipid interaction with a large electrostatic component, a GAPDH-lipid interaction with comparable electrostatic and hydrophobic components, and a polyhistidine-lipid interaction that is solely electrostatic.     

Self-assembled monolayers on polymer surfaces

The method of forming self-assembled monolayers on polymer surfaces is reviewed. It is shown that the alkylsiloxane monolayers formed on polymer surfaces are structurally similar to the analogous monolayers formed on silicon wafers. These monolayers can be post-derivatized in order to introduce a number of hydrophobic, reactive and polar functionalities, some of which may be useful model systems for studying biological interactions at surfaces.     

Development of a quantitative tool for the comparison of the prebiotic effect of dietary oligosaccharides

AIMS: To develop a quantitative equation [prebiotic index (PI)] to aid the analysis of prebiotic fermentation of commercially available and novel prebiotic carbohydrates in vitro, using previously published fermentation data. METHODS: The PI equation is based on the changes in key bacterial groups during fermentation. The bacterial groups incorporated into this PI equation were bifidobacteria, lactobacilli, clostridia and bacteroides. The changes in these bacterial groups from previous studies were entered into the PI equation in order to determine a quantitative PI score. PI scores were than compared with the qualitative conclusions made in these publications. In general the PI scores agreed with the qualitative conclusions drawn and provided a quantitative measure. CONCLUSIONS: The PI allows the magnitude of prebiotic effects to be quantified rather than evaluations being solely qualitative. SIGNIFICANCE AND IMPACT OF THE STUDY: The PI equation may be of great use in quantifying prebiotic effects in vitro. It is expected that this will facilitate more rational food product development and the development of more potent prebiotics with activity at lower doses.     

The study on the interaction between phytosterols and phospholipids in model membranes

Sterols are one of the major components of cellular membranes. Although in mammalian membranes cholesterol is a predominant sterol, in the human organism plant sterols (phytosterols) can also be found. Phytosterols, especially if present in concentrations higher than normal (phytosterolemia), may strongly affect membrane properties. In this work, we studied phytosterol-phospholipid interactions in mixed Langmuir monolayers serving as model membranes. Investigated were two phytosterols, beta-sitosterol and stigmasterol and a variety of phospholipids, both phosphatidylethanolamines and phosphatidylcholines. The phospholipids had different polar heads, different length and saturation of their hydrocarbon chains. The interactions between molecules in mixed sterol/phospholipid films were characterized with the mean area per molecule (A(12)) and the excess free energy of mixing (DeltaG(Exc)). The effect of the sterols on the molecular organization of the phospholipid monolayers was analyzed based on the compression modulus values. It was found that the incorporation of the phytosterols into the phospholipid monolayers increased their condensation. The plant sterols revealed higher affinity towards phosphatidylcholines as compared to phosphatidylethanolamines. The phytosterols interacted more strongly with phospholipids possessing longer and saturated chains. Moreover, both the length and the saturation of the phosphatidylcholines influenced the stoichiometry of the most stable complexes. Our results, compared with those presented previously for cholesterol/phospholipid monolayers, allowed us to draw a conclusion that the structure of sterol (cholesterol, beta-sitosterol, stigmasterol) does not affect the stoichiometry of the most stable complexes formed with particular phospholipids, but influences their stability. Namely, the strongest interactions were found for cholesterol/phospholipids mixtures, while the weakest for mixed systems containing stigmasterol.     

Study of P-glycoprotein effect on the lipid monolayer properties by the Langmuir-Blodgett technique

Expression of the P-glycoprotein (Pgp) is proved to be one of the main reasons for the development of the multidrug resistance (MDR) phenotype by cancer cells. The effect of Pgp on the properties of lipid monolayers was studied using membrane fractions of sensitive and Pgp over-expressing multidrug resistance cancer cells containing 11, 24 or 32% of Pgp relative to the total content of membrane proteins. The effect of the Pgp membrane concentration on the properties of monolayers prepared from the membrane fractions was analyzed by the Langmuir-Blodgett method. The subphase composition was found to play a critical role in the stability of monolayers at any Pgp concentration. The optimal subphase comprised 10 mM tris-HCl buffer, pH 6.5, which made it possible to create very stable monolayer films with the pressure of collapse of about 30-40 mN/m. Monolayers prepared from membrane fractions of sensitive cells and cells containing the maximum (32%) amount of Pgp were found to be much more stable compared with fractions comprising 11 or 24% of Pgp. The analysis of monolayer compression dynamics revealed three distinct stages: (1) the self-organization of lipid molecules, which is characterized by an abrupt change of surface potential; (2) the compression of Pgp molecules at the constant potential of monolayers; and (3) the compression of lipid molecules, which is characterized by a quasilinear increase of both pressure and surface potential. It was shown that the specific surface areas of monolayers formed from sensitive and Pgp-enriched membranes containing 11 or 24% of Pgp are very similar, whereas the surface area of the monolayer formed from membranes containing 32% of Pgp is nearly 1.5-fold greater. This fact may reflect the effect of the threshold rearrangement of the structure of lipid molecules or cooperative modifications of lipid-Pgp interactions induced by the increase in the Pgp content from 24 to 32%. The effect of verapamil, a well-known Pgp modulator, on the properties of monolayers was studied. It was show that verapamil is able to induce changes of the surface of Pgp-containing monolayers, and these modifications are maximal at the Pgp:verapamil 1:1 molar ratio. The data present the first experimental evidence for the possible intervention of Pgp modulator into the processes of lipid-lipid or lipid-Pgp cooperative interactions within Pgp-enriched membranes.     

Interactions of the lipopeptide antifungal iturin A with lipids in mixed monolayers

The miscibility and the interactions of the antifungal lipopeptide iturin A with lipids, DMPC and cholesterol, are studied in monolayers at the air/water interface and a comparison of the respective behaviour of iturin A and the biologically inactive methylated derivative MeTyr-iturin A is made. Each lipopeptide is miscible with anyone of the lipids. This behaviour is revealed by the dependence of the transition pressure upon composition and by deviations from the additivity rule of the mean molecular area. The thermodynamic properties of the mixed systems are studied by the method of Goodrich. The mixed monolayers are always more stable than the two separate components, subsequently there are interactions between the components. However, the excess free energy of mixing delta Gexm is positive for the iturin A/DMPC system which is an indication that the interactions between lipopeptide and lipid molecules are weaker than the interactions between the pure components themselves. This is compatible with the presence of self-associated lipopeptide molecules. However, delta Gexm is highly negative for the iturin A/cholesterol system giving evidence of the formation of a specific complex between iturin A and cholesterol which is not the case with the methylated derivative. These data are analysed in connection with previous results concerning the pore-forming properties of these lipopeptides and the lack of biological activity of MeTyr-iturin A.     

Interaction of chitosan with cell membrane models at the air-water interface

In this paper we employed phospholipid Langmuir monolayers as membrane models to probe interactions with chitosan. Using a combination of surface pressure--area and surface potential--area isotherms and rheological measurements with the pendent drop technique, we observed that chitosan interacts with phospholipid molecules at the air-water interface. We propose a model in which chitosan interacts with the phospholipids mainly through electrostatic interactions, but also including H-bonding and hydrophobic forces, depending on the phospholipid packing density. At large areas per molecule, chitosan in the subphase adsorbs onto the monolayer, expanding it. At small areas per molecule, chitosan is located in the subsurface. Indeed, a mixed chitosan-phospholipid monolayer can be transferred onto solid supports, even at high surface pressures. The effects of chitosan on the viscoelastic properties of phospholipid monolayers may be taken as evidence for the ability of chitosan to disrupt cell membranes.