Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function

The great variety of different lipids in membranes, with modifications to the hydrocarbon chains, polar groups and backbone structure suggests that many of these lipids may have unique roles in membrane structure and function. Acidic groups on lipids are clearly important, since they allow interaction with basic groups on proteins and with divalent cations. Another important property of certain lipids is their ability to interact intermolecularly with other lipids via hydrogen bonds. This interaction occurs through acidic and basic moieties in the polar head groups of phospholipids, and the amide moiety and hydroxyl groups on the acyl chain, sphingosine base and sugar groups of sphingo- and glycolipids. The putative ability of different classes of lipids to interact by intermolecular hydrogen bonding, the molecular groups which may participate and the effect of these interactions on some of their physical properties are summarized in Table IX. It is frequently questioned whether intermolecular hydrogen bonding could occur between lipids in the presence of water. Correlations of their properties with their molecular structures, however, suggest that it can. Participation in intermolecular hydrogen bonding increases the lipid phase transition temperature by approx. 8-16 Cdeg relative to the electrostatically shielded state and by 20-30 Cdeg relative to the repulsively charged state, while having variable effects on the enthalpy. It increases the packing density in monolayers, possibly also in the liquid-crystalline phase in bilayers, and decreases the lipid hydration. These effects can probably be accounted for by transient, fluctuating hydrogen bonds involving only a small percentage of the lipid at any one time. Thus, rotational and lateral diffusion of the lipids may take place but at a slower rate, and the lateral expansion is limited. Intermolecular hydrogen bonding between lipids in bilayers may be significantly stabilized, despite the presence of water, by the fact that the lipids are already intermolecularly associated as a result of the hydrophobic effect and the Van der Waals' interactions between their chains. The tendency of certain lipids to self-associate, their asymmetric distribution in SUVs, their preferential association with cholesterol in non-cocrystallizing mixtures, their temperature-induced transitions to the hexagonal phase and their inhibitory effect on penetration of hydrophobic residues of proteins partway into the bilayer can all be explained by their participation in intermolecular hydrogen bonding interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interhelical hydrogen bonding drives strong interactions in membrane proteins

Polar residues in transmembrane alpha-helices may strongly influence the folding or association of integral membrane proteins. To test whether a motif that promotes helix association in a soluble protein could do the same within a membrane, we designed a model transmembrane helix based on the GCN4 leucine zipper. We found in both detergent micelles and biological membranes that helix association is driven strongly by asparagine, independent of the rest of the hydrophobic leucine and/or valine sequence. Hydrogen bonding between membrane helices gives stronger associations than the packing of surfaces in glycophorin A helices, creating an opportunity to stabilize structures, but also implying a danger that non-specific interactions might occur. Thus, membrane proteins may fold to avoid exposure of strongly hydrogen bonding groups at their lipid exposed surfaces.     

Permeability of bilayer lipid membranes in phase transition. The significance of intermolecular phosphate-phosphate hydrogen bonding

The effect of pH on the phase transition temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) and 1,2-dipalmitoyl-sn-glycero-3-thionphosphate (thion-DPPA) has been investigated. The phase transition was detected using the jump like increase effect in the conductance of the planar bilayer membrane. It is shown that the steepness of pH-dependence of the phase transition temperature differs for these two kinds of lipids in the pH range of 3.5-8. This result is explained in terms of decreased intermolecular hydrogen bonding between the head groups of thion-DPPA. Calculations taking into account the ability of DPPA molecules to intermolecular phosphate-phosphate hydrogen bonding were made. The results of calculations are in good agreement with the experimental data obtained in this study.     

Ethanol-induced modifications to membrane lipid structure: effect on phospholipase A2-membrane interactions

Chronic ethanol intoxication leads to the development of a resistance to lipid disordering by ethanol, a phenomenon known as "membrane tolerance". In the absence of the added ethanol, the lipid order, as measured by ESR and fluorescence techniques, does not necessarily change as a result of chronic ethanol ingestion (as in liver microsomes, for example). This suggests that the spectroscopic techniques detect tolerance somewhat indirectly, in that the modification responsible may reside in a region distinct from that being probed and also raises the question of whether membrane tolerance is necessarily associated with an alteration in the membrane lipid structure. Here we show that liver microsomes from rats treated chronically with ethanol are rendered relatively resistant to the hydrolytic action of exogenous phospholipase A2, compared to preparations from control animals. This resistance persists in reconstituted lipid vesicles prepared from extracted phospholipids. Since the same substrate (1-palmitoyl-2-N-(4-nitrobenzo-2-oxa-1,3-diazole)amino caproylphosphatidylcholine) was used in both membranes from ethanol-treated animals and controls, the modification appears to reside in the structure and/or organization of the membrane. Further evidence that the lipid structure is modified by chronic ethanol treatment is provided by the observation that perturbance of the membrane structural integrity by increasing levels of oleic acid led to a progressive loss of the ethanol-induced relative resistance to hydrolysis by phospholipase A2. The results of this study support the idea that membrane tolerance involves a modification to lipid structure probably at the bilayer surface. The use of exogenous phospholipase A2 provides a new method for probing the structural modifications induced by chronic ethanol ingestion.     

Lindane-induced modifications to membrane lipid structure: Effect on membrane fluidity after subchronic treament

Chronic lindane intoxication by injecting subcutaneously the toxicant, resulted in an altered lipid pattern in rat ventral prostate membranes. An increase of membrane fluidity was also observed using a fluorescence polarization technique. Whenin vitro experiments were carried out with both treated and untreated rats, an interesting lack of parallelism was found, which could indicate the development of a resistance to membrane disordering by lindane. The observed changes in cholesterol and phospholipid composition are also consistent with the hypothesis that lindane perturbs the lipid matrix of membranes, possibly inducing complex compensatory changes in the membrane lipid composition.     

Tubulin modifications and their cellular functions

All microtubules are built from a basic alpha/beta-tubulin building block, yet subpopulations of microtubules can be differentially marked by a number of post-translational modifications. These modifications, conserved throughout evolution, are thought to act individually or in combination to control specific microtubule-based functions, analogous to how histone modifications regulate chromatin functions. Here we review recent studies demonstrating that tubulin modifications influence microtubule-associated proteins such as severing proteins, plus-end tracking proteins, and molecular motors. In this way, tubulin modifications play an important role in regulating microtubule properties, such as stability and structure, as well as microtubule-based functions, such as ciliary beating, cell division, and intracellular trafficking.     

Kirromycin-induced modifications facilitate the separation of EF-Tu species and reveal intermolecular interactions

A simplified method for the separation of a kirromycin-sensitive tufB-encoded elongation factor Tu (EF-TuBs) from a kirromycin-resistant tufA product (EF-TuAr) was obtained by exploiting the specific increase of negative [corrected] charges induced by the antibiotic, resulting in a retarded elution of kirromycin-bound EF-TuBs on ionic chromatography. The kirromycin-free EF-TuBs is active in poly(Phe) synthesis and shows similar properties to EF-TuAsBs. As expected for these two distinct species, the dissociation of the EF-TuArBs.GTP complex in the presence of kirromycin shows a biphasic curve; in contrast, a monophasic GTP dissociation rate was found for a combination of two mutated EF-Tu species, EF-TuArBo, revealing the existence of intermolecular interactions. These observations prove for the first time the existence of cooperative phenomena between EF-Tu species in vitro, as suggested earlier by in vivo experiments.     

Theoretical study of hydrogen bonding interactions on MDI-based polyurethane

Hydrogen bonding among hard–hard segments and hard–soft segments in 4,4′-diphenylmethane diisocyanate (MDI)-based polyurethane was investigated theoretically by density functional theory (DFT). Both B3LYP/6-31G* and B3PW91/6-31G* methods gave good structures, reasonable Mulliken charges, binding energies, dipole moments, and good infrared (IR) spectra trends in predicting hydrogen bonding. Bond distances R(N–H?O), which were in the range of 3.005–3.028 Å for the carbonyl bonded hydrogen-bond, and 3.074–3.075 Å for the ester bonded hydrogen-bond, are in reasonable agreement with experimental values. Most of the carbonyl oxygen in polyurethane exists in a hydrogen-bonded form. Complex (c), with two carbonyl hydrogen bonds, features the largest dipole moment, while complex (d) with two ester hydrogen bonds, possesses the smallest dipole moment, i.e., lower than that of the isolated monomer, which may be due to the symmetry of the two monomers. These results confirm that the DFT method is a good tool with which to study weak interactions, and indicate that hydrogen bonds are indeed formed between carbonyl and N-H, or ester and N-H, with the former being stronger.

Peptide nucleic acids and their structural modifications

Peptide (polyamide) analogues of nucleic acids (PNAs) make very promising groups of natural nucleic acid (NA) ligands and show many other interesting properties. Two types of these analogues may be highlighted as particularly interesting: the first, containing a polyamide with alternating peptide/pseudopeptide bonds as its backbone, consisting of N-(aminoalkyl)amino-acid units (type I), with nucleobases attached to the backbone nitrogen with the carboxyalkyl linker; and the second, containing a backbone consisting of amino-acid residues carrying the nucleobases in their side chains (type II). So far, these two groups have been studied most intensively. The paper describes main groups of peptide nucleic acids, as well as various other amino acid-derived nucleobase monomers or their oligomers, which were either studied in order to determine their hybridisation to nucleic acids, or only discussed with respect to their potential usefulness in the oligomerisation and nucleic acids binding.     

alpha-Synuclein membrane interactions and lipid specificity

With the discovery of missense mutations (A53T and A30P) in alpha-synuclein (alpha-Syn) in several families with early onset familial Parkinson's disease, alpha-Syn aggregation and fibril formation have been thought to play a role in the pathogenesis of alpha-synucleinopathies, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. As previous reports have suggested that alpha-Syn plays a role in lipid transport and synaptic membrane biogenesis, we investigated whether alpha-Syn binds to a specific lipid ligand using thin layer chromatography overlay and examined the changes in its secondary structure using circular dichroism spectroscopy. alpha-Syn was found to bind to acidic phospholipid vesicles and this binding was significantly augmented by the presence of phosphatidylethanolamine, a neutral phospholipid. We further examined the interaction of alpha-Syn with lipids by in situ atomic force microscopy. The association of soluble wild-type alpha-Syn with planar lipid bilayers resulted in extensive bilayer disruption and the formation of amorphous aggregates and small fibrils. The A53T mutant alpha-Syn disrupted the lipid bilayers in a similar fashion but at a slower rate. These results suggest that alpha-Syn membrane interactions are physiologically important and the lipid composition of the cellular membranes may affect these interactions in vivo.     

Recognition of thymine by triazine fluorescent probe through intermolecular multiple hydrogen bonding

In order to design a fluorescent probe with a long wavelength and multiple hydrogen bond sites for thymine, 3-(4-chloro-6-methylamino-1,3,5-triazinylamino)-7-dimethylamino-2-methylphenazine is synthesized by subsequently reacting neutral red and methylamine with cyanuric chloride. Its recognition behavior for thymine and its spectroscopic properties in different solvents are studied. The probe's fluorescence can be selectively quenched by thymine instead of guanine, indicating that fully complementary hydrogen bonding plays a key role in such recognition processes. In addition, the recognition mechanism of the probe for thymine and the bathochromic shift of its fluorescence spectra with the solvent polarity are also discussed.     

Relationships between the orientation and the structural properties of peptides and their membrane interactions

Physical properties of membranes, such as fluidity, charge or curvature influence their function. Proteins and peptides can modulate those properties and conversely, the lipids can affect the activity and/or the structure of the former. Tilted peptides are short hydrophobic protein fragments characterized by an asymmetric distribution of their hydrophobic residues when helical. They were detected in viral fusion proteins and in proteins involved in different biological processes that need membrane destabilization. Those peptides and non lamellar lipids such as PE or PA appear to cooperate in the lipid destabilization process by enhancing the formation of negatively-curved domains. Such highly bent lipidic structures could favour the formation of the viral fusion pore intermediates or that of toroidal pores. Structural flexibility appears as another crucial property for the interaction of peptides with membranes. Computational analysis on another kind of lipid-interacting peptides, i.e. cell penetrating peptides (CPP) suggests that peptides being conformationally polymorphic should be more prone to traverse the bilayer. Future investigations on the structural intrinsic properties of tilted peptides and the influence of CPP on the bilayer organization using the techniques described in this chapter should help to further understand the molecular determinants of the peptide/lipid inter-relationships.     

Lipids and lipid modifications in the regulation of membrane traffic

Lipids play a multitude of roles in intracellular protein transport and membrane traffic. While a large body of data implicates phosphoinositides in these processes, much less is known about other glycerophospholipids such as phosphatidic acid, diacylglycerol, and phosphatidylserine. Growing evidence suggests that these lipids may also play an important role, either by mediating protein recruitment to membranes or by directly affecting membrane dynamics. Although membrane lipids are believed to be organized in microdomains, recent advances in cellular imaging methods paired with sophisticated reporters and proteomic analysis have led to the formulation of alternative ideas regarding the characteristics and putative functions of lipid microdomains and their associated proteins. In fact, the traditional view that membrane proteins may freely diffuse in a large 'sea of lipids' may need to be revised. Lastly, modifications of proteins by lipids or related derivatives have surprisingly complex roles on regulated intracellular transport of a wide range of molecules.     

Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties

Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery.     

Effect of hydrogen peroxide on sugar transport inSchizosaccharomyces pombe. Absence of membrane lipid peroxidation

Stationary unaerated cells ofS. pombe containing endogenous substrates but not energized by any exogenous ones take up 2-deoxy-d-glucose, 6-deoxy-d-glucose,d-xylose andd-arabinose actively over diffusion equilibrium. The active uptake is inhibited by 20–100 mmol/L H₂O₂ which causes an increase inK _T but has no effect onJ _max. This “competitive inhibition” indicates that H₂O₂ affects directly the sugar binding sites of the transporters. The ATP-binding site of the plasma membrane H⁺-ATPase is also affected by 100 mmol/L H₂O₂; theK _T decreases 7-fold,J _max about 2.5-fold. These effects are not likely to be mediated by membrane lipid peroxidation which appears to be lacking inS. pombe, and this lack may be one of the reasons for the high resistance of this yeast to H₂O₂. Because of thisS. pombe represents a suitable system for studying direct effects of oxidants on membrane proteins.     

Alcohols dehydrate lipid membranes: an infrared study on hydrogen bonding.

The effects of alcohols (methanol, ethanol, and n-butanol) on the hydrogen bonding of dipalmitoylphosphatidylcholine (DPPC) were studied by Fourier-transform infrared spectroscopy (FTIR) in water-in-oil (carbon tetrachloride) reversed micelles. The bound O-H stretching mode of water, bonded to DPPC, appeared as a broad band at around 3400 cm-1. The O-H bending mode of this complex appeared as a weak broad band at 1644 cm-1. No free O-H signal was observed. When alcohols were added, a part of DPPC-bound water was replaced by the alcohols. The released 'free' water appeared at 3680 cm-1. This free O-H stretching band represents water-alcohol complex. A new broad band of O-H stretching appeared at 3235 cm-1, which represents the alcohol molecules bound to the phosphate moiety of DPPC. When the alcohol concentration was increased, the intensities of the free O-H stretching and bending bands increased. The P = O- antisymmetric stretching band at 1238 cm-1 became broader and shifted to lower frequencies. This means that alcohols interacted with the phosphate moiety and replaced the bound water. In the deconvoluted spectra of the C = O stretching mode, the ratio between the free sn-2 and the hydrogen-bonded sn-2 bands increased; a part of the bound water at the sn-2 carbon in the glycerol skeleton is also released and the free sn-2 signal increased. From the change in the intensity of the P = O- stretching band, the partition coefficients of alcohols between the phosphate region of DPPC and water were estimated: methanol 7.8, ethanol 16.7 at 22.0 degrees C in mole fraction bases. In molality, these values translates into methanol 0.21 and ethanol 0.45. These results indicate that short-chain alcohols interact with lipid membranes at the phosphate moiety at the hydrophilic head, weaken the membrane-water interaction, and destabilize membranes.