Theoretical conformational analysis of phospholipids II. Role of hydration in the gel to liquid crystal transition of phospholipids

To obtain a satisfactory agreement between computed transition temperatures and those determined experimentally, we introduce explicitly water molecules which hydrate the polar headgroup of dipalmitoylphosphatidylethanolamine molecules. The calculated free energy curves as a function of the intermolecular interchain distance and the degree of hydration of the polar groups permit the determination of the transition of the phospholipid system from the gel to the liquid crystalline phase. The detailed structure of the hydration shell is defined using the supermolecular approach.     

Theoretical conformational analysis of phospholipids. II. Role of hydration in the gel to liquid crystal transition of phospholipids

To obtain a satisfactory agreement between computed transition temperatures and those determined experimentally, we introduce explicitly water molecules which hydrate the polar headgroup of dipalmitoylphosphatidylethanolamine molecules. The calculated free energy curves as a function of the intermolecular interchain distance and the degree of hydration of the polar groups permit the determination of the transition of the phospholipid system from the gel to the liquid crystalline phase. The detailed structure of the hydration shell is defined using the supermolecular approach.     

Study of the transverse diffusion of spin-labeled phospholipids in biological membranes. II. Inner mitochondrial membrane of rat liver: use of phosphatidylcholine exchange protein.

Spin-labeled phosphatidylcholine was incorporated into the membrane of isolated "inner membrane+matrix" particles of rat liver mitochondria by incubation with sonicated spin-labeled phosphatidylcholine vesicles at 22 degrees C. When the spin label was on the acyl chain the incorporation of phosphatidylcholine into the membrane was stimulated by the presence of the phosphatidylcholine exchange protein extracted from rat or beef liver. On the other hand no stimulation was observed when the nitroxide was on the polar head-group. When spin-labeled phosphatidycholine was incorporated into the mitochondrial membrane in the absence of phosphatidylcholine exchange protein, ascorbate treatment at 0 degrees C reduced the EPR signal of the spin-labeled membranes by approximately 50%, indicating that fusion incorporates molecules equally on both sides of the membrane. On the other hand when spin-labeled phosphatidylcholine was incorporated in the presence of the exchange protein most of the EPR signal could be destroyed by the ascorbate treatment at 0 degrees C, indicating that the spin-labeled phosphatidylcholine had been selectively incorporated in the outer layer of the membrane. Finally when the label is on the polar head-group the inner content of mitochondria reduces the label facing the matrix, thus creating again an anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the mitochondrial membrane was found to be stable at 25 degrees C for more than 2 h. It is therefore concluded that the rate of outside-inside and inside-outside transitions are extremely slow (half-life greater than 24 h).     

Distributions of water around amino acid residues in proteins

The atomic co-ordinates from 16 high-resolution (less than or equal to 1.7 A = 0.1 nm), non-homologous proteins have been used to study the distributions of water molecule sites around the 20 different amino acid residues. The proportion of residues whose main-chain atoms are in contact with water molecules was fairly constant (between 40% and 60%), irrespective of the nature of the side-chain. However, the proportion of residues whose side-chain atoms were in contact with water molecules showed a clear (inverse) correlation with the hydrophobicity of the residue, being as low as 14% for leucine and isoleucine but greater than 80% for asparagine and arginine. Despite the problems in determining accurate water molecule sites from X-ray diffraction data and the complexity of the protein surface, distinct non-random distributions of water molecules were found. These hydration patterns are consistent with the expected stereochemistry of the potential hydrogen-bonding sites on the polar side-chains. The water molecules around apolar side-chains lie predominantly at van der Waals' contact distances, but most of these have a primary, shorter contact with a neighbouring polar atom. Further analysis of these distributions, combined with energy minimization techniques, should lead to improved modelling of protein structures, including their primary shells of hydration.     

Study of the transverse diffusion of spin-labeled phospholipids in biological membranes. II. Inner mitochondrial membrane of rat liver: Use of phosphatidylcholine exchange protein

Spin-labeled phosphatidylcholine was incorporated into the membrane of isolated “inner membrane+matrix” particles of rat liver mitochondria by incubation with sonicated spin-labeled phosphatidylcholine vesicles at 22°C. When the spin label was on the acyl chain the incorporation of phosphatidylcholine into the membrane was stimulated by the presence of the phosphatidylcholine exchange protein extracted from rat or beef liver. On the other hand no stimulation was observed when the nitroxide was on the polar head-group.When spin-labeled phosphatidylcholine was incorporated into the mitochondrial membrane in the absence of phosphatidylcholine exchange protein, ascorbate treatment at O°C reduced the EPR signal of the spin-labeled membranes by approximately 50%, indicating that fusion incorporates molecules equally on both sides of the membrane. On the other hand when spin-labeled phosphatidylcholine was incoporated in the presence of the exchange protein most of the EPR signal could be destroyed by the ascorbate treatment at 0°C, indicating that the spin-labeled phosphatidylcholine had been selectively incorporated in the outer layer of the membrane. Finally when the label is on the polar head-group the inner content of mitochondria reduces the label facing the matrix, thus creating again an anisotropy of the labeling.The anisotropic distribution of spin-labeled phosphatidylcholine in the mitochondrial membrane was found to be stable at 25°C for more than 2 h. It is therefore concluded that the rate of outside-inside and inside-outside transitions are extremely slow (half-life greater than 24 h).     

The liquid condensed diffusional transition of dipalmitoylphosphoglycerocholine in monolayers

The fine details of the phase transition of dipalmitoylphosphoglycerocholine (DPPC) monolayers at air/NaCl solution interfaces were investigated at 21 +/- 1 degrees C by using the fluorescence after photobleaching technique employing 12-(9-anthroyloxy)stearic acid as fluorescent probe. The mode of compression of the monolayer (i.e., continuous compression or successive additions of the lipid at fixed area) together with the ionic strength of the subphase (0.1 or 1.0 M NaCl) were particularly studied. The photobleaching results show that the lateral diffusion coefficient of the probe molecules decreases drastically within the liquid-condensed phase, i.e., from the end of the liquid-expanded-liquid-condensed phase transition to the beginning of the solid-condensed phase. The molecular areas at which the phase transition occurs under the various experimental conditions, together with a parallel analysis of the hydration states and related molecular areas of the DPPC molecules in multilayers, strongly suggest that the steric hindrance associated with the hydration water of the polar head of DPPC molecules in the monolayer is responsible for the drastic decrease in diffusion coefficient in the liquid-condensed phase. Furthermore, the fluorescence characteristics of the probe molecules also show that, together with the aforementioned reorganization of the polar head, a structural reorganization of the aliphatic chains of the lipid molecules also takes place in the liquid-condensed phase. The liquid-condensed phase therefore appears as a transition region from liquid to solid phases in which the lipid molecules present a significant decrease in their lateral diffusion related to a structural reorganization of both their polar and aliphatic components.     

Apolar and polar solvation thermodynamics related to the protein unfolding process.

Thermodynamics related to hydrated water upon protein unfolding is studied over a broad temperature range (5-125 degrees C). The hydration effect arising from the apolar interior is modeled as an increased number of hydrogen bonds between water molecules compared with bulk water. The corresponding contribution from the polar interior is modeled as a two-step process. First, the polar interior breaks hydrogen bonds in bulk water upon unfolding. Second, due to strong bonds between the polar surface and the nearest water molecules, we assume quantization using a simplified two-state picture. The heat capacity change upon hydration is compared with model compound data evaluated previously for 20 different proteins. We obtain good correspondence with the data for both the apolar and the polar interior. We note that the effective coupling constants for both models have small variations among the proteins we have investigated.     

Influence of hydration on segmental chain librations and dynamical transition in lipid bilayers

Continuous wave electron paramagnetic resonance spectroscopy of chain-labeled phospholipids is used to investigate the effects of hydration on the librational oscillations and the dynamical transition of phospholipid membranes in the low-temperature range 120–270 K. Bilayers of dipalmitoylphostatidiycholine (DPPC) spin-labeled at the first acyl chain segments and at the methyl ends and prepared at full, low, and very low hydration are considered. The segmental mean-square angular amplitudes of librations, 〈α²〉, are larger in the bilayer interior than at the polar/apolar interface and larger in the fully and low hydrated than in the very low hydrated membranes. For chain segments at the beginning of the hydrocarbon region, 〈α²〉-values are markedly restricted and temperature independent in DPPC with the lowest water content, whereas they increase with temperature in the low and fully hydrated bilayers, particularly at the highest temperatures. For chain segments at the chain termini, the librational amplitudes increase progressively, first slowly and then more rapidly with temperature in bilayers at any level of hydration. From the temperature dependence of the mean-square librational amplitude, the dynamical transition is detected around 240 K at the polar/apolar interface in fully and low hydrated DPPC and at around 225 K at the inner hydrocarbon region for bilayers at any hydration condition. At the dynamical transition the bilayers cross low energy barriers of activation energy in the range 10–20 kJ/mol. The results highlight biophysical properties of DPPC bilayers at low-temperature and provide evidence of the effects of the hydration on the dynamical transition in bilayers.     

Distance measurements using paramagnetic ion-induced relaxation in the saturation transfer electron spin resonance of spin-labeled biomolecules: Application to phospholipid bilayers and interdigitated gel phases

The saturation transfer electron spin resonance (STESR) spectra of spin-labeled phosphatidylcholines in gel phase lipid bilayers are shown to be sensitive to dipolar spin-spin interactions with paramagnetic ions in the aqueous phase. The reciprocal integrated intensity of the STESR spectrum is linearly dependent on aqueous Ni²⁺ ion concentration, hence, confirming the expectation that the STESR intensity is directly proportional to the spin-lattice relaxation time of the spin label. The gradient of the relaxation rate with respect to Ni²⁺ ion concentration decreases strongly with the position of the nitroxide group down the sn-2 chain of the spin-labeled lipid and is consistent with a 1/R³ dependence on the distance, R, from the bilayer surface. The values derived for the dimensions of the bilayer and lipid molecules in the case of dipalmitoyl phosphatidylcholine (DPPC) are in good agreement with those available from x-ray diffraction studies. Allowance for the multibilayer nature of the DPPC dispersions gives an estimate of the water layer thickness that is also consistent with results from x-ray diffraction. The profile of the paramagnetic ion-induced relaxation is drastically changed with DPPC dispersions in glycerol for which the lipid chains are known to be interdigitated in the gel phase. The terminal methyl groups of the lipid chains are located approximately in register with the C-3 atoms of the sn-2 chain of the oppositely oriented lipid molecules in the interdigitated phase. The thickness of the lipid layer and the effective thickness of the lipid polar group are reduced by ~40% in the interdigitated phase as compared with the bilayer phase. The calibrations of the distance dependence established by use of spin labels at defined chain positions should be applicable to STESR measurements on other biological systems.     

The kinetics of formation and structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine

A combination of differential scanning calorimetry (DSC) and X-ray diffraction have been used to study the kinetics of formation and the structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine (18:0-lysoPC). For water contents greater than 40 weight %, DSC shows a sharp endothermic transition at 27 degrees C (delta H = 6.75 kcal/mol) corresponding to a low-temperature phase----micelle transition. This sharp transition is not reversible, but is regenerated in a time and temperature-dependent manner. For example, with incubation at 0 degrees C the maximum transition enthalpy (delta H = 6.75 kcal/mol) is generated in about 45 min after an initial slow nucleation process of approx. 20 min. The kinetics of formation of the low-temperature phase is accelerated at lower temperatures and may be related to the disruption of 18:0-lysoPC micelles by ice crystal formation. X-ray diffraction patterns of 18:0-lysoPC recorded at 10 degrees C over the hydration range 20-80% are characteristic of a lamellar gel phase with tilted hydrocarbon chains with the bilayer repeat distance increasing from 47.6 A at 20% hydration to a maximum of 59.4 A at 39% hydration. At this maximum hydration, approx. 19 molecules of water are bound per molecule of 18:0-lysoPC. Electron density profiles show a phosphate-phosphate distance of 30 A, indicating an interdigitated lamellar gel phase for 18:0-lysoPC at all hydration values. The angle of chain tilt is calculated to be between 20 and 30 degrees. For water contents greater than 40%, this interdigitated lamellar phase converts to the micellar phase at 27 degrees C in a kinetically fast process, while the reverse (micelle----interdigitated bilayer) transition is a kinetically slower process (see also Wu, W. and Huang, C. (1983) Biochemistry 22, 5068-5073).     

Theoretical conformational analysis of phospholipids. I. Study of the interactions between phospholipid molecules by use of semi-empirical methods with the explicit introduction of polar headgroup interactions

We present a theoretical conformational analysis of a system composed of seven dipalmitoylphosphatidylethanolamine molecules in interaction. The combined use of classical semi-empirical methods for the polar headgroup region with mechanical statistical calculations for the aliphatic chains permits the evaluation of the free energy for a phospholipids molecule. The free energy variation in function of the mean intermolecular interchain distance gives information about the main lipid bilayer phase transition. It appears, however, necessary to take into account the hydration of the polar headgroups.     

Theoretical conformational analysis of phospholipids: I. Study of the interactions between phospholipid molecules by use of semi-empirical methods with the explicit introduction of polar headgroup interactions

We present a theoretical conformational analysis of a system composed of seven dipalmitoylphosphatidylethanolamine molecules in interaction. The combined use of classical semi-empirical methods for the polar headgroup region with mechanical statistical calculations for the aliphatic chains permits the evaluation of the free energy for a phospholipids molecule. The free energy variation in function of the mean intermolecular interchain distance gives information about the main lipid bilayer phase transition. It appears, however, necessary to take into account the hydration of the polar headgroups.     

The mechanism of the type III antifreeze protein action: a computational study

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of differential distortions of the water-water hydrogen bonding angle in the first hydration shell. This method of hydration analysis is applied here to study the hydration of the wild type III thermal hysteresis protein from eel pout and three mutations at residue 16. Wild type and one mutant have full activity, the other two mutants have little or no anti-freeze (thermal hysteresis) activity. The analysis reveals significant differences in the hydration structure of the ice-binding site (centered on residue 16) among four proteins. For the A16T and A16Y mutants with reduced activity, polar groups have a typical polar-like hydration. For the wild type and mutant A16C with 100% of the wild type activity, polar groups have unusual, very apolar-like hydration. In the latter case, hydrating water molecules form a more ice-like pattern of hydrogen bonding on the ice-binding face, while in the former case water-water H-bonds are more distorted and more heterogenous. Overall, the binding surface of active protein strongly enhances the water tetrahedral structure, i.e. promotes ice-like hydration. It is concluded that the specific shape, residue size and clustering of both polar/apolar groups are essential for the binding surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.     

Site-Specific Peptide Probes Detect Buried Water in a Lipid Membrane

Transmembrane peptides contain polar residues in the interior of the membrane, which may alter the electrostatic environment and favor hydration in the otherwise nonpolar environment of the membrane core. Here, we demonstrate a general, nonperturbative strategy to probe hydration of the peptide backbone at specific depths within the bilayer using a combination of site-specific isotope labels, ultrafast two-dimensional infrared spectroscopy, and spectral modeling based on molecular dynamics simulations. Our results show that the amphiphilic pH-low insertion peptide supports a highly heterogeneous environment, with significant backbone hydration of nonpolar residues neighboring charged residues. For example, a leucine residue located as far as 1 nm into the hydrophobic bulk reports hydrogen-bonded populations as high as ∼20%. These findings indicate that the polar nature of these residues may facilitate the transport of water molecules into the hydrophobic core of the membrane.     

Interaction of intestinal brush border membrane vesicles with small unilamellar phospholipid vesicles. Exchange of lipids between membranes is mediated by collisional contact

The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions.     

Hydration of B-DNA: comparison between the water network around poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) on the basis of Monte Carlo computations

A computational method is elaborated for studying the water environment around regular polynucleotide duplexes; it allows rigorous structural information on the hydration shell of DNA to be obtained. The crucial aspect of this Monte Carlo simulation is the use of periodical boundary conditions. The output data consists of local maxima of water density in the space near the DNA molecule and the properties of one- and two-membered water bridges as function of pairs of polar groups of DNA. In the present paper the results for poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) are presented. The differences in their hydration shells are of a purely structural nature and are caused by the symmetry of the polar groups of the polymers under study, the symmetry being reflected by the hydration shell. The homopolymer duplex hydration shell mirrors the mononucleotide repeat. The water molecules contacting the polynucleotide in the minor groove are located nearly in the plane midway between the planes of successive base pairs. One water molecule per base pair forms a water bridge facing two polar groups of bases from adjacent base pairs and on different strands making a "spine"-like structure. In contrast, the major groove hydration is stabilized exclusively by two-membered water bridges; the water molecules deepest in the groove are concentrated near the plane of the corresponding base pair. The alternating polymer is characterized by a marked dyad symmetry of the hydration shell corresponding to the axis between two successive base pairs. The minor groove hydration of the dCpdG step resembles the characteristic features of the homopolymer, but the bridge between the O2 oxygens of the other base-stacking type is formed by two water molecules. The major groove hydration is characterized by high probability of one-membered water bridges and by localization of a water molecule on the dyad axis of the dGpdC step. The found structural elements are discussed as reasonable invariants of a dynamic hydration shell.     

High-affinity binding of water by proteins is similar in air and in organic solvents

Published data for water adsorption by proteins suspended in organic solvents (of interest as enzyme reaction mixtures) have been converted to a basis of thermodynamic water activity (aw). The resulting adsorption isotherms have been compared with those known for proteins equilibrated with water from a gas phase. This comparison can show any effects of the solvent on the interaction between the protein and water at the molecular level. At lower water contents (aw less than about 0.4), similar adsorption isotherms are found in each solvent and in the gas phase; differences are probably less than the likely errors. Hence, it may be concluded that the presence of an organic solvent has little effect on the interaction between proteins and tightly bound water; on a molecular scale there is probably little penetration of the primary hydration layer by solvent molecules, even fairly polar ones such as EtOH. At higher aw values, there are differences between the isotherms which probably are significant. Nonpolar solvents increase the amount of water bound by the enzyme (at fixed aw), while polar solvents (mainly EtOH) may reduce the amount of water bound by the enzyme, presumably by occupying part of the secondary hydration layers in place of water.     

Protein-water interactions in ribonuclease A and angiogenin: a molecular dynamics study

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein-water interactions have been carried out on two members of the ribonuclease A superfamily-RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein-water networks are investigated. The locations of such networks are identified for both proteins.     

Study of the transverse diffusion of spin labelled phospholipids in biological membranes. I. Human red blood cells

Spin labeled analogs of phosphatidylcholine were used to study the transverse diffusion (flip-flop) of phospholipids in the erythrocyte membrane. The nitroxide spin label was placed either on the β acyl chain or on the choline group. These labeled phosphatidylcholine molecules were incorporated into the membrane by incubation of the red cells at 22°C with sonicated spin-labeled phosphatidylcholine vesicles from which all traces of free fatty acids and lyso derivatives were carefully removed by bovine serum albumin treatment. This incorporation did not provide any change in the morphology of the cell as indicated by scanning electron microscopy. When spin-labeled phosphatidylcholine, having a nitroxide on the β chain but near the polar head-group, was incorporated into the erythrocyte membrane, ascorbate treatment at 0dgC allows selective reduction of the signal coming from the outer layer of the membrane. When the label was on the polar head-group, the inner content of the erythrocyte rapidly reduced the label facing the cytoplasm, thus creating a spontaneous anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the erythrocyte membrane was found to be stable at 22 and 37°C for more than 4 h. It is therefore concluded that the rate of outside-inside and inside-outside transition is so slow that the anisotropic distribution of the phospholipids in the erythrocyte membrane can be maintained during cell life.