Rotational Dynamics of Water at the Phospholipid Bilayer Depending on the Head Groups Studied by Molecular Dynamics Simulations.

Hydration states are a crucial factor that affect the self-assembly and properties of soft materials and biomolecules. Although previous experiments have revealed that the hydration state strongly depends on the chemical structure of lipid molecules, the mechanisms at the molecular level remain unknown. Classical and density-functional tight-binding (DFTB) molecular dynamics (MD) simulations are employed to determine the mechanisms underlying dissimilar water dynamics between lipid membranes with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) head groups. The classical MD simulation shows that rotational relaxations of water are faster on the PE lipid than on the PC lipid, which is consistent with a previous experimental study using terahertz spectroscopy. Furthermore, DFTB-MD simulation of N(CH3)4+ and NH4+ ions, which correspond to the different head groups in PC and PE, respectively, shows qualitative agreement with the classical MD simulation. Remarkably, the PE lipids and the NH4+ ions break hydrogen bonds between water molecules in the secondary hydration shell. In contrast, the PC lipids and the N(CH3)4+ ions bind water molecules weakly in both the primary and secondary hydration shells and increase hydrogen bonds between water. Our atomistic simulations show that these changes in the hydrogen-bond network of water molecules cause the different rotational relaxation of water molecules between the two lipids.

Reversible Supra-Folding of User-Programmed Functional DNA Nanostructures on Fuzzy Cationic Substrates.

We report that user-defined DNA nanostructures, such as two-dimensional (2D) origamis and nanogrids, undergo a rapid higher-order folding transition, referred to as supra-folding, into three-dimensional (3D) compact structures (origamis) or well-defined µm-long ribbons (nanogrids), when they adsorb on a soft cationic substrate prepared by layer-by-layer deposition of polyelectrolytes. Once supra-folded, origamis can be switched back on the surface into their 2D original shape through addition of heparin, a highly charged anionic polyelectrolyte known as an efficient competitor of DNA-polyelectrolyte complexation. Orthogonal to DNA base-pairing principles, this reversible structural reconfiguration is also versatile; we show in particular that 1) it is compatible with various origami shapes, 2) it perfectly preserves fine structural details as well as site-specific functionality, and 3) it can be applied to dynamically address the spatial distribution of origami-tethered proteins.

Multimodal Multiphoton Imaging of the Lipid Bilayer by Dye-Based Sum-Frequency Generation and Coherent Anti-Stokes Raman Scattering.

Coherent anti-Stokes Raman scattering (CARS) imaging is widely used for imaging molecular vibrations inside cells and tissues. Lipid bilayers are potential analytes for CARS imaging due to their abundant CH2 vibrational bonds. However, identifying the plasma membrane is challenging since it possesses a thin structure and is closely apposed to lipid structures inside the cells. Since the plasma membrane provides the most prominent asymmetric location within cells, orientation sensitive sum-frequency generation (SFG) imaging is a promising technique for selective visualization of the plasma membrane labeled by a nonfluorescent and SFG-specific dye, Ap3, when using a CARS microscope system. In this study, we closely compare the characteristics of lipid bilayer imaging by dye-based SFG and CARS using giant vesicles (GVs) and N27 rat dopaminergic neural cells. As a result, we show that CARS imaging can be exploited for the visualization of whole lipid structures inside GVs and cells but is insufficient for identification of the plasma membrane, which instead can be achieved using dye-based SFG imaging. In addition, we demonstrate that these unique properties can be combined and applied to the live-cell tracking of intracellular lipid structures such as lipid droplets beneath the plasma membrane. Thus, multimodal multiphoton imaging through a combination of dye-based SFG and CARS can serve as a powerful chemical imaging tool to investigate lipid bilayers in GVs and living cells.

Phase Transition from the Interdigitated to Bilayer Membrane of a Cationic Surfactant Induced by Addition of Hydrophobic Molecules.

Although the transition between a bilayer and an interdigitated membrane of a surfactant and lipid has been widely known for long, its mechanism remains unclear. This study reveals the transition mechanism of a cationic surfactant, dioctadecyldimethylammonium chloride (DODAC), through experiments and theoretical calculations. Experimentally, the transition from the interdigitated to bilayer structure in the gel phase of DODAC is found to be induced by adding hydrophobic molecules such as n-alkane and its derivatives. Further addition induces a different transition to another bilayer phase. Our theory, considering the competition of the electrostatic interaction between cationic headgroups and the hydrophobic interaction emerging at the alkyl-chain ends exposed to water, reproduces these two phase transitions. In addition, changes in alkyl-chain packing in the membranes at these transitions are reproduced. The underlying mechanism is that the interdigitated membrane is formed at a small additive content due to electrostatic repulsion. As the energetic disadvantage with respect to the hydrophobic interaction becomes dominant as the content increases, the transition to the bilayer occurs at a specific content. The bilayer-bilayer transition at a higher content is induced by the change in the balance of these interactions. Based on a similar concept, we suggest the mechanism of the additive-induced bilayer-interdigitated transition of phospholipids, i.e., neutrally charged (zwitterionic) surfactants.

Interleaflet coupling of n-alkane incorporated bilayers.

The relationship between the membrane bending modulus (κ) and compressibility modulus (KA) depends on the extent of coupling between the two monolayers (leaflets). Using neutron spin echo (NSE) spectroscopy, we investigate the effects of n-alkanes on the interleaflet coupling of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. Structural studies with small-angle X-ray and neutron scattering (SAXS and SANS) showed that the bilayer thickness increased with increasing n-alkane length, while NSE suggested that the bilayers became softer. Additional measurements of the membrane thickness fluctuations with NSE suggested that the changes in elastic moduli were due to a decrease in coupling between the leaflets upon addition of the longer n-alkanes. The decreased coupling with elongating n-alkane length was explained based on the n-alkane distribution within the bilayers characterized by SANS measurement of bilayers composed of protiated DPPC and deuterated n-alkanes. A higher fraction of the incorporated long n-alkanes were concentrated at the central plane of the bilayers and decreased the physical interaction between the leaflets. Using NSE and SANS, we successfully correlated changes in the mesoscopic collective dynamics and microscopic membrane structure upon incorporation of n-alkanes.

Phase separation of a ternary lipid vesicle including n-alkane: Rugged vesicle and bilayer flakes formed by separation between highly rigid and flexible domains.

We investigate the phase separation of a ternary lipid bilayer including n-alkane and construct the ternary phase diagram. When a certain proportion of a long n-alkane is mixed with a binary mixture of lipids, which exhibit the disordered liquid-crystalline phase and the ordered gel phase at room temperature, we observed the characteristic morphology of bilayers with phase separation. The ordered bilayer forms flat and rigid domains, which is connected or rimmed with flexible domains in the disordered phase. The asymmetric emergence of the phase separation region close to the ordered phase side is interpreted based on the almost equal distribution of the n-alkane to the ordered and disordered phase domains.

Examination of molecular packing in orthogonal smectic liquid crystal phases: a guide for molecular design of functional smectic phases.

The reported layer spacings (dsmectic) of six homologues of mesogens exhibiting orthogonal smectic phases (SmE, SmB, and SmA phases) are reexamined. The slopes of the linear dependences on chain length (n, the number of carbon atoms in the hydrocarbon chain) are clearly categorized into two groups: 1.9 Å (CH2)-1 and 1.4 Å (CH2)-1. It is clarified that in the former the molecules take a rod-like form (rod-form; category-I), whereas in the latter the molecules are bent around the connection between the core and chain moieties (bent-form; category-II). The average relative positions of adjacent molecules within the smectic structures are deduced from the intercept of the linear functions of dsmectic against n. The relation between and the features of molecules belonging to the two categories are discussed for molecular design of functional smectic liquid crystals.

Structure and molecular packing in smectic BCr and Ad phases of Schiff base liquid crystal compounds through the analyses of layer spacing, entropy and crystal structure.

Single-crystal structural analyses and heat capacity measurements were performed on two Schiff base liquid crystal compounds, 5CBAA (4-chlorobenzylidene-4'-pentyloxyaniline) and 5ABCA (4-pentyloxybenzylidene-4'-chloroaniline). The alkyloxy-chain of a 5CBAA molecule was conformationally ordered in the crystal at room temperature. While that of 5ABCA was partially disordered in the room temperature phase but ordered in a low-temperature phase at 100 K. The structural phase transition involving the disordering of the conformation was observed at 107 K in the heat capacity of 5ABCA. Both compounds showed two liquid crystalline phases, SmBCr and SmAd. The net entropy change associated with the chain disordering was essentially the same in them despite the difference in the orientation of their central -CH[double bond, length as m-dash]N- moiety. The layer-spacings of SmBCr and SmAd phases were analyzed for their chain-length dependence in both series of mesogens (nCBAA and nABCA), as well as in the case of nBBAA (4-bromobenzylidene-4'-alkyloxyaniline). The results reveal that these smectic structures are composed of alternately stacked core- and chain-layers with an antiparallel arrangement of cores and a bent-form of molecules.

Cell-quintupling: Structural phase transition in a molecular crystal, bis(trans-4-butylcyclohexyl)methanol.

A structural phase transition at 151.6 K of the title compound [bis(trans-4-butylcyclohexyl)methanol] is examined by X-ray diffraction crystallography, Fourier-transform infrared spectroscopy, and adiabatic calorimetry. A general consideration on possible superstructures indicates that a single modulation wave is sufficient to drive this cell-quintupling transition. The entropy of transition determined calorimetrically indicates that two conformations are dominant in the room-temperature phase in contrast to the fivefold disorder expected from the structure of the low-temperature phase.

Photoinduced Bilayer-to-Nonbilayer Phase Transition of POPE by Photoisomerization of Added Stilbene Molecules.

The photocontrol of a bilayer-to-nonbilayer phase transition (the liquid-crystalline Lα phase to the inverted hexagonal HII phase) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) by the photoisomerization of incorporated stilbene molecules was examined by utilizing differential scanning calorimetry, small-angle X-ray diffraction, ultraviolet (UV)/visible absorption, and attenuated total reflectance Fourier transform infrared spectroscopies. cis-Stilbene lowered the transition temperature, Th, to a greater extent than did trans-stilbene, and the difference was at most ca. 10 °C. At temperatures higher than the Th of POPE/cis-stilbene but lower than that of POPE/trans-stilbene, the photoisomerization from the trans to the cis form of the stilbene molecules by irradiation with UV light caused a Lα-HII phase transition. The UV irradiation partially induced the HII phase at a constant temperature because of the incomplete photoisomerization of stilbene (ca. 60%). The reduction in Th by the incorporation of stilbenes was caused mainly by the reduction in the spontaneous radius of curvature of the lipid monolayer, R0. The greater bulkiness of cis-stilbene as compared to the trans form resulted in a more effective reduction in R0 and stabilization of the HII phase.

Contrasting Effects of a Rigid Core and an Alkyl Chain in nCB on the Phase Behavior of Lipid Bilayers.

Molecules incorporated into biomembranes often bear both a core and an alkyl chain in a single molecule (e.g., sterols). To clarify the effects of these two parts of a molecule, the phase behavior of a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer containing 4-n-alkyl-4'-cyanobiphenyl (nCB) (n = 0-8) was investigated. The trends of the main transition temperature (Tm) with respect to n and of the pretransition temperature (Tp) with respect to nCB content changed at n = 3. It was therefore suggested that the two parts of the molecule had opposing effects on the phase behavior of DPPC bilayers. The core appears to perturb molecular ordering in the gel phase and lowers Tm (like cholesterol), while alkyl chains appear to order the lipids in the gel phase and raise Tm (like n-alkanes). In addition, Tm exhibits the so-called odd-even effect based on the alkyl chain length of the minor component, nCB. Depending on the value of n, the variation in Tp was dependent on the additive content, although the pretransition was rarely observed at high contents.

Communication: Rigidification of a lipid bilayer by an incorporated n-alkane.

Towards a greater understanding of the effects of organic molecules in biomembranes, the effects of a flexible alkyl chain on the morphologies of phospholipid vesicles are investigated. Vesicles composed of 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine and tetradecane (TD) rupture during cooling from the liquid-crystalline phase to the gel phase. A model calculation based on the size-dependent rupture probability indicates that the bending rigidity of the bilayer in the gel phase is more than 10 times higher than that without TD, resulting in the rupture arising from elastic stress. The rigidification is caused by the denser molecular packing in the hydrophobic region by TD. There is little change of the rigidity in the liquid-crystalline phase. Additionally, the rigidification produces a characteristic morphology of the ternary giant vesicles including TD. Reported thermal behaviors imply that molecules with a linear and long alkyl chain, such as trans fatty acids, universally exhibit a similar effect, in contrast to rigid and bulky molecules, such as cholesterol.

A possible critical point for nematic order on the basis of Landau free energy having dual instabilities for nano-segregated smectic liquid crystals.

Landau expansion of free energy assuming dual instabilities for the nano-segregated SmA phase is analyzed. In addition to known phase sequences (on cooling, disordered isotropic liquid → nematic phase → smectic phase, and disordered isotropic liquid → smectic phase), a new sequence (disordered isotropic liquid → density wave with subsidiary nematic order → smectic phase) and the existence of a critical point are demonstrated in the case where the instability for density wave formation occurs at a higher temperature.

Communication: Salt-induced water orientation at a surface of non-ionic surfactant in relation to a mechanism of Hofmeister effect.

The behavior of water molecules at the surface of nonionic surfactant (monomyristolein) and effects of monovalent ions on the behavior are investigated using the heterodyne-detected vibrational sum frequency generation spectroscopy. It is found that water molecules at the surface are oriented with their hydrogen atoms pointing to the bulk, and that the degree of orientation depends on the anion strongly but weakly on the cation. With measured surface potentials in those saline solutions, it is concluded that the heterogeneous distribution of anions and cations in combination with the nonionic surfactant causes the water orientation. This heterogeneous distribution well explains the contrasting order of anions and cations with respect to the ion size in the Hofmeister series.

Salt effects on lamellar repeat distance depending on head groups of neutrally charged lipids.

Change in lamellar repeat distances of neutrally charged lipids upon addition of monovalent salts was measured with small-angle X-ray scattering for combinations of two lipids (PC and PE lipids) and six salts. Large dependence on lipid head group is observed in addition to those on added cation and anion. The ion and lipid dependences have little correlation with measured surface potentials of lipid membranes. These results indicate that the lamellar swelling by salt is not explained through balance among interactions considered previously (van der Waals interaction, electrostatic repulsion emerged by ion binding, etc.). It is suggested that effect of water structure, which is affected by not only ions but also lipid itself, should be taken into account for understanding membrane-membrane interactions, as in the Hofmeister effect.

Correlation between Hydration States and Self-assembly Structures of Phospholipid and Surfactant Studied by Terahertz Spectroscopy.

Phospholipids and surfactants form membranes and other self-assembled structures in water. However, it is not fully understood how the surrounding water (hydration water) is involved in their structure formation. In this paper, I summarize the results of our investigation of the long-range hydration state of phospholipids and surfactants at their surfaces by means of terahertz spectroscopy. By observing the collective rotational dynamics of water in the picosecond time scale, this technique allows us to observe not only the water directly bound to the solute, but also the weakly affected water outside of it. For example, PC phospholipids inhibit water dynamics over long distances, whereas PE phospholipids make water more mobile than bulk water. The causes of this difference in hydration and how it is involved in the structural formation of the membrane are reviewed.

Rotational Dynamics of Water near Osmolytes by Molecular Dynamics Simulations.

The behavior of water molecules around organic molecules has attracted considerable attention as a crucial factor influencing the properties and functions of soft matter and biomolecules. Recently, it has been suggested that the change in protein stability upon the addition of small organic molecules (osmolytes) is dominated by the change in the water dynamics caused by the osmolyte, where the dynamics of not only the directly interacting water molecules but also the long-range hydration layer affect the protein stability. However, the relation between the long-range structure of hydration water in various solutions and the water dynamics remains unclear at the molecular level. We performed density-functional tight-binding molecular dynamics simulations to elucidate the varying rotational dynamics of water molecules in 15 osmolyte solutions. A positive correlation was observed between the rotational relaxation time and our proposed normalized parameter obtained by dividing the number of hydrogen bonds between water molecules by the number of nearest-neighbor water molecules. For the 15 osmolyte solutions, an increase or a decrease in the value of the normalized parameter for the second hydration shell tended to result in water molecules with slow and fast rotational dynamics, respectively, thus illustrating the importance of the second hydration shell for the rotational dynamics of water molecules. Our simulation results are anticipated to advance the current understanding of water dynamics around organic molecules and the long-range structure of water molecules.

Reassessment of structure of smectic phases: nano-segregation in smectic E phase in 4-n-alkyl-4'-isothiocyanato-1,1'-biphenyls.

Based on new diffraction data from aligned samples of smectic E (SmE) phase of 4-n-alkyl-4'-isothiocyanato-1,1'-biphenyls, systematics against the alkyl chain length n is analyzed. In order to perform the analysis, the molecular form factor approximated by a box-shaped distribution is calculated while taking the rounding of the distribution at corners into account. The analysis clearly shows the nano-segregated layered structure, which does not fit to the traditional structural view of SmE phase but does fit to the model the present authors proposed recently. Some implications of this conclusion are discussed in relation to the importance of the molten state of alkyl chains in most of real mesogens revealed previously through thermodynamic analyses.

Contrasting Changes in Strongly and Weakly Bound Hydration Water of a Protein upon Denaturation.

Water is considered integral for the stabilization and function of proteins, which has recently attracted significant attention. However, the microscopic aspects of water ranging up to the second hydration shell, including strongly and weakly bound water at the sub-nanometer scale, are not yet well understood. Here, we combined terahertz spectroscopy, thermal measurements, and infrared spectroscopy to clarify how the strongly and weakly bound hydration water changes upon protein denaturation. With denaturation, that is, the exposure of hydrophobic groups in water and entanglement of hydrophilic groups, the number of strongly bound hydration water decreased, while the number of weakly bound hydration water increased. Even though the constraint of water due to hydrophobic hydration is weak, it extends to the second hydration shell as it is caused by the strengthening of hydrogen bonds between water molecules, which is likely the key microscopic mechanism for the destabilization of the native state due to hydration.

Quasi-elastic neutron scattering reveals the relationship between the dynamical behavior of phospholipid headgroups and hydration water.

The dynamics of hydration water (HW) in 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) was investigated by means of quasi-elastic neutron scattering (QENS) and compared with those observed in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The headgroup dynamics of DMPE was investigated using a mixture of tail-deuterated DMPE and D2O, and the QENS profiles were interpreted as consisting of three modes. The fast mode comprised the rotation of hydrogen atoms in -NH3+ and -CH2- groups in the headgroup of DMPE, the medium-speed mode comprised fluctuations in the entire DMPE molecule, and the slow mode comprised fluctuations in the membrane. These interpretations were confirmed using molecular dynamics (MD) simulations. The HW dynamics analysis was performed on a tail-deuterated DMPE and H2O mixture. The QENS profiles were analyzed in terms of three modes: (1) a slow mode, identified as loosely bound HW in the DMPC membrane; (2) a medium-speed mode similar to free HW in the DMPC membrane; and (3) a fast mode, identified as rotational motion. The relaxation time for the fast mode was approximately six times shorter than that of rotational water in DMPC, consistent with the results of terahertz time-domain spectroscopy. The activation energy of medium-speed HW in DMPE differed from that of free HW in DMPC, suggesting the presence of different hydration states or hydrogen-bonded networks around the phosphocholine and phosphoethanolamine headgroups.