Teruhiko Baba

Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers

The lipid membranes found in archaea have high bilayer stability and low permeability. The molecular structure of their constituent lipids is characterized by ether-linked, branched hydrophobic chains, whereas the conventional lipids obtained from eukaryotic or eubacterial sources have ester linked straight chains. In order to elucidate the influence of the ether linkage, instead of an ester one, on the physical properties of the lipid bilayers, we have carried out comparative 10 ns molecular dynamics simulations of diphytanyl phosphatidylcholine (ether-DPhPC) and diphytanoyl phosphatidylcholine (ester-DPhPC) bilayers in water, respectively. We analyze bilayer structures, hydration of the lipids, membrane dipole potentials, and free energy profiles of water and oxygen across the bilayers. We observe that the membrane dipole potential for the ether-DPhPC bilayer, which arises mainly from the ether linkage, is about half of that of the ester-DPhPC. The calculated free energy barrier for a water molecule in the ether-DPhPC bilayer system is slightly higher than that in the ester-DPhPC counterpart, which is in accord with experimental data.

Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids.

The formability, current-voltage characteristics and stability of the planar lipid bilayer membranes from the synthetic phytanyl-chained glycolipids, 1, 3-di-O-phytanyl-2-O-(beta-glycosyl)glycerols (Glc(Phyt)(2), Mal(N)(Phyt)(2)) were studied. The single bilayer membranes were successfully formed from the glycolipid bearing a maltotriosyl group (Mal(3)(Phyt)(2)) by the folding method among the synthetic glycolipids examined. The membrane conductance of Mal(3)(Phyt)(2) bilayers in 100 mM KCl solution was significantly lower than that of natural phospholipid, soybean phospholipids (SBPL) bilayers, and comparable to that of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers. From the permeation measurements of lipophilic ions through Mal(3)(Phyt)(2) and DPhPC bilayers, it could be presumed that the carbonyl groups in glycerol backbone of the lipid molecule are not necessarily required for the total dipole potential barrier against cations in Mal(3)(Phyt)(2) bilayer. The stability of Mal(3)(Phyt)(2) bilayers against long-term standing and external electric field change was rather high, compared with SBPL bilayers. Furthermore, a preliminary experiment over the functional incorporation of membrane proteins was demonstrated employing the channel proteins derived from octopus retina microvilli vesicles. The channel proteins were functionally incorporated into Mal(3)(Phyt)(2) bilayers in the presence of a negatively charged glycolipid. From these observations, synthetic phytanyl-chained glycolipid bilayers are promising materials for reconstitution and transport studies of membrane proteins.

Hydration and molecular motions in synthetic phytanyl-chained glycolipid vesicle membranes.

Proton permeation rates across membranes of a synthetic branch-chained glycolipid, 1,3-di-O-phytanyl-2-O-(beta-D-maltotriosyl)glycerol (Mal3(Phyt)2) as well as a branch-chained phospholipid, diphytanoylphosphatidylcholine (DPhPC) were lower than those of straight-chained lipids such as egg yolk phosphatidylcholine (EPC) by a factor of approximately 4 at pH 7.0 and 25 degrees C. To examine whether degrees of water penetration and molecular motions in Mal3(Phyt)2 membranes can account for the lower permeability, nanosecond time-resolved fluorescence spectroscopy was applied to various membranes of branch-chained lipids (Mal3(Phyt)2, DPhPC, and a tetraether lipid from an extremely thermoacidophilic archaeon Thermoplasma acidophilum), as well as straight-chained lipids (EPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and digalactosyldiacylglycerol (DGDG)) using several fluorescent lipids. Degrees of hydration of glycolipids, Mal3(Phyt)2, and DGDG were lower than those of phospholipids, EPC, POPC, and DPhPC at the membrane-water interfaces. DPhPC showed the highest hydration among the lipids examined. Meanwhile, rotational and lateral diffusive motions of the fluorescent phospholipid in branch-chained lipid membranes were more restricted than those in straight-chained ones. The results suggest that the restricted motion of chain segments rather than the lower hydration accounts for the lower proton permeability of branch-chained lipid membranes.

Formation of stable nanodiscs by bihelical apolipoprotein A-I mimetic peptide.

Nanodiscs are composed of scaffold protein or peptide such as apolipoprotein A‐I (apoA‐I) and phospholipids. Although peptide‐based nanodiscs have an advantage to modulate the size of nanodiscs by changing phospholipid/peptide ratios, they are usually less stable than apoA‐I‐based nanodiscs. In this study, we designed a novel nanodisc scaffold peptide (NSP) that has proline‐punctuated bihelical amphipathic structure based on apoA‐I mimetic peptides. NSP formed α‐helical structure on 1‐palmitoyl‐2‐oleoyl phosphatidylcholine (POPC) nanodiscs prepared by cholate dialysis method. Dynamic light scattering measurements demonstrated that diameters of NSP nanodiscs vary depending upon POPC/NSP ratios. Comparison of thermal unfolding of nanodiscs monitored by circular dichroism measurements demonstrated that NSP forms much more stable nanodiscs with POPC than monohelical peptide, 4F, exhibiting comparable stability to apoA‐I‐POPC nanodiscs. Intrinsic Trp fluorescence measurements showed that Trp residues of NSP exhibit more hydrophobic environment than that of 4 F on nanodiscs, suggesting the stronger interaction of NSP with phospholipids. Thus, the bihelical structure of NSP appears to increase the stability of nanodiscs because of the enhanced interaction of peptides with phospholipids. In addition, NSP as well as 4F spontaneously solubilized POPC vesicles into nanodiscs without using detergent. These results indicate that bihelical NSP forms nanodiscs with comparable stability to apoA‐I and has an ability to control the size of nanodiscs simply by changing phospholipid/peptide ratios. Copyright

Physicochemical Studies of Bacteriorhodopsin Reconstituted in Partially Fluorinated Phosphatidylcholine Bilayers

A membrane protein bacteriorhodopsin (bR) that is successfully reconstituted in liposome of a novel partially fluorinated analog of dimyristoylphosphatidylcholine (DMPC) with the perfluorobutyl segments in the myristoyl groups, diF4H10-PC, has been investigated by some spectroscopic and X-ray diffraction techniques to clarify effects of substitution of nine hydrogen atoms by fluorine atoms on structural and physical properties of the membrane protein by comparison with the previous results on proteoliposome of bR and DMPC. Below the gel-to-liquid crystalline phase transition of diF4H10-PC bilayer, bR molecules adopt the two-dimensional lattice structure of trimers as the structural unit and show a photocycle very similar to that of native purple membrane like reconstituted bR in DMPC liposome in the gel phase. Even upon heating up to temperatures well above the phase transition, the nativelike functional reconstitution and higher structural stability of bR molecules in diF4H10-PC liposome are retained, which strikingly contrasts with lipid phase transition-induced disaggregation of protein molecules and light-induced denaturation in DMPC liposome. Greater membrane rigidity and low affinity between bR and fluorinated lipid molecules are proposed as a driving force for keeping nativelike properties of bR molecules in diF4H10-PC liposome even in the fluid phase.

pH and salt-induced reversible aggregation of nonionic synthetic glycolipid vesicles

Abstract Salt and pH can induce reversible aggregation of vesicles composed of a nonionic synthetic glycolipid, 1,3-di-o-phytanyl-2-o-(β-maltotriosyl) glycerol. The aggregation of the vesicles appears reversible with respect to a change in the pH value of the medium as seen from the reversible change in the turbidity. It was also found that in an acidic region (pH 4–6), the size of the aggregated vesicles are well above 1000 nm, an indication of vesicle aggregation. But in an alkaline region (pH 8–10), the sizes are 110–130 nm, close to their original size of 100–110 nm, which strongly suggests the reversible disaggregation and also confirms the lack of vesicle fusion. The ζ-potentials of vesicles are measured in the presence of NaCl with the pH changes of the vesicle suspension. It is seen that the ζ-potential of vesicles changes with the pH value. The surface charges of the Mal3(Phyt)2 vesicles arise from two independent mechanisms; one is excess ‘adsorption’ of OH− ions at the vesicle–water interfaces and the other is dissociation of hydroxyl groups in a high pH region (pH >11). The changes of the surface charges are thought to be the major factor which induces the aggregation and disaggregation of this nonionic glycolipid vesicle.

Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes

Background: The N-terminal fragment of amyloidogenic apoA-I mutants deposits as fibrils by unknown mechanisms. Results: The G26R mutation partially prevents helix formation of the N-terminal fragment upon lipid binding, thereby facilitating β-transition and fibril formation. Conclusion: Membrane binding modulates fibril formation of apoA-I through partially destabilized helical conformation. Significance: The results reveal a new pathway for amyloid fibril formation by apoA-I. The N-terminal amino acid 1–83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1–83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8–33 and 8–33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1–83 fragment and 8–33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1–83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.

Effect of perfluoroalkyl chain length on monolayer behavior of partially fluorinated oleic acid molecules at the air-water interface.

A series of oleic acid (OA) analogs containing terminal perfluoroalkyl groups (CF3, C2F5, n-C3F7, n-C4F9 or n-C8F17) was synthesized to clarify how the fluorinated chain length affects the stability and molecular packing of liquid-expanded OA monolayers at the air-water interface. Although the substitution of terminal CF3 group for CH3 in OA had no effect on monolayer stability, further fluorination led to a gradual increase in monolayer stability at 25 °C. Surface pressure-area isotherm revealed that partially fluorinated OA analogs form more expanded monolayers than OA at low surface pressures, and that the monolayer behavior of OA analogs with the even-carbon numbered fluorinated chain is almost the same as that of OA upon monolayer compression, whereas the behavior of OA analogs with the odd-carbon numbered fluorinated chain significantly differs from that of OA. These results indicate: (i) the terminal short part (at least C2 residue) in OA predominantly determines the liquid-expanded monolayer stability; (ii) the molecular packing state of OA may be perturbed by the substitution of a short odd-carbon numbered fluorinated chain; (iii) hence, OA analogs with even-carbon numbered chain are considered to be preferable as hydrophobic building blocks for the synthesis of fluorinated phospholipids.

Heparin promotes fibril formation by the N-terminal fragment of amyloidogenic apolipoprotein A-I

Glycosaminoglycans are known to be associated with extracellular amyloid deposits of various amyloidogenic proteins. In this study, we found that the glycosaminoglycan heparin greatly accelerates the elongation step in fibril formation by the N‐terminal 1–83 fragment of human apolipoprotein A‐I (apoA‐I), especially in the amyloidogenic W50R variant. Using fragment peptides, we demonstrate that heparin significantly promotes β‐transition and fibril formation of the highly amyloidogenic region spanning residues 44–65 and colocalizes with fibrils formed by the W50R variant. These results suggest the possible role of glycosaminoglycans in fibril formation by amyloidogenic apoA‐I variants.

Effect of the fluorination degree of hydrophobic chains on the monolayer behavior of unsaturated diacylphosphatidylcholines bearing partially fluorinated 9-octadecynoyl (stearoloyl) groups at the air–water interface

The effect of the fluorination degree of hydrophobic chains on the monolayer behavior of unsaturated diacylphosphatidylcholines (PCs) was examined by employing a series of PCs bearing partially fluorinated 9-octadecynoyl (stearoloyl) groups (DFnStPCs, n: the number of fluorinated carbon atoms in a stearoloyl group; n=1, 2, 4, 8), including their hydrophobic parts--partially fluorinated stearolic acids (FnStAs)--at the air-water interface. π-A isotherm measurements and Brewster angle microscope observations revealed: (i) all DFnStPCs including FnStAs form monolayers of liquid character at 25 °C; (ii) they form more expanded monolayers than their non-fluorinated counterparts, distearoloyl-PC (DStPC) and stearolic acid, while the monolayer stability increases with n; (iii) compared with DStPC and DF8StPC, DFnStPCs (n=1, 2, 4) in the low-π region tend to show a weakening in their self-aggregation property and an increase in the work required for monolayer compression; (iv) although DF8StPC forms the most expanded monolayer, the behavior of DF8StPC resembles that of DStPC rather than that of DFnStPCs (n=1, 2, 4). The monolayer behavior of DFnStPCs (n=1, 2, 4) is explained by postulating a flatly-lying conformation of hydrophobic chains, in which three polar parts (ester group, triple bond, CF2-CH2 linkage) in chains are immersed in the subphase at large areas. DStPC and DF8StPC lacking a CF2-CH2 linkage, however, do not likely adopt such a conformation.