Comparison of functionality and structural stability of bacteriorhodopsin reconstituted in partially fluorinated dimyristoylphosphatidylcholine liposomes with different perfluoroalkyl chain lengths.

Amphiphilic molecules with one or more perfluoroalkyl groups (Rf, CnF2n+1), which show peculiar interfacial properties, are attracting much attention in membrane protein science. We recently have developed a partially fluorinated dimyristoylphosphatidylcholine (DMPC) with a perfluorobutyl group in the hydrophobic chain terminal (F4-DMPC) and demonstrated that F4-DMPC is a promising material for incorporating membrane proteins. Moreover, we have found out that membrane properties of a series of partially fluorinated DMPCs with different Rf chain lengths (Fn-DMPCs) vary in a significant Rf chain length-dependent manner. In the present study, structural and functional properties of a membrane protein bacteriorhodopsin (bR) in the Fn-DMPC (n = 4, 6, and 8) membranes (bR/Fn-DMPC) are investigated using several physicochemical techniques. Regardless of the Rf chain lengths, bR/Fn-DMPCs retain native-like structural and functional properties at 30 °C, unlike bR molecules in DMPC vesicles. In particular, bR/F6-DMPC, which is in the fluid phase at 30 °C, shows flash-induced transient absorption changes very similar to the native purple membrane (PM) and very high thermal stability of bR trimers comparable to the PM. Structural and functional properties of bR/Fn-DMPCs are discussed compared to the PM and bR/DMPC.

Regular-triangle trimer and charge order preserving the Anderson condition in the pyrochlore structure of CsW2O6.

Since the discovery of the Verwey transition in magnetite, transition metal compounds with pyrochlore structures have been intensively studied as a platform for realizing remarkable electronic phase transitions. We report on a phase transition that preserves the cubic symmetry of the ß-pyrochlore oxide CsW2O6, where each of W 5d electrons are confined in regular-triangle W3 trimers. This trimer formation represents the self-organization of 5d electrons, which can be resolved into a charge order satisfying the Anderson condition in a nontrivial way, orbital order caused by the distortion of WO6 octahedra, and the formation of a spin-singlet pair in a regular-triangle trimer. An electronic instability due to the unusual three-dimensional nesting of Fermi surfaces and the strong correlations of the 5d electrons characteristic of the pyrochlore oxides are both likely to play important roles in this charge-orbital-spin coupled phenomenon.

Giant isotropic negative thermal expansion in Y-doped samarium monosulfides by intra-atomic charge transfer.

Stimulated by strong demand for thermal expansion control from advanced modern industries, various giant negative thermal expansion (NTE) materials have been developed during the last decade. Nevertheless, most such materials exhibit anisotropic thermal expansion in the crystal lattice. Therefore, strains and cracks induced during repeated thermal cycling degrade their performance as thermal-expansion compensators. Here we achieved giant isotropic NTE with volume change exceeding 3%, up to 4.1%, via control of the electronic configuration in Sm atoms of SmS, (4 f)6 or (4 f)5(5d)1, by partial replacement of Sm with Y. Contrary to NTE originating from cooperative phenomena such as magnetism, the present NTE attributable to the intra-atomic phenomenon avoids the size effect of NTE and therefore provides us with fine-grained thermal-expansion compensators, which are strongly desired to control thermal expansion of microregions such as underfill of a three-dimensional integrated circuit. Volume control of lanthanide monosulfides via tuning of the 4 f electronic configuration presents avenues for novel mechanical functions of a material, such as a volume-change driven actuator by an electrical field, which has a different drive principle from those of conventional strain-driven actuators such as piezostrictive or magnetostrictive materials.

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers.

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.

Structural change of bacteriorhodopsin in the purple membrane above pH 10 decreases heterogeneity of the irreversible photobleaching components.

Kinetic investigations of irreversible photobleaching of bacteriorhodopsin (bR) in purple membrane (PM) at high temperature have previously shown two kinds of bR species upon light illumination. The bR species consist of kinetically fast- and slow-denatured components, whose proportions were dependent upon structural changes in dark, as shown by CD. In order to elucidate electrostatic contribution on the heterogeneous stability and the bR structure in PM, photobleaching behaviour and structural changes over a wide pH range were investigated by kinetics as well as various spectroscopic techniques. Kinetics revealed that photobleaching below pH 9 obeyed double-exponential functions, whereas measurements above pH 10 were characterized by a single-decay component. FT-IR deconvoluted spectra showed a alpha(II)-to-alpha(I) transition in the transmembrane helices around pH 10. Near-IR Raman scattering spectra demonstrated the equilibrium shift of retinal isomers from all trans to 13-cis form. Near-UV CD spectra suggested configurational changes in the aromatic residues around the retinal pocket. An exciton-to-positive transition in visible CD spectrum was observed. This indicates disorganization in the 2D-crystalline lattice of PM, which occurred concomitantly with the changes above pH 10. A model for the changes in kinetic behaviour and molecular structure around pH 10 is discussed, focusing on changes in charge distribution upon alkalinization.

Inhomogeneous stability of bacteriorhodopsin in purple membrane against photobleaching at high temperature.

Heterogeneity in the state of bacteriorhodopsin in purple membrane was studied through temperature jump experiments carried out in darkness and under illumination with visible light. The thermal denaturation, the irreversible component of spectral change at high temperature, had two decay components, suggesting that bacteriorhodopsin in purple membrane has heterogeneous stability. The temperature dependence of kinetic parameters under illumination revealed that the fast-decay component gradually increased at above 60 degrees C, indicating that the proportion of unstable bacteriorhodopsin increased. Significant change in the visible circular dichroism (CD) spectra was observed in darkness in the same temperature range as the increase of the fast-decay component under illumination. Denaturation experiments for C-terminal-cleaved bacteriorhodopsin showed that the C-terminal segment had some effect on the structural stability of bacteriorhodopsin under illumination. Dynamic and static models of the inhomogeneous stability of bacteriorhodopsin in purple membrane are discussed on the basis of the results of the denaturation kinetics and the visible CD spectra.

Irreversible photobleaching of bacteriorhodopsin in a high-temperature intermediate state.

The photo-intermediate state of bacteriorhodopsin is a metastable state that spontaneously transforms to the ground state over the energy barrier of a local minimum. As the recovery of the photocycle to the ground state and irreversible photobleaching to the denatured state may occur from the same local energy minimum, depending on the temperature, the structural stability of bacteriorhodopsin under illumination at high temperature was measured in order to study the intra- and inter-molecular interactions that contribute to the recovery of the ground state. Visible CD spectra of bacteriorhodopsin began to change at 60 degrees C from a bilobed to positive type in accordance with an appearance of an absorption peak at 470 nm. Irreversible photobleaching, the light-induced denaturation, also started to occur at 60 degrees C, suggesting some correlation between irreversible photobleaching and the structural change to the high-temperature intermediate state. However, bacteriorhodopsin in the dark was stable up to 70 degrees C, suggesting that there is some additional factor that lends structural stability to bacteriorhodopsin in the dark. The contribution of protein-protein interactions to stability is discussed on the basis of the difference in the denaturation behaviors between light and dark conditions.

Dependence of purple membrane bump curvature on pH and ionic strength analyzed using atomic force microscopy combined with solvent exchange.

Purple membrane (PM), which is a membrane patch formed by the self-assembly of the membrane protein bacteriorhodopsin (bR) with archaeal lipids, is a good subject for studying the mechanism for the supramolecular structural formation of membrane proteins. Several studies have suggested that PM is not simply planar but that it has a curvature. Atomic force microscopy (AFM) studies also indicate the presence of dome-like structures (bumps) on the cytoplasmic surface of PM. PM must have a curvature to form the bump structures; therefore, bump formations will be related to a mechanism for supramolecular structural formation via self-assembly. To elucidate the effect of an asymmetric distribution of charged residues between two aqueous domains on the bump curvature, AFM topography of identical PM sheets were examined with variation of the solvent ionic strength and pH using a newly constructed solvent circulation system. The radius and height distributions of the bumps on the identical PM sheets indicated a linear correlation. The bump curvature, which was simply estimated by the slope of the distribution, became smaller with increasing KCl concentration, which suggests that tension at the cytoplasmic surface caused by electrostatic repulsive force between negatively charged amino acid residues becomes weaker by the electrostatic shielding effect. AFM observations revealed that the bump curvature remained even at high KCl concentration where the Debye length is within a few Angstroms; therefore, the contribution of the intrinsic difference between the domain sizes of bR between two sides was confirmed. Interestingly, the bump curvature was significantly increased by the addition of CaCl2 and then decreased with a similar dependency to KCl at higher CaCl2 concentration. The effect of pH on the bump curvature was also examined, where the curvature increased and reached a maximum at pH 9, while it decreased above pH 10, at which point the two-dimensional crystalline lattice of bR began to disassemble. These experimental results indicate that the bump curvature is strongly influenced by electrostatic interactions. A plausible model for bump structure formation by electrostatic repulsive force is presented based on these results.

Multiple membrane interactions and versatile vesicle deformations elicited by melittin.

Melittin induces various reactions in membranes and has been widely studied as a model for membrane-interacting peptide; however, the mechanism whereby melittin elicits its effects remains unclear. Here, we observed melittin-induced changes in individual giant liposomes using direct real-time imaging by dark-field optical microscopy, and the mechanisms involved were correlated with results obtained using circular dichroism, cosedimentation, fluorescence quenching of tryptophan residues, and electron microscopy. Depending on the concentration of negatively charged phospholipids in the membrane and the molecular ratio between lipid and melittin, melittin induced the "increasing membrane area", "phased shrinkage", or "solubilization" of liposomes. In phased shrinkage, liposomes formed small particles on their surface and rapidly decreased in size. Under conditions in which the increasing membrane area, phased shrinkage, or solubilization were mainly observed, the secondary structure of melittin was primarily estimated as an α-helix, ß-like, or disordered structure, respectively. When the increasing membrane area or phased shrinkage occurred, almost all melittin was bound to the membranes and reached more hydrophobic regions of the membranes than when solubilization occurred. These results indicate that the various effects of melittin result from its ability to adopt various structures and membrane-binding states depending on the conditions.

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.

Structural changes in bacteriorhodopsin in purple membranes induced by irreversible photobleaching with heterogeneous and homogeneous stability.

Kinetic studies of irreversible photobleaching of bacteriorhodopsin (bR) in purple membrane (PM) at neutral pH have previously indicated the existence of two kinds of species which differ in their structural stability. bR was shown to have kinetically slow- and fast-decayed components with the faster one increasing with changes in intra- and intermolecular structures in darkness. However, our recent work reported that photobleaching kinetics above pH 10 were characterized by a single-decay component. In order to elucidate the factors responsible for the heterogeneous or homogeneous stability of photobleaching, we conducted investigations into the structural changes in bR in PM induced by photobleaching at pH 7 and 11 by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. ATR-FTIR spectra of bR photobleached at pH 7 and 11 showed that an increase in IR peak intensity at 1656 cm(-1) occurred simultaneously with decreases at 1666 cm(-1), indicating an alpha(II)-to-alpha(I) transition in transmembrane helices during photobleaching. The most significant change in IR spectra occurred at 1626 cm(-1) for samples photobleached at pH 7, and was attributed to structures formed between adjacent molecules. The origin of the heterogeneity of photobleaching is discussed on the basis of structural characteristics found in the bleached membranes.

Effect of lipid phase transition on molecular assembly and structural stability of bacteriorhodopsin reconstituted into phosphatidylcholine liposomes with different acyl-chain lengths.

Previous studies on the correlation between bacteriorhodopsin (bR) disassembly and photobleaching suggested that a weakening of intermolecular interactions is responsible for irreversible photobleaching (Mukai, Y.; Kamo, N.; Mitaku, S. Protein Eng. 1999, 12, 755-759; Yokoyama, Y.; Sonoyama, M.; Mitaku, S. J. Biochem. 2002, 131, 785-790). In order to reveal the role of the lipid matrix in bR assembly and photobleaching, we reconstituted bR into diacylphosphatidylcholine (diacylPC) vesicles with three different saturated acyl-chain lengths. Visible circular dichroism (CD) spectra collected upon photobleaching showed an exciton-to-positive transition for bR reconstituted into dimyristoyl-, dipalmitoyl-, and distearoyl-PC vesicles around 17, 35, and 50 °C, respectively. These transition temperatures were close to the main transition temperature of reconstituted vesicles measured by calorimetry, indicating that the lipid phase transition brought about protein disaggregation. Absorption spectra of reconstituted bR exhibited a blue-shifted retinal absorption during protein disaggregation in the ground state. Absorption spectra collected from samples exposed to continuous illumination revealed an accumulation of M-intermediate state, and the absorption band around 410 nm underwent a blue shift through the visible CD change, indicating conformational perturbations due to protein disassembly. Irreversible photobleaching started to occur at the same temperature range as the change in the visible CD spectrum, clarifying the correlation between bR disassembly and photobleaching. In contrast, no thermal bleaching was observed below 60 °C for any sample kept in the dark. A plausible model for irreversible photobleaching is presented, on the basis of these experimental results.