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The interaction of proteins with hydrophobic ligands in biological membranes is an important research topic in the life sciences. The hydrophobic nature of ligands, especially their lack of water solubility, often makes it difficult to experimentally investigate their interactions with proteins, thus hampering quantitative evaluation based on thermodynamic parameters. The fatty acid-binding proteins, particularly FABP3, discussed in this review can recognize fatty acids, a primary component of membrane lipids, with high affinity. The precise three-dimensional structure of fatty acids and related ligands bound in FABP3 and their interaction with the binding pocket will contribute to the understanding of accurately determining physicochemical factors that cause the expression of affinity between protein surfaces and lipids in biological membranes. During the research of FABP3, we encountered many of the problems that were widely implicated in experiments dealing with hydrophobic ligands. To address these issues, we developed experimental methodologies using X-ray crystallography, calorimetry, and surface plasmon resonance. Using these methods and computational approaches, we have obtained several insights into the interaction of hydrophobic ligands with protein binding sites. Structural and functional studies of FABP potentially lead to a better understanding of the interaction between lipids and proteins, and thus, this protein may provide one of the model systems for investigating substance transport across cell membranes and inner membrane systems.

A comparative study on the thermotropic transition of phospholipid bilayer membrane is carried out for a series of novel partially fluorinated dimyristoylphosphatidylcholines with different perfluo...

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.

Perfluoroalkyl (Rf) chains have a specific helical conformation due to the steric repulsion between the adjacent CF2 units. Although Rf chains have no chiral center, two chiral structures, i.e., the right-handed (R) and left-handed (L) helices, are available as the most stable conformations, which are atropisomers to each other. According to the stratified dipole array (SDA) theory, the helical structure about the chain axis plays a key role in the spontaneous molecular aggregation of Rf chains in a two-dimensional manner, and the Rf chains having the same chirality tend to be aggregated spontaneously to generate molecular domains. This implies that an Rf compound in a solid state should be a mixture of the R and L domains, and each domain should exhibit distinguishable optical activity. To identify molecular domains with different atropisomers, in this study, Raman optical activity (ROA) measurements were performed on a Raman imaging spectrometer. Through the ROA measurements of recrystallized solid samp...

Fluorous properties represented by water-and-oil repellency are perfluoroalkyl (Rf) compound-specific characteristics, which are widely used for surface coating of glass, electronic devices and textiles for preventing water and grease fouling. According to the stratified dipole-arrays (SDA) theory, the minimum Rf length of (CF₂)₇ is theoretically necessary for realizing fluorous properties. Unfortunately, however, production of compounds involving this chemical unit is strictly banned because of concerns of environmental pollution, which is a big dilemma. Here, we show that the fluorous properties can be realized by self-assembled monolayer (SAM) even with a short Rf-containing compound, since the SAM technique makes the best use of the self-aggregation property of the Rf groups, and it readily makes the molecules immobile.

3-Methyl-4-nitrophenol (3M4NP) is formed in soil as a hydrolysis product of fenitrothion, one of the major organophosphorus pesticides. A Pseudomonas strain was isolated as a 3M4NP degrader from a crop soil and designated TSN1. This strain utilized 3M4NP as a sole carbon and energy source. To elucidate the biodegradation pathway, we performed transposon mutagenesis with pCro2a (mini-Tn5495) and obtained three mutants accumulating a dark pink compound(s) from 3M4NP. Rescue cloning and sequence analysis revealed that in all mutants, the transposon disrupted an identical aromatic compound meta-cleaving dioxygenase gene, and a monooxygenase gene was located just downstream of the dioxygenase gene. These two genes were designated mnpC and mnpB, respectively. The gene products showed high identity with the methylhydroquinone (MHQ) monooxygenase (58%) and the 3-methylcatechol 2,3-dioxygenase (54%) of a different 3M4NP degrader Burkholderia sp. NF100. The transposon mutants converted 3M4NP or MHQ into two identical metabolites, one of which was identified as 2-hydroxy-5-methyl-1,4-benzoquinone (2H5MBQ) by GC/MS analysis. Furthermore, two additional genes (named mnpA1 and mnpA2), almost identical to the p-nitrophenol monooxygenase and the p-benzoquinone reductase genes of Pseudomonas sp. WBC-3, were isolated from the total DNA of strain TSN1. Disruption of mnpA1 resulted in the complete loss of the 3M4NP degradation activity, demonstrating that mnpA1 encodes the initial monooxygenase for 3M4NP degradation. The purified mnpA2 gene product could efficiently reduce methyl p-benzoquinone (MBQ) into MHQ. These results suggest that strain TSN1 degrades 3M4NP via MBQ, MHQ, and 2H5MBQ in combination with mnpA1A2 and mnpCB, existing at different loci on the genome.

Pore size distributions in hydrogels immobilizing purple membrane (PM) are elucidated by focusing on a relationship with PM stacking. PM stacking, which is useful for engineering applications, occurred spontaneously in porous hydrogels. Recently, it is suggested that porous gel network generations are essential for PM stacking. Here, to clarify a relationship with pore size, optical microscopic observations for hydrogels immobilizing PM were carried out. The results showed that PM stacking occurred at porous gels with a larger pore size than PM long axis length, while it did not occur in the smaller pores. These strongly suggest the appropriate pore size for PM stacking.

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.

The CF2 symmetric stretching vibration (νs(CF2)) band of a perfluoroalkyl (Rf) group in an infrared (IR) spectrum exhibits a unique character, i.e., an apparent high wavenumber shift with increasing the chain length, which is an opposite character to that of the CH stretching vibration band of a normal alkyl chain. To reveal the mechanism of the unusual IR band shift, two vibrational characters of an Rf chain are focused: (1) a helical conformation of an Rf chain, (2) the carbon (C) atoms having a smaller mass than the fluorine (F) atom dominantly vibrate as a coupled oscillator leaving F atoms stay relatively unmoved. These indicate that a ‘coupled oscillation of the skeletal C atoms’ of an Rf chain should be investigated considering the helical structure. In the present study, therefore, the coupled oscillation of the Rf chain dependent on the chain length is investigated by Raman spectroscopy, which is suitable for investigating a skeletal vibration. The Raman active νs(CF2) band is found to be split into two bands, and the splitting is readily explained by considering the helical structure and length with respect to group theory, and the unusual IR peak shift is concluded to be explained by the helical length.

Fluorinated surfactants (Shepherd and Holzenburg 1995; Popot 2010; Frotscher et al. 2015) and phospholipids (Santaella et al. 1991; Yoder et al. 2007; Takai et al. 2008; Gagnon et al. 2018) are now attracting more and more attention in biophysical fields because the incorporation of fluorine atoms provides distinctive physical properties to amphiphilic molecules, which would open up a new way of structural and functional characterization of membrane proteins (MPs). In particular, a novel partially fluorinated analog of dimyristoylphosphatidylcholine (DMPC), F4-DMPC (Fig. 1, m = 9; diF4H10-PC was used as the former abbreviation in the previous papers) (Yoshino et al. 2012; Takahashi et al. 2013; Okamoto et al. 2016), is of great promise for biophysical studies of MPs. Bacteriorhodopsin (bR) molecules in the F4-DMPC bilayer membrane form the native purple membrane (PM)–like two-dimensional crystals of trimers as the structural unit in the liquid-crystalline phase as well as in the gel phase (Yoshino et al. 2013; Takahashi et al. 2019), which shows a striking contrast with phase transition–induced disassembly of bR trimers to monomers upon reconstitution into vesicles of the corresponding hydrogenated DMPC (Cherry et al. 1978; Heyn et al. 1981; Sonoyama et al. 2009; Yokoyama et al. 2010). As a further attempt to extend the novel partially fluorinated phosphatidylcholine (PF-PC) F4-DMPC to phospholipids with longer acyl chains, we have recently developed a novel analog of dipalmitoylphosphatidylcholine (DPPC) with the perfluorobutyl group like F4-DMPC, F4-DPPC (Fig. ​(Fig.1,1, m = 11). In this short report, comparison of F4-DPPC with F4-DMPC on structural and physical properties of the phospholipid bilayer membrane and reconstituted bR is discussed. Open in a separate window Fig. 1 Chemical structure of F4-DMPC (m = 9) and F4-DPPC (m = 11)

The plasma membranes of archaea are abundant in macrocyclic tetraether lipids that contain a single or double long transmembrane hydrocarbon chains connecting the two glycerol backbones at both ends. In this study, a novel amacrocyclic bisphosphatidylcholine lipid bearing a single membrane-spanning octacosamethylene chain, 1,1’-O-octacosamethylene-2,2′-di-O-tetradecyl-bis-(sn-glycero)-3,3′-diphosphocholine (AC-(di-O-C14PC)2), was synthesized to elucidate effects of the interlayer cross-linkage on membrane properties based on comparison with its corresponding diether phosphatidylcholine, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DTPC), that forms bilayer membrane. Several physicochemical techniques demonstrated that while AC-(di-O-C14PC)2 monolayer, which adopts a particularly high-ordered structure in the gel phase, shows remarkably high thermotropic transition temperature compared to DTPC bilayer, the fluidity of both phospholipids above the transition temperature is comparable. Nonetheless, the fluorescent dye leakage from inside the AC-(di-O-C14PC)2 vesicles in the fluid phase is highly suppressed. The origin of the membrane properties characteristic of AC-(di-O-C14PC)2 monolayer is discussed in terms of the single long transmembrane hydrophobic linkage and the diffusional motion of the lipid molecules.

The effects of a gel network structure on purple membrane (PM) spontaneous stacking are discussed, with particular emphasis on the relationship between the PM periodic pre-structures before gelation. Such peculiar intermembrane pre-structures with a periodic distance of ∼20 nm have recently been found to provide an important key to PM stacking, which is critical for the engineering applications of PM. Here, we investigated how the PM stacking differs depending on the gel network of a poly(vinyl alcohol) gel. On the basis of the results, we propose a plausible interplay between the PM periodic pre-structures and the type of gel network for PM stacking. The results indicate that a porous gel network, in addition to the PM periodic pre-structure in solutions, is necessary for PM stacking in immobilized gel samples.

Perfluoroalkyl compounds are known to exhibit a hydrophobic character on the surface of the material, although the CF bond has a large dipole, which should make the molecular surface polar and hydrophilic. This inconsistency has long been a chemical matter to be solved. Herein, a stratified dipole-arrays model is proposed: the molecular polar surface can be fully hidden by forming a two-dimensional aggregate of perfluoroalkyl (Rf) groups; this aggregate is spontaneously induced by dipole–dipole interaction arrays owing to the helical structure of the Rf group. In this model, a ‘short’ Rf group should play the role of a single Rf group with a hydrophilic character, whereas a ‘long’ Rf group should spontaneously form a hexagonal aggregate. To examine this model, Rf-containing myristic acids with various Rf lengths have been synthesized and their aggregation properties are analyzed by using the Langmuir monolayer technique aided by precise IR spectroscopic analysis.

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.

Perfluoroalkyl (Rf) compounds containing a normal alkyl group often yield highly complicated infrared (IR) spectra especially in the C–F stretching vibration (νC–F) region. To reveal the reason behind this, the IR p-polarized multiple-angle incidence resolution spectrometry (pMAIRS) is employed to measure a monolayer of CF3(CF2)9(CH2)3COOH deposited on a silicon substrate. The compound is known to spontaneously aggregate to form a molecular assembly with the closest packing, in which the molecules are oriented perpendicular to the substrate. The IR pMAIRS spectra apparently prove that the complexity of the νC–F region is due to the normal alkyl part directly connected to the Rf group because the carbons in the Rf group are vibrated as a coupled oscillator, and the oscillation of the alkyl part propagates to the Rf part along the molecular axis.

The mixing behavior of hydrated dimyristoylphosphatidylcholine and its partially fluorinated analogue, di(nonafluorotetradecanoyl)phosphatidylcholine, where all hydrogens of the terminal n -butyl segments in two hydrophobic chains are replaced by fluorines, has been investigated by calorimetry and X-ray diffraction. The analysis of the calorimetric data by using the regular solution theory has revealed that the phase diagram of the mixtures is a eutectic type with a miscibility gap. The non-ideality parameters of the gel and liquid-crystalline phases are estimated to be 5.7 ± 0.1 and 4.2 ± 0.1 kJ/mol, respectively. These large values indicate that the mixing is significantly non-ideal. X-ray diffraction has shown that a phase separation occurs in the miscibility gap.

Fluorinated lipids and surfactants are attractive biomimetic materials for the extraction and reorganization of membrane proteins because of the biological inertness of fluorocarbons. We investigated the fundamental physical properties of a partially fluorinated phospholipid (F4-DMPC), such as phase transition, area thermal expansion, and lateral lipid diffusion, to evaluate the intermolecular interaction of F4-DMPC in the hydrophobic region quantitatively on the basis of free-volume theory. Fluorescence microscope observation of the supported lipid bilayer (SLB) of F4-DMPC showed that the phase transition between the liquid crystalline and gel phases occurred at 5 °C and that the area thermal expansion coefficient was independent of the temperature near the phase transition temperature. We performed a single particle tracking of the F4-DMPC-SLB on a SiO2/Si substrate, to measure the diffusion coefficient and its temperature dependence. The apparent activation energy (E′a) of lateral lipid diffusion, whic...

A discriminative study of a single perfluoroalkyl (Rf) group from a bulk material is recently recognized to be necessary toward the total understanding of Rf compounds based on a primary chemical structure. The single molecule and the bulk matter have an interrelationship via an intrinsic two-dimensional (2D) aggregation property of an Rf group, which is theorized by the stratified dipole-arrays (SDA) theory. Since an Rf group has dipole moments along many C–F bonds, a single Rf group would possess a hydrophilic-like character on the surface. To reveal the hydration character of a single Rf group, in the present study, surface potential (ΔV) measurements are performed for Langmuir monolayers of Rf-containing compounds. From a comparative study with a monolayer of a normal hydrocarbon compound, the hydration/dehydration dynamics of a lying Rf group on water has first been monitored by ΔV measurements, through which a single Rf group has been revealed to have a unique “dipole-interactive” character, which enables the Rf group interacted with the water ‘surface.’ In addition, the SDA theory proves to be useful to predict the 2D aggregation property across the phase transition temperature of 19 °C by use of the ΔV measurements.

Extended proteins such as calmodulin and troponin C have two globular terminal domains linked by a central region that is exposed to water and often acts as a function-regulating element. The mechanisms that stabilize the tertiary structure of extended proteins appear to differ greatly from those of globular proteins. Identifying such differences in physical properties of amino acid sequences between extended proteins and globular proteins can provide clues useful for identification of extended proteins from complete genomes including orphan sequences. In the present study, we examined the structure and amino acid sequence of extended proteins. We found that extended proteins have a large net electric charge, high charge density, and an even balance of charge between the terminal domains, indicating that electrostatic interaction is a dominant factor in stabilization of extended proteins. Additionally, the central domain exposed to water contained many amphiphilic residues. Extended proteins can be identified from these physical properties of the tertiary structure, which can be deduced from the amino acid sequence. Analysis of physical properties of amino acid sequences can provide clues to the mechanism of protein folding. Also, structural changes in extended proteins may be caused by formation of molecular complexes. Long-range effects of electrostatic interactions also appear to play important roles in structural changes of extended proteins.

The flagellar filament of Salmonella is an assembly of a single protein, flagellin. There are different helical and straight shape filaments from wild type and mutants. Raman and Raman optical activity (ROA) spectroscopies were employed to characterize the structure of flagellar filaments. The Raman spectra of normal and two straight filaments were almost same, but in the ROA spectra, intensive ROA peaks at amide I, amide III and skeletal vibrational regions were observed from only one of two straight filaments, L-type straight filaments. When the filaments were depolymerized or fragmented, the ROA bands disappeared on the scale of the large ROA intensities. It is thought that the ROA bands of L-type flagellar filaments are related to their shape, length and possible higher level chiral organization.

All amino acid sequences derived from 248 prokaryotic genomes, 10 invertebrate genomes (plants and fungi) and 10 vertebrate genomes were analysed by the autocorrelation function of charge sequences. The analysis of the total amino acid sequences derived from the 268 biological genomes showed that a significant periodicity of 28 residues is observable for the vertebrate genomes, but not for the other genomes. When proteins with a charge periodicity of 28 residues (PCP28) were selected from the total proteomes, we found that PCP28 in fact exists in all proteomes, but the number of PCP28 is much larger for the vertebrate proteomes than for the other proteomes. Although excess PCP28 in the vertebrate proteomes are only poorly characterized, a detailed inspection of the databases suggests that most excess PCP28 are nuclear proteins.

G-protein coupled receptors (GPCRs) bind with G-proteins upon activation by ligands. Understanding the mechanisms of specific binding between GPCRs and G-proteins is one of the most important issues in bioinformatics research. In this study, the physical properties of various regions were analyzed in order to classify GPCRs by G-protein family and to better understand binding specificity. We focused on cytoplasmic loops (IL1, IL2 and N/C-terminus of IL3), extracellular loops (NTL, EL1 and N/C-terminus of EL2) and cytoplasmic termini of transmembrane helices, except for helices that connect to C-terminus loops. The distribution of hydrophobicity, charge density, lysine and arginine densities, and loop length enabled discrimination of GPCRs with more than 90% accuracy.

The human genome includes more than 36,000 open reading frames that are translated to amino acid sequences of proteins. When the charge distribution in amino acid sequences from the total human genome was analyzed by the autocorrelation function, a surprisingly sharp periodicity of 28 residues was observed. Every protein with the charge periodicity of 28 residues (PCP28) could be discriminated by a simple algorithm, and the number of PCP28 amounted to about 3% of the total open reading frames of the human genome. The net charge of most PCP28 was highly positive. The possible structural and functional features of this type of protein were discussed in terms of the electric repulsion within molecules.

Proteins with a charge periodicity of 28 residues (PCP28) were found recently in the human proteome, and many of the annotated PCP28 were located in the nucleus (Ke et al., Jpn. J.Appl.Phys. 2007). The physical properties of the amino acid sequences were analyzed to detect the difference in the physicochemistry between the nuclear and cytoplasmic PCP28 and develop a software system to classify the two types of PCP28. A significant difference in the global parameters from the entire sequence and the local parameters around a segment with the highest positive charge density was found between the nuclear and cytoplasmic PCP28. The global classification score included the densities of proline and cysteine, and the nagative charge density, while the local score included the symmetry of the charge distribution, the density of cysteine, and the positive charge density. A prediction system was developed using the global and local scores, which possessed a sensitivity and specificity of 92％ and 88％, respectively. The mechanism of translocation of proteins to the nucleus is discussed using the parameters relevant to the predictive system., ヒトプロテオームにおいて電荷の28残基周期性を持ったタンパク質（PCP28)が発見され、PCP28の多くは核に局在していることが分かった（Ke et al., Jpn. J. Allp. Phys.2007)。核PCP28と細胞質PCP28のアミノ酸配列の物理化学的性質を解析し、物性の差からそれら二つのPCP28を分類するシステムを開発した。核PCP28と細胞質PCP28の間で、配列全体からのグローバルパラメータと高い電荷密度を持つセグメント周辺のローカルパラメータにおいて大きな違いが見られた。グローバルスコアではプロリン密度、システイン密度、負電荷密度に差があり、ローカルスコアでは電荷分布のシンメトリ、システイン密度、正電荷密度に差が見られた。これら二つのスコアを用いて予測システムを開発した結果、予測精度は感度が92％、特異性が97％であった。また、核へのタンパク質の輸送メカニズムを、予測システムに関連するパラメータから議論する。

More than 36,000 open reading frames (ORFs) from the human genome were previously analyzed by the autocorrelation function of electric charge distribution, revealing the existence of many proteins with a charge periodicity of 28 residues (PCP28) (Ke et al., Jpn. J. Appl. Phys. 2007). The major component of PCP28 was located in the nucleus, and the nuclear PCP28 of ten vertebrate and seven invertebrate organisms were predicted with a novel software system (Sakiyama et al., CBI Journal 2007) for revealing the biological significance of nuclear PCP28. Retrieval of the features of the human nuclear PCP28 in Swiss-Prot revealed that almost 90% of nuclear PCP28 functions in transcriptional regulation, including hypothetical transcription factors. To study how nuclear PCP28 is increased in eukaryote genomes, we compared the number of all nuclear PCP28 in vertebrate and invertebrate genomes. The results showed that nuclear PCP28 is specifically increased in vertebrate genomes and that the ratio of other types of PCP28 is almost constant in all eukaryote genomes. These findings strongly suggest that nuclear PCP28 is an essential protein for vertebrate organisms.

Reconstituted membranes with diverse diacylphospholipids were prepared by using bacteriorhodopsin (bR) in which the intrinsic lipid content was decreased to 24% of the original while the trimeric structure and photocycle of bR were retained. Four phospholipids with a different headgroup, phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylserine (PS), were adopted for reconstitution. By varying the lipid-protein ratios, the interactions of these phospholipids with bR, as a boundary lipid, were evaluated by solid state (2)H/(31)P NMR, circular dichroism (CD), and laser-flash photolysis. The (31)P NMR results revealed that the headgroup of acidic phosphatidylglycerol (PG) interacts more strongly with bR than that of phosphatidylcholine (PC). CD analysis indicated that the trimetric structure of bR was retained in all the phospholipid-bR preparations at low and medium lipid contents. Acidic lipids PA, PG and PS restored the photocycle activity of bR to an extent comparable to (or slightly lower than) that of the purple membrane while PC caused a marked reduction of the bR photocycle efficiency. Among PGs with different fatty acyl groups, those with mono- and di-unsaturated lipids tended to preserve the photocycle efficiency, whereas the fully saturated lipid did not. These results show that acidic unsaturated phospholipids, particularly dioleoylphosphatidylglycerol (DOPG), have higher affinity for bR and efficiently restore its trimetric structure. The present study suggests that bR reconstituted in DOPG bilayers may possibly be used as a model system for spectroscopic investigations of the lipid-bR interactions with the membrane-integral α-helices, and potentially for a similar type of membrane proteins.

The aggregation and reaction of 17,19-hexatriacontadiyne molecules are studied on a Au(111) surface. The molecular orientation and arrangement are elucidated by infrared reflection absorption spectroscopy (IRAS) and scanning tunneling microscopy (STM). A vapor-deposited monolayer and a multilayered film formed by adsorption from the solution provide IRA spectra with bands due to the antisymmetric and symmetric stretching of methylenes in the gauche conformation. After the adsorbed film is rinsed with the solvent, however, the spectrum loses the gauche bands and is characterized by the enhanced C-H(distal) and C-H(proximal) stretching bands, which means that all-trans molecules are laid flat. Only STM images for the rinsed film display columnar structures on the herringbones of the reconstructed Au(111) surface; the alkyl chain direction is found to be parallel to the Au atom row. The results indicate that an ordered monolayer is formed first at the liquid-solid interface, and then, disordered overlayers with the gauche conformation are grown but removed by a rinse. Upon exposure to UV light, thus obtained monomer columns are converted into oligomers with flexible backbones and an increased gauche population in the alkyl chains, which resemble red phase polydiacetylenes in LB films.

In the current studies, we examined the effects of hexagonal lattice formation with lipid membranes on the structural stability of native bacteriorhodopsin (bR). Denaturation kinetic measurements for bR solubilized with the mild nonionic detergent Triton X-100 (TX100) were performed in the dark and under illumination by visible light. The solubilized bR was stable in the dark over a wide concentration range of TX100 (1 to 200 mM). In purple membranes, a bilobed band was observed in visible circular dichroism spectra due to interactions between neighboring chromophores. At all concentrations of TX100, this was replaced by a single positive band. Upon illumination with visible light, TX100-solubilized bR clearly showed photobleaching to bacterioopsin. These experimental results suggest that photobleaching is due to a lack of intermolecular interactions inside the purple membrane lattice. Extensive kinetic measurements further revealed that the rate constant of photobleaching is strongly dependent on the detergent concentration, although the activation energy for photobleaching does not significantly change with the TX100 concentration. The mechanism of photobleaching for the solubilized bR is discussed with respect to detergent micelle properties.

Secretory proteins function as agents for numerous cell-cell interactions and determine the survival strategies adopted by organisms. Using the SOSUI system for membrane proteins (Hirokawa et al., Bioinformatics, 1988) and SOSUIsignal for signal peptides (Gomi et al., CBIJ, 2004), we undertook predictive analyses of secretory proteins from 248 prokaryota using all of the amino acid sequences coded by their respective genomes. The number of secretory proteins exhibited a strong positive correlation with the number of total open reading frames, with analysis of these correlations revealing that prokaryotic organisms could be placed into several groups. Symbiotic or obligate parasitic organisms in eukaryotic cells with less than 1200 open reading frames exhibited a single linear relationship between the number of secretory proteins and the total number of open reading frames. Conversely, free-living organisms with more than 2500 open reading frames could be grouped into three linear relationships. The intercept with the axis of the number of open reading frames in the linear relationships was approximately 300 genes for the survival of symbiotic or obligate parasitic organisms and approximately 700 for the free-living organisms. The factor responsible for distinguishing between the different categories of organisms appeared to be G＋C content and the number of open reading frames. The roles of secretory proteins and membrane proteins were discussed on the basis of the ratios of those proteins. The list of all predicted secretory proteins for 248 prokaryota is available through the internet at the URL: http://bp.nuap.nagoya-u.ac.jp/sosuisignal/SOSUIsignalDB/., 分泌タンパク質は細胞間のやり取りを橋渡しする役目を持っており、生物種の生存戦略と密接な関わりがある。 膜タンパク質予測システムSOSUIとシグナルペプチド予測システムSOSUIsignalを用いて248種の原核生物ゲノムから分泌タンパク質の予測を行った。 分泌タンパク質の数とゲノムのORF数には正の相関がみられた。 詳細な解析より、原核生物をいくつかのグループに分けることができた。 ORF数が1200以下の生物は真核生物細胞内共生、寄生生物で、一つの線形近似が可能だった。 2500以上の生物種は独立的に生存できる生物であり、3つのカテゴリに分けられた。 線形近似のORF数の軸への切片は共生ー寄生生物種では300、独立生存グループでは700であった。 また、GC含量もORF数と同様に、生物のカテゴリを分ける主な要因であった。 最後に原核生物の生存戦略を分泌タンパク質の解析に基いて議論した。

High sensitivity differential scanning calorimetry is applied to the problem of interaction between phospholipids and cholesterol in bilayers, i.e., whether or not cholesterol shows a preferential interaction with sphingomyelins over glycerophospholipids. The simplest system yet used for exploring this problem is designed–one composed of 1,2-dimyristoyl- sn -glycero-3-phosphatidylcholine (DMPC), chemically and stereochemically pure N -palmitoyl-sphingomyelin ( pure -C16:0-SM), and cholesterol, based on a belief that chemically and stereochemically purity excludes chain length mismatch and chiral interaction that may affect the interaction from this system. Incremental addition of cholesterol to mixtures of DMPC and pure -C16:0-SM with a ratio of 6:4 or 4:6 leads to a continuous shift in endotherm due to DMPC toward a lower temperature. Such results indicate that cholesterol favors pure -C16:0-SM.

The structural properties of bacteriorhodopsin (bR) over a wide temperature range of 30−80 °C have been examined through the hydroxylamine reactivity of the Schiff base inside the molecule and thro...

The edge-on orientation of alkyl chains, in which the carbon zigzag plane is perpendicular but the chain direction is parallel to the substrate surface, has been detected for specially designed 9,9-dioctadecylfluorene (DOF) molecules on an iodine-covered Au(1 1 1) surface by infrared reflection absorption spectroscopy (IRAS). Using p-polarized light, the methylene symmetric stretching vibrational mode with transition dipole moment μ parallel to the carbon plane has been observed predominantly at 2852 cm −1 compared to the methylene antisymmetric stretching mode with μ perpendicular to the plane.

The structure of bacteriorhodopsin (bR) solubilized using a mild non-ionic detergent, n -octyl-β-gulucoside (OG), which undergoes irreversible photobleaching by irradiation of visible light, was investigated by FT-Raman and FT-IR spectroscopy. Retinal isomerization to the 13- cis configuration along with α II -to-α I conformational changes were observed during solubilization. Hydrogen–deuterium (H–D) exchange in the peptide group, to which bR embedded in purple membrane is extremely resistant, was remarkably enhanced upon solubilization with the detergent. These data suggest that the non-native structure of and acceleration of water molecule penetration into the internal region of bR molecules are related to irreversible photobleaching induced by light illumination in the solubilized state.

The 13C CP/MAS NMR, X-ray diffraction, and Raman spectroscopies were used for monitoring the structural transition of Samia cynthia ricini (S. c. ricini) silk fibroin induced by stretching. Here th...

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.

We describe a novel method for predicting a signal peptide of which three-domain (tripartite) structure is recognized by three modules of the software system. The first module numerates hydrophobic segment in N-terminal 100 residues, the second predicts signal sequences including both signal peptides and signal anchors, and the third discriminates signal peptides. Two novel indexes, SS- and SP-indexes, were developed for the discrimination of signal sequences and signal peptides, respectively, by calculating the relative propensities of amino acids at the carboxyl-terminal end of the hydrophobic region. The number of adjustable parameters in the whole system was only five. When three groups of data (917 signal peptides, 103 signal anchors and 544 non-signal sequences) were analyzed, signal peptides of eukaryotes could be discriminated with the Matthews correlation coefficient of 0.89. The signal peptide predictor SOSUIsignal is available at the web site: http://bp.nuap.nagoya-u.ac.jp/sosui/sosuisignal/sosuisignal_submit.html. This system has the advantage of very fast calculation.

A novel partially fluorinated analog of DMPC, diF4H10-PC, was synthesized, and its thermal and interfacial properties were compared with those of DMPC. By fluorine substitution in the terminal buty...

The thermal phase transition of dl-norleucine (crystalline) has been analyzed via time-resolved Fourier transform infrared (FT-IR) spectroscopy by using a double-scores plot calculated from partial least-squares regression (PLSR). In a previous study, the temperature-dependent spectra collected by time-resolved infrared spectroscopy suggested that another minor thermal phase transition would be present prior to the well-known major transition at ∼118 °C. Eigenvalue analysis by principal component analysis (PCA) suggested, however, that there were four distinguishable signal components, which was not consistent with the evaluation by the absorbance-change plot. In the present study, it has been noticed that a non-systematic response to the temperature increase is involved in the systematic spectral changes. To discriminate the temperature-independent (non-systematic) change from the temperature-related (systematic) changes, two scores of X- and Y-variables in the PLSR modeling have been comparatively used. The new evaluation has readily discriminated a non-systematic factor from basis factors, and the non-systematic factor has been attributed to fringes due to the optical setup used for the measurements.

We observed Raman spectra of Silk I and Silk II powders of (Ala-Gly)15, which is a model peptide of crystalline region of Bombyx mori silk fibroin. Additionally Raman spectra of Ac-(Gly-Ala)4-NHMe and Ac-(Gly-Ala)3-NHMe with Silk I and Silk II forms were calculated according to the repeated β-turn type II structural model and β-sheet model, respectively. The ab initio molecular orbital calculation was used for the theoretical calculation. The calculated results could reproduce the spectra. Especially, in silk I form, observation of splitting of amide I and III bands was in agreement with the predicted bands according to the repeated β-turn type II structural model.

We previously discovered the correlation between light-induced chromophore color change of a photo-receptor membrane protein bacteriorhodopsin (bR) and its two-dimensional crystalline state in the membrane. To apply this phenomenon to a novel optical memory device, it is necessary that bR molecules are immobilized as maintaining their structure and functional properties. In this work, a poly(vinyl alcohol) (PVA) hydrogel with physical cross-linkages (hydrogen bonds between PVA chains) that resulted from repeated freezing-and-thawing (FT) cycles was used as an immobilization medium. To investigate the effects of physically cross-linked PVA gelation on the structure and function of bR in purple membranes (PMs), spectroscopic techniques were employed against PM/PVA immobilized samples prepared with different FT cycle numbers. Visible circular dichroism spectroscopy strongly suggested PM stacking during gelation. X-ray diffraction data also indicated the PM stacking as well as its native-like crystalline latt...

Solubilization and structural stability of a membrane protein bacteriorhodopsin (bR) with n-octyl-β-thioglucoside (OTG) was investigated in comparison with a previous study on bR solubilized with n-octyl-β-glucoside (OG). Highly efficient and stable solubilization of bR with OTG was accomplished above the OTG concentration of about 15 mM. In comparison with OG-solubilized bR, the structural stability of OTG-solubilized bR was high in the dark and under light illumination. These results indicate that OTG is a detergent superior to OG for solubilizing bR molecules.

In order to gain an obvious insight into the role of water in the mechanism of the excimer laser ablation of the cornea, we have macroscopically investigated the ablation behavior of collagen gel in the swelled state by direct photoetching using an ArF excimer laser with time-resolved photography, and furthermore, the thermal effects on the microscopic structures of the collagen molecules by FTIR–ATR spectroscopy. The hydrated collagen film (HF) has a smaller threshold fluence than the dried collagen film (DF). From the time-resolved photographs, the ejected materials were detected only for HF. It was predicted that the effect of bubble formation for HF contributes to the etching. The FTIR–ATR spectroscopic results revealed that the existence of the water suppressed the denaturation of the collagen to gelatin on the surface in the irradiated region. Overall, it was inferred that during the ablation process for HF, the laser energy would be mostly consumed as the latent heat of evaporation of water, that is, the water in the gel matrix would contribute to the suppression of the increment in the temperature in the irradiated region.