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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.

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.

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.

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。

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 secondary structure of the manganese-stabilizing protein of the thermophilic cyanobacterium Synechococcus elongatus in solution was investigated by Fourier-transform infrared (FT-IR) and circular dichroism (CD) spectroscopies. Both methods showed a high proportion of disordered structure (40-43%) and a relatively small amount of beta-sheet (23-24%) and alpha-helix (17-19%). The conformation of the protein remained essentially unchanged at temperatures up to 70 degrees C. Unfolding of the protein occurred at higher temperatures and FT-IR spectroscopy revealed that beta-sheet was more strongly unfolded than alpha-helix at 76 degrees C. The protein largely lost the ordered secondary structures at 90 degrees C, but, when cooled down to 30 degrees C, regained its original conformation. Thus, the cyanobacterial protein is very thermostable and its denaturation at an extremely high temperature is reversible.

Secondary structure formation in the disordered terminal regions of flagellin were studied by circular dichroic (CD) spectroscopy, Fourier transform infrared spectroscopy, and x-ray diffraction. The terminal regions of flagellin are known to form alpha-helical bundles upon polymerization into flagellar filaments. We found from comparative CD studies of flagellin and its F40 tryptic fragment that a highly alpha-helical conformation can be induced and stabilized in the terminal regions in 2,2,2-trifluoroethanol (TFE) containing solutions, which is known to promote intra-molecular hydrogen bonding. Two oligopeptides, N(37-61) and C(470-494), each corresponding to a portion of terminal regions and predicted to have a high alpha-helix forming potential, were synthesized and studied. Both peptides were disordered in an aqueous environment, but they showed a strong tendency to assume alpha-helical structure in solutions containing TFE. On the other hand, peptides were found to form transparent gels at high concentrations (> 15 mg/ml) and all three methods confirmed that the peptides become ordered into a predominantly beta structure upon gel formation. Our results show that large segments of the disordered terminal regions of flagellin can adopt alpha-helical as well as beta structure depending on the environmental conditions. This high degree of conformational adaptability may be reflecting some unique characteristics of the flagellin termini, which are involved in self-assembly and polymorphism of flagellar filament.

The numbers of membrane proteins in the current genomes of various organisms provide an important clue about how the protein world has evolved from the aspect of membrane proteins. Numbers of membrane proteins were estimated by analyzing the total proteomes of 248 prokaryota, using the SOSUI system for membrane proteins (Hirokawa et al., Bioinformatics, 1998) and SOSUI-signal for signal peptides (Gomi et al., CBIJ, 2004). The results showed that the ratio of membrane proteins to total proteins in these proteomes was almost constant: 0.228. When amino acid sequences were randomized, setting the probability of occurrence of all amino acids to 5%, the membrane protein/total protein ratio decreased to about 0.085. However, when the same simulation was carried out, but using the amino acid composition of the above proteomes, this ratio was 0.218, which is nearly the same as that of the real proteomic systems. This fact is consistent with the birth, death and innovation (BDI) model for membrane proteins, in which transmembrane segments emerge and disappear in accordance with random mutation events.

Measurements of regeneration kinetics were performed in order to investigate the regeneration mechanisms of bacteriorhodopsin (bR) from thermally unfolded bacterio-opsin (bO) and all-trans retinal. Regeneration kinetics data were successfully fitted to a single exponential function when regeneration was performed at 25 degrees C after incubation at high temperatures. Conversely, the process of regeneration after the addition of retinal to bO at high temperatures occurred at two different rate constants. These findings strongly suggest that the slower regeneration of bR at high temperatures occurs as a result of dynamic structural fluctuation of bO, whereas the faster process corresponds to regeneration from bO, which retains a native structure capable of retinal binding.

A software system, SOSUI, was previously developed for discriminating between soluble and membrane proteins and predicting transmembrane regions (Hirokawa et al., Bioinformatics, 14 (1998) 378–379). The performance of the system was 99% for the discrimination between two types of proteins and 96% for the prediction of transmembrane helices. When all of the amino acid sequences from 15 single-cell organisms were analyzed by SOSUI, the proportion of predicted polytopic membrane proteins showed an almost constant value of 15–20%, irrespective of the total genome size. However, single-cell organisms appeared to be categorized in terms of the preference of the number of transmembrane segments: species with small genomes were characterized by a significant peak at a helix number of approximately six or seven; species with large genomes showed a peak at 10 or 11 helices; and species with intermediate genome sizes showed a monotonous decrease of the population of membrane proteins against the number of transmembrane helices.

Ultrasonic relaxation measurement was employed for confirmation of the interaction between dimyristoylphosphatidylcholine (DMPC) membrane and a soluble protein, carbonic anhydrase II (CA II). The enhancement of the fluctuation of DMPC membrane structure was observed in the presence of CA II under acidic condition, pH 3.6–4, indicating the interaction between DMPC and CA II. The pyrene fluorescence spectrum of CA II solution clearly showed that this protein adopted an unfolding intermediate called the molten globule state under the low pH condition, in which CA II interacted with DMPC. However, CA II in the molten globule state did not cause membrane lysis in contrast to the high lytic activity of α-lactalbumin on DMPC liposomes.