Search

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.

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.

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.

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。

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.

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.

A short-chain phosphatidylcholine, diheptanoyl PC (diC7 PC), was employed for solubilizing a membrane protein bacteriorhodopsin (bR). Highly stable and efficient solubilization of bR was accomplish...

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.