细菌视紫红质中质子泵出的分子机理

细菌视紫红质(Bacteriorhodopsin,或bR)是盐生嗜盐菌(Halobacterium salinarium)等细菌的跨膜蛋白质,其色基视黄醛的光致异构化作用触发细菌视紫红质的一系列结构变化,把质子从细胞质泵到细胞外空间。对细菌视紫红质中质子泵出分子机理进行了描述。     

Tol-Pal系统在细菌分裂中的作用机制

二分裂是细菌繁殖的重要方式,细菌细胞膜各层于时空上协调内陷在细菌二分裂过程中发挥着关键作用。细菌细胞质膜层和肽聚糖层的内陷由细菌分裂体来驱动;而革兰阴性菌(Gram-negative bacteria,简称G~-菌)外膜的内陷则是通过与肽聚糖层连接的外膜脂蛋白而被动牵拉形成。质子动力偶联的Tol-Pal系统,不仅在G~-菌外膜稳定性中发挥作用,而且在细菌细胞分裂中也发挥着关键性的作用。Tol-Pal系统构成了G~-菌中分裂装置的动态复合体,G~-菌分裂收缩时,在质子动力的驱动下Tol-Pal复合体促使肽聚糖相关脂蛋白Pal在分裂位点聚集,建立瞬时跨膜连接,牵拉分裂位点的外膜随着肽聚糖层和内膜层中部的收缩而内陷。现就Tol-Pal系统在细菌分裂中的作用作一总结,以便深入理解tol-pal突变菌的不同表型,为细菌分裂抑制剂的研发提供理论依据。     

微生物纳米导线的导电机制及功能

微生物种间直接电子传递是指在厌氧条件下,一种微生物将电子直接传递给另外一种微生物,将两种不同微生物的代谢途径耦合在一起,以达到互养共生的目的。细菌-古菌之间的直接电子传递是其物质转换与能量代谢的新途径和新调控机制,直接参与甲烷的合成以及与硫酸盐还原耦合的厌氧甲烷氧化,在驱动碳和硫的地球化学转化与循环中起着十分重要的作用。目前研究结果认为细菌-古菌之间的直接电子传递主要是由含多个血红素的C型细胞色素介导的,这些细胞色素能形成不间断的胞外电子传递途径,以电子多步跃迁机制在细菌和古菌的细胞质膜之间传递电子。     

“正常”膜蛋白取向的细菌视紫红质脂质体制备及其瞬态光学响应

细菌视紫红质 (bR)是嗜盐菌质膜上的一种跨膜蛋白质 ,有其独特的光驱质子泵功能 ,可以被定向组装到磷脂脂质体膜上 ,并且表现出和细胞膜上相反的取向。通过细菌培养和细胞膜分离 ,获得了含bR蛋白质的紫膜悬浮体系 ,在pH =2 .5时将bR悬浮液和两亲性的DPPC磷脂混合、通过自组装的方式形成了含bR膜蛋白的磷脂脂质体 ,并通过瞬态光学响应测量考察了bR的取向和质子泵生物活性。结果表明 ,bR膜蛋白可以被整合到DPPC的脂质体膜上 ;蛋白质的质子泵运行规律的测量进一步验证了在酸性条件下所制备的脂质体上bR保持了不寻常的择优取向 ,与细胞膜上的“正常”取向一致 ,而与绝大多数文献报道的中性条件下制备的脂质体质子泵取向相反。     

氧化磷酸化中质子的转运机制介绍和讨论

氧化磷酸化过程中电子传递和磷酸化所伴随的质子（H＋）跨线粒体内膜转运,是生物化学教学中的一个重点和难点。该文介绍参与H＋跨膜（线粒体内膜或细菌质膜）转运的复合体Ⅰ（又称为NADH-Q还原酶或NADH脱氢酶）、复合体Ⅲ（又称为细胞色素还原酶或细胞色素bc1复合体）、复合体Ⅳ（又称为细胞色素氧化酶或细胞色素c氧化酶）和复合体Ⅴ（又称为F1F0-ATP合酶）跨膜转运H＋的机制。     

猪眼视紫红质膜液晶态结构特征

光敏色素视紫红质(Rhodopsin)是一种以生色团为辅基的色素蛋白。近年来的研究表明视紫红质分子是在脊椎动物视网膜光感受器的片层膜内或膜上,它在光-电转换机能中起着重要作用。光感受器膜类似于一般生物膜,也处于有序、多变的液晶态。在自我有序的液晶相-片层结构中,分子的长轴基本平行,形成分子层。这种表面可用于简单的有机反应,如异构化、酶的氧化、还原以及脱氢作用等。这些结构对能量的变化也很容易发生反应。因此研究视紫红质在片层膜上的液晶态结构,对进一步探索光感受器功能是有重要意义。     

小麦根H^＋—ATPase与脂质体的重组方法研究

液泡是植物细胞中的大型细胞器,除了维持细胞的渗透压和贮存代谢的中间产物外,其内部还含有多种水解酶、pH值偏酸且具有类似溶酶体的功能。液泡膜ATPase是一种新类型的质子泵。由质子泵作用形成的跨液泡膜质子电化学梯度和质子驱动力为各种溶质(如阳、阴离子、氨基酸和糖类等)分子的主动跨膜转运提供了动力,使液泡成为植物细胞内离子平衡的调节器。液泡膜ATPase和线粒体膜ATPase都具有泵质子的功能,而且都受阴离子激活,其生化性质有许多相似     

质子漏及其在基础代谢中的作用

“质子漏”是指电子传递链跨膜泵出的质子通过不涉及ATP合成的途径而跨膜扩散流回基质的过程,它的出现形成了由呼吸链驱动的质子泵出和质子回漏的无效循环通路.质子漏的耗氧在呼吸速率中占有重要的比重,对细胞呼吸有很强的控制作用,可以调节能量偶联系数,同时质子漏也是重要的产热过程,它承担了基础代谢产热的20%～30%.质子漏的生理功能有产热、增加代谢调节潜能、清除有害自由基和调节碳流等.     

光遗传技术中的两种主要光敏蛋白及其在阿尔茨海默病研究中的应用

阿尔茨海默病(Alzheimer’s disease, AD)是一种常见的中枢神经系统退行性疾病,但是,AD的发病机制还有待进一步的阐明。近年来,光遗传技术因其在时间和空间上的高精确性而逐渐被应用于AD的研究中。Ⅰ型光敏蛋白中的视紫红质通道蛋白-2 (channelrhodopsin-2, ChR2)和盐碱古菌嗜盐细菌视紫红质蛋白(Natronomonas halorhodopsin, NpHR)可以通过将光信号快速转换成跨膜离子电流来调节神经元的活动,因此,已经被应用于光遗传技术中。现对ChR2和NpHR在光遗传技术中的应用进行介绍,并总结其在AD研究中的应用进展。     

酰化菌紫质的动力学光谱及光电特性研究

用人工双分子膜(BLM)技术及动力学光谱研究了赖氨酸残基在紫膜的结构和功能中所起的作用.酰化基团与菌紫质(bR)分子中的赖氨酸残基的ε氨基作用,使光照后bR的跨膜质子迁移信号及膜的充放电速度变慢,光循环中间产物M412的产量下降,半衰期延长.但UV/Vis吸收光谱表明酰化并未破坏bR中视黄醛的构象环境.在高pH或高盐浓度下,酰化的影响降低.这些结果表明:赖氨酸残基并不是泵出质子的提供者,没有直接参与质子的跨膜输运,而是通过表面电位来影响bR的质子泵功能.     

Electrostatic Environment of Proteorhodopsin Affects the pKa of Its Buried Primary Proton Acceptor

The protonation state of embedded charged residues in transmembrane proteins (TMPs) can control the onset of protein function. It is understood that interactions between an embedded charged residue and other charged or polar residues in the moiety would influence its pKa, but how the surrounding environment in which the TMP resides affects the pKa of these residues is unclear. Proteorhodopsin (PR), a light-responsive proton pump from marine bacteria, was used as a model to examine externally accessible factors that tune the pKa of its embedded charged residue, specifically its primary proton acceptor D97. The pKa of D97 was compared between PR reconstituted in liposomes with different net headgroup charges and equilibrated in buffer with different ion concentrations. For PR reconstituted in net positively charged compared to net negatively charged liposomes in low-salt buffer solutions, a drop of the apparent pKa from 7.6 to 5.6 was observed, whereas intrinsic pKa modeled with surface pH calculated from Gouy-Chapman predictions found an opposite trend for the pKa change, suggesting that surface pH does not account for the main changes observed in the apparent pKa. This difference in the pKa of D97 observed from PR reconstituted in oppositely charged liposome environments disappeared when the NaCl concentration was increased to 150 mM. We suggest that protein-intrinsic structural properties must play a role in adjusting the local microenvironment around D97 to affect its pKa, as corroborated with observations of changes in protein side-chain and hydration dynamics around the E-F loop of PR. Understanding the effect of externally controllable factors in tuning the pKa of TMP-embedded charged residues is important for bioengineering and biomedical applications relying on TMP systems, in which the onset of functions can be controlled by the protonation state of embedded residues.     

L105K mutant of proteorhodopsin

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria. Thousands of PRs are classified into blue-absorbing (λ(max) ~ 490 nm) and green-absorbing (λ(max) ~ 525 nm) PR, and the color determinant is known to be at position 105, where blue-absorbing and green-absorbing PR possess Gln and Leu, respectively. Position 105 is in contact with the retinal chromophore in the hydrophobic region of the cytoplasmic side. In this paper, we have introduced a positively charged lysine group at position 105, which is the first report of the introduction of a positively charged group into the hydrophobic cytoplasmic domain in microbial rhodopsins. The L105K mutant PR shows an ~21 nm red shift (λ(max) ~ 549 nm) at pH 7.0, and the pK(a) of the counterion (7.2) does not change significantly compared to that of wild-type PR (6.8). The analysis of thermal stability shows that the mutation causes some destabilization of structure, but the mutant is more stable toward hydroxylamine reaction than the wild type. The flash photolysis measurement at pH 9.0 shows that the decay of the M intermediate of L105K is ~3 times slower than that of the wild type. The slow M decay possibly originates from the perturbation of the proton donor (Glu108) and the retinal Schiff base due to positioning of a positively charged lysine group in the proton transfer pathway. The perturbation of proton transport is also observed when we measure light-induced proton pumping. The rate of proton transport in L105K mutant is 6 times slower than that of the wild type, which corroborates our flash photolysis result.     

Proteorhodopsin: Characterisation of 2D crystals by electron microscopy and solid state NMR

Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in γ-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by ¹⁵N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee-Goldberg cross-polarisation has been used to probe protein backbone mobility.     

Proteorhodopsin: characterisation of 2D crystals by electron microscopy and solid state NMR

Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in gamma-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by 15N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee-Goldberg cross-polarisation has been used to probe protein backbone mobility.     

Characterization of the chimeric seven-transmembrane protein containing conserved region of helix C–F of microbial rhodopsin from Ganges River

Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria. Recently, many PR-like genes were found in non-marine environments. The goal of this study is to explore the function of rhodopsins that exist only as partial proteo-opsin genes using chimeras with marine green PR (GPR). We isolated nine partial genes of PR homologues using polymerase chain reaction (PCR) and chose three homologues of GPR from the surface of the Ganges River, which has earned them the name “CFR, Chimeric Freshwater Rhodopsin.” In order to characterize the proteins, we constructed the cassette based on GPR sequence without helices C to F and inserted the isolated conserved partial sequences. When expressed in E. coli, we could observe light-driven proton pumping activity similar to proteorhodopsin, however, photocycle kinetics of CFRs are much slower than proteorhodopsin. Half-time decay of O intermediates of CFRs ranged between 143 and 333 ms at pH 10; their absorption maxima were between 515 and 522 nm at pH 7. We can guess that the function of native rhodopsin, a retinal protein of fresh water bacteria, may be a light-driven proton transport based on the results from chimeric freshwater rhodopsins. This approach will enable many labs that keep reporting partial PCR-based opsin sequences to finally characterize their proteins.     

Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes

Proteorhodopsin (PR), a light-driven proton pump from marine proteobacteria, exhibits photocycle characteristics similar to bacteriorhodopsin (BR) at neutral pH, including an M-like photointermediate. However, at acidic pH, spectroscopic evidence for an M-like species was absent, and the vectoriality of proton pumping was inverted. To gain further insight into this unusual property, we examined the voltage dependence of stationary and laser flash-induced photocurrents of PR under different pH conditions upon expression in Xenopus oocytes. The current-voltage curves were linear under all conditions tested, and photocurrent reversal potentials distinctly depended on the pH gradient. PR mutants D97N and D97T exhibited transient and stationary inward currents already at neutral pH, showing that neutralization of the proton acceptor abolishes forward pumping and permits only inward proton transport. Mutation E108G, which disrupts the donor site for Schiff base (SB) reprotonation, resulted in largely reduced photocurrents, which could be strongly stimulated by azide, similar to previous observations on BR mutant D96G. When PR and BR photocurrents in response to blue or green laser flashes during or after continuous illumination were compared, direct electrical evidence for the occurrence of an M-like intermediate at neutral pH could only be obtained when reprotonation of the SB was slowed down by PR mutation E108G. For PR at acidic pH, laser flashes only produced inwardly directed photocurrents, independent from background illumination, thus precluding electrical identification of an M-like species. However, when visible absorption spectroscopy was carried out at low temperatures, occurrence of an M-like species was robustly observed at low pH. This indicates that SB deprotonation and reprotonation occur during the PR photocycle also at low pH. Our results corroborate the conclusion that in PR, the direction of proton pumping can be switched by changes in pH and membrane potential, with the protonation state of Asp-97 being the key determinant for selecting between transport modes.     

Diversity and functional analysis of proteorhodopsin in marine Flavobacteria

Proteorhodopsin (PR) genes are widely distributed among marine prokaryotes and functions as light-driven proton pump when expressed heterologously in Escherichia coli, suggesting that light energy passing through PR may be substantial in marine environment. However, there are no data on PR proton pump activities in native marine bacteria. Here, we demonstrate light-driven proton pump activity (c. 124 H(+) PR(-1) min(-1) ) in recently isolated marine Flavobacteria. Among 75 isolates, 38 possessed the PR gene. Illumination of cell suspensions from all eight tested strains in five genera triggered marked pH drops. The action spectrum of proton pump activity closely matched the spectral distribution of the sea surface green light field. Addition of hydroxylamine to a solubilized membrane fraction shifted the spectrum to a form characteristic of PR photobleached into retinal oxime, indicating that PRs in flavobacterial cell membranes transform the photon dose in incident radiation into energy in the form of membrane potential. Our results revealed that PR-mediated proton transport can create the sufficient membrane potential for the ATP synthesis in native flavobacterial cells.     

Three-Dimensional Solid-State NMR Study of a Seven-Helical Integral Membrane Proton Pump—Structural Insights

Proteorhodopsin (PR) is a recently discovered ubiquitous eubacterial retinal-binding light-driven proton pump. Almost 1000 PR variants are widely distributed in species of marine and freshwater bacteria, suggesting PR's important photobiological role. PR is a typical seven-transmembrane α-helical membrane protein and as such poses a significant challenge to structural studies. Attempts to crystallize PR have not been successful, and its three-dimensional structure remains unknown. We show that PR reconstituted in lipids gives well-resolved magic-angle spinning NMR spectra of high signal-to-noise ratio. We report sequential assignment of ¹³C and ¹⁵N backbone and side-chain chemical shifts for 103 of 238 residues in PR, achieved by three-dimensional chemical shift correlation experiments performed on two samples with different patterns of reverse labeling. The chemical shift analysis gives a number of important structural insights not available from other studies: we have established protonation states of several carboxylic acids, identified the boundaries and distortions of transmembrane α-helices, and detected secondary structure elements in the loops. We confirmed that internal Asp227, which was proposed to form part of the Schiff base counterion, is ionized, while Glu142, which is located close to the extracellular surface, is neutral, in agreement with earlier predictions. We infer that, similar to bacteriorhodopsin's structure, PR has a proline kink in helix C, a non-proline kink in helix G, a short β-turn in the B-C loop, and a short α-helical segment in the E-F loop.