蜂毒对菌紫质(bR)光循环中间体M_(412)和质子泵的影响

蜂毒对菌紫质(bR)光循环中间体M_(412)及质子泵的动力学过程有较大影响.表现在M_(412)慢衰减组分(M_(412~S))和质子的半衰期增大及产出量的减小,说明蜂毒分子的介入抑制了M_(412)的生成和衰减过程,同时也阻碍了紫膜的质子泵功能.但似乎对M_(412)快衰减组分影响不大.以上结果支持了M_(412~S)与质子泵功能有关的说法.实验结果同时反映出蜂毒除与膜脂分子存在强烈作用外还与bR本身直接作用.因此,蜂毒是一种研究膜脂蛋白相互作用及膜蛋白功能的较好材料.     

水在菌紫质光循环和质子泵中的作用

用圆二色仪和闪光动力学先谱仪分别测量了空气干燥紫膜薄层的圆二色谱及紫膜LB膜的M_(412)的衰减过程.在于燥紫膜的圆二色谱上出现412nm的正峰,它是光循环中间体M_(412)的特征峰.在无水介质中,紫膜LB膜中的BR仍能进行先化学循环而检测到中间体M_(412),但M_(412)的衰减速度减慢,产生M_(412)的堆积,质子化过程受阻.在有水的介质中,只要有足够的H~+存在,紫膜LB膜中的BR的中间体M_(412)的衰减速度明显加快.说明水介质的H~+是完成正常光化学循和质子化过程必不可少的.     

两种化学修饰菌紫质的光化循环和光电位

本文分别用两种氮氧自由基对菌紫质(bR)中的两种氨基酸残基——赖氨酸和丝氨酸进行了修饰,并对修饰后的bR光循环中间产物M_(412)动力学过程、质子泵效率及光电响应信号进行了测量。通过与正常紫膜进行比较,可以看出:赖氨酸被修饰后,M_(412)快、慢组分的衰减及质子的吸收过程均减慢,M_(412)和质子的产额、跨膜光电位信号均有不同程度的提高;丝氨酸被修饰后,M_(412)和质子的动力学过程的变化与赖氨酸基本相同,但M_(412)和质子的产额及跨膜光电位提高很大,且光电位出现缓慢反向现象。结果表明赖氨酸不大可能直接参与质子的传递过程,其对紫膜质子泵的影响主要是通过其所带电荷对表面电位的贡献;而丝氨酸却似乎对bR的功能影响很大并对维持质子通道构象环境的稳定起着很大作用。     

酶切菌紫质(bR)C端对紫膜光循环和质子泵效率的影响

本文研究紫膜悬浮液经低剂量木瓜蛋酶处理去掉菌紫质(Bacteriorhodopsin简写bR)分子C-末端后。其光循环产物和质子泵效率的变化。实验发现经酶切后,M_(412)产物中慢衰减组份M_(412)降低了20％,O_(640)降低了50％,而质子泵效率降低了70％。双光脉冲实验表明酶解作用并不影响光循环周期。这些事实说明了去C-端所引起的质子泵效率降低,不是通过光循环的途径而产生的。介质中离子强度对正常紫膜和酶解紫膜的质子泵效率有明显不同的影响 说明了C端在不同盐浓度中的构象对质子泵行为有很重要的作用。     

离子强度、温度及表面活性剂Triton X-100对紫膜质子泵效率的影响

用毫秒级闪光动力学光谱仪研究了离子强度、温度及表面活性剂Triton X-100对紫膜质子泵效率的影响。结果表明:紫膜溶液中适量离子的加入可使其质子泵效率(H~+/M_(412))显著提高(纯水中,H~+/M_(412)—0.56,而在150mM KCl中可达—1.3),高价离子的影响显然远大于低价离子;在5℃—50℃的较大温度范围内,紫膜的质子泵效率变化不大,但当温度升至60℃以上时,紫膜的质子泵效率迅速下降直至为零;非离子表面活性剂Triton X-100的加入对紫膜的质子泵效率影响不大,仅随Triton量的增加而略微下降。以上实验现象,直接或间接地说明了离子在紫膜质子泵功能中的重要作用。就此对离子及脂在紫膜质子泵中的作用机制进行了进一步的探讨。     

菌紫质光循环中间体的时间分辨谱

利用毫微秒时间分辨的吸收变化和微秒的闪光动力学光谱研究了室温下菌紫质光循环中间体的形成过程及衰减过程。实验结果表明,室温下,K_(610)和M_(412)中间体同样出现,L_(550)中间体还不能得到证实,有待于进一步研究。     

Triton X-100对细菌视紫红质中间产物M_(412)的效应

本文研究用非离子表面活性剂Triton X-100处理后的细菌视紫红质(BR Bacteriorhodo-psin)光循环中间产物M_(412)动力学过程的变化.实验结果表明,用不同浓度的Triton处理pH=6.5的BR体系时,其中间产物M_(412).快衰减成分的半衰期(τ_(1/2)~f)在Triton浓度为0.05%(w/w)附近突然变慢,随着Triton浓度的加大,τ_(1/2)~f又逐渐加快;慢衰减部分的半衰期(τ_(1/2)~s)则随Tri-ton浓度的增加逐渐变慢.BR的生色团峰发生蓝移.说明不同浓度的Triton在水溶液中聚集状态不同,可不同程度地破坏膜脂的液晶态结构,从而导致镶嵌在其中的BR发生构象的变化,使转运质子的氢键通道受到不同程度的影响,故质子泵转运通道发生改变、致使M_(412)的衰减速率改变.     

肼对蓝膜的影响

利用紫外可见吸收光谱和动力学光谱法研究了无水肼对蓝膜的影响,研究结果表明:无水肼可以使蓝膜转化为紫膜,同时光循环也得到恢复,但是光循环中间体M412的衰减加快,这与金属阳离子加入到蓝膜溶液中时的现象是完全不同的(这个过程中M412的衰减是减慢的).同时研究了pH和温度对无水肼与蓝膜之间相互作用的影响.在无水肼加入到蓝膜溶液中时,重组反应的灵敏度是pH和温度依赖的.在pH4.8到pH2之间,灵敏度随酸性的增加而降低.在20～40℃之间,无水肼与蓝膜溶液的反应灵敏度随着温度的升高而降低.     

水份含量对紫膜薄层某些光学性质的影响

本文研究在乾燥紫膜薄层中不同水合程度对光循环中间体M衰减速率,对园二色谱(CD)和蛋白紫外荧光光谱的影响.在相对湿度小于70%时,M衰减速率相对稳定,不随湿度改变而变化;但当湿度大于70%时,则衰减速率急剧上升.从可见区的CD光谱中,也发现了在相对湿度大于70%后,旋转强度急剧上升.蛋白紫外荧光光谱随湿度增加未发现有明显变化.实验结果说明一定水份的含量对紫膜完成质子泵功能是必不可少的,它能改变色素团的微环境,从而改变色素团和蛋白间的相互作用.     

紫膜在含水凝胶中的定向和状态

用几种光谱学方法研究了紫膜凝胶的定向度、生色团视黄醛的状态以及其光循环中间产物M的动力学过程。结果表明:用通常采用的制备方法所得到的紫膜凝胶虽然能得到光电响应信号,但其定向度并不理想,生色团视黄醛的构象受到较大扰动,光循环中间产物M的产出及衰减也受到抑制,部分样品甚至由于其视黄醛的脱落而完全失去颜色,其质子泵功能也随之丧失。这说明虽然紫膜凝胶是目前研究紫膜质子泵机理(光电响应测量)和构造分子电子器件较好的和有希望的人工膜系统之一,但由于该系统对紫膜结构和功能的扰动仍然较大,紫膜的定向较难控制,所以,我们仍需在凝胶的形成体系及方法的改进上作大量工作。     

Proton transfer reactions in native and deionized bacteriorhodopsin upon delipidation and monomerization

We have investigated the role of the native lipids on bacteriorhodopsin (bR) proton transfer and their connection with the cation-binding role. We observe that both the efficiency of M formation and the kinetics of M rise and decay depend on the lipids and lattice but, as the lipids are removed, the cation binding is a much less important factor for the proton pumping function. Upon 75% delipidation using 3-[(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS), the M formation and decay kinetics are much slower than the native, and the efficiency of M formation is approximately 30%-40% that of the native. Upon monomerization of bR by Trition X-100, the efficiency of M recovers close to that of the native (depending on pH), M formation is approximately 10 times faster, and M decay kinetics are comparable to native at pH 7. The same results on the M intermediate are observed if deionized blue bR (deI bbR) is treated with these detergents (with or without pH buffers present), even though deionized blue bR containing all the lipids has no photocycle. This suggests that the cation(s) has a role in native bR that is different than in delipidated or monomerized bR, even so far as to suggest that the cation(s) becomes unimportant to the function as the lipids are removed.     

Molecular dynamics study of the M412 intermediate of bacteriorhodopsin.

Molecular dynamics simulations have been carried out to study the M412 intermediate of bacteriorhodopsin's (bR) photocycle. The simulations start from two simulated structures for the L550 intermediate of the photocycle, one involving a 13-cis retinal with strong torsions, the other a 13,14-dicis retinal, from which the M412 intermediate is initiated through proton transfer to Asp-85. The simulations are based on a refined structure of bR568 obtained through all-atom molecular dynamics simulations and placement of 16 waters inside the protein. The structures of the L550 intermediates were obtained through simulated photoisomerization and subsequent molecular dynamics, and simulated annealing. Our simulations reveal that the M412 intermediate actually comprises a series of conformations involving 1) a motion of retinal; 2) protein conformational changes; and 3) diffusion and reconfiguration of water in the space between the retinal Schiff base nitrogen and the Asp-96 side group. (1) turns the retinal Schiff base nitrogen from an early orientation toward Asp-85 to a late orientation toward Asp-96; (2) disconnects the hydrogen bond network between retinal and Asp-85 and tilts the helix F of bR, enlarging bR's cytoplasmic channel; (3) adds two water molecules to the three water molecules existing in the cytoplasmic channel at the bR568 stage and forms a proton conduction pathway. The conformational change (2) of the protein involves a 60 degrees bent of the cytoplasmic side of helix F and is induced through a break of a hydrogen bond between Tyr-185 and a water-side group complex in the counterion region.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning alpha-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of "the cation" in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca2+, Mg2+ and Mn2+) and monovalent metal cations (Li+, Na+, K+ and Cs+), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation相似文献   

Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin

Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable.     

Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles.

In this study, we have investigated effects of volatile anesthetics on absorption spectra, proton pumping activity and decay of photointermediate M of bacteriorhodopsin (bR) in differently aggregated states. Anesthetics used in this study are ether-type general anesthetics; enflurane and sevoflurane. The observed effects on bR depend not only on variety or concentration of anesthetics but also strongly on the aggregation state of bR molecules in the membrane. In purple membrane (PM), bR having maximum light absorption at 567 nm (bR567) is formed in the presence of sevoflurane or a small amount of enflurane, while a species absorbing maximally at 480 nm (bR480) is formed upon the addition of large amounts of enflurane. X-ray diffraction studies show that the former species maintains crystallinity of PM, but the latter does not. In reconstituted vesicles where bR molecules exist as monomer, even sevoflurane forms bR480. Flash photolysis experiments show that bR567 contains a shorter-lived M intermediate absorbing maximally at 412 nm in the photoreaction cycle than bR does and that bR480 contains at least two long-lived M intermediates which seem to absorb maximally near and at lower than 380 nm. The measurements of light-induced pH changes of the whole cells and of the reconstituted vesicles in the presence of the anesthetics indicate that bR567 has a enhanced proton pumping efficiency, while bR480 has a quite low or no activity. No significant difference was observed in the anesthetic action between two inversely pumping vesicles. These observations suggest that on the formation of bR480, anesthetics enter into the membrane and affect the protein-lipid interaction.     

Effect of partial delipidation of purple membrane on the photodynamics of bacteriorhodopsin

The effect of lipid-protein interaction on the photodynamics of bacteriorhodopsin (bR) was investigated by using partially delipidated purple membrane (pm). When pm was incubated with a mild detergent, Tween 20, the two major lipid components of pm, phospholipids and glycolipids, were released in different ways: the amount of phospholipids released was proportional to the logarithm of the incubation time; the release of glycolipids became noticeable after the release of approximately 2 phospholipids/bR, but soon leveled off at approximately 50% of the initial content. It was found that the thermal decay of the photocycle intermediate N560 was inhibited by the removal of less than 2 phospholipids per bR. This inhibition was partly explained by an increase in the local pH near the membrane surface. More significant changes in the bR photoreactions were observed when greater than 2 phospholipids/bR were removed: (1) the extent of light adaptation became much smaller, and this reduction correlated with the release of glycolipids; (2) N560 became difficult to detect; (3) the M412 intermediate, which is characterized by a pH-insensitive lifetime, was replaced by a long-lived M-like photoproduct with a pH-sensitive lifetime. The heavy delipidation apparently altered the mechanism by which the deprotonated Schiff base receives a proton. An important conformational change in the protein moiety is suggested to take place during the M412 state, this conformational change being inhibited in the rigid lipid environment.     

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.     

Sensitive detection of protein-lipid interaction change on bacteriorhodopsin using dodecyl β-D-maltoside

A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl β-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR.