菌紫质膜对315nm辐照的光反应

利用仪器本身的测量光束３１５ｎｍ光照对紫膜薄膜中菌紫质的光反应的影响的ＣＤ谱研究说明：３１５ｎｍ的近紫外光可以激发薄膜中菌紫质的光反应,３１５ｎｍ与４０８ｎｍ、３３５ｎｍ光激发的光反应变化类型一致,但与５６８ｎｍ光激发的反应变化类型不一致;３１５ｎｍ光激发的光反应与菌紫质的初始样品状态有关,与菌紫质所处的分子状态的分布有关,而不是直接与初始样品状态存在的表现条件有关。结果认为利用包括近ＵＶ光在内的不同光照条件来调控ＢＲ的光反应是有可能的。     

菌紫质D96N样品的光暗适应型及其转化机制

通过对菌紫质D96N基因突变形成的薄膜样品在光照下动态光谱的测定和分析,研究了此样品的光适应型、暗适应型特性及其之间的转化机制。实验证实,样品受光照激发后很快从暗适应型（D态）光适应型（B态）进入光循环,经过一系列的光化学中间体到边较稳定的M态。M态经过约1小时热驰豫完全回到光适应型B态,B态再经过24小时缓慢热驰豫完全回到暗适应型D态。B态受光则进入光循环,不受光则转化为D态,从而认为B态是菌紫质光循环路径中一个重要的分支节点。据此,提出了该样品在光照下B,D和M态之间转化的模型。     

菌紫质光循环后半程的动力学过程

用动力学光谱仪测量了不同条件下菌紫质照光后在４１０ｎｍ和５７０ｎｍ处的吸收值随时间的变化,并将得到的曲线用多种方程进行拟合,以找到其最适合的动力学方程式。再与不同类型的反应动力学方程相比较,从而得到有关菌紫质的毫秒量级的先循环后半程的信息。实验结果支持有关先循环的如下模型：中间产物Ｍ有两个并列的组分Ｍｆ和Ｍｓ其中Ｍｓ经Ｎ（还有Ｏ）返回到ｂＲ５７０,其中不排除返回反应存在的可能性。     

三种因素对紫膜（PM）薄膜中菌紫质（BR）光反应类型的影响

由中性和碱性ｐＨ成膜液形成的ＰＭ薄膜在不同ＲＨ时ＣＤ语及其不同波长预光照产生的ＤＣＤ谱的研究发现,不同波长预光照产生的ＤＣＤ谱,按照在７００－４６０ｎｍ间的特征可分为全正型、长正短负型和短正长负型三大类。无论成膜液的ｐＨ是中性还是碱性,也不管ＰＭ薄膜的ＲＨ如何,≤４２８ｎｍ的近紫外光照都产生全正型ＤＣＤ谱,而≥６０ｎｍ的光照都产生短正长负型ＤＣＤ谱,但是５０６ｎｍ－５９０ｎｍ间的光照产生的ＤＣＤ谱却不仅与成膜液的ｐＨ有关,而且也与ＰＭ薄膜的ＲＨ有关,成膜液ｐＨ为１０．０的ＰＭ薄膜在ＲＨ７５％时未见有长正短负型ＤＣＤ谱。在近紫外的Ｍ峰区第一、三类ＤＣＤ谱无正峰,而在第二类中为正峰。这些特征都是ＰＭ薄膜中不同结构状态的ＢＲ分子配分浓度分布变化的反映。结果认为,成膜液的ｐＨ、膜所处的ＲＨ和预光照的波长均对ＢＲ的光循环反应有调控作用。这些信息可用于ＢＲ的调控研究,对ＢＲ分子器件的研制开发有重要的参考价值。     

蜂毒突变体对紫膜质子泵功能的影响

具有光驱动质子泵功能的嗜盐菌紫膜是一种被广泛研究的生物膜系统。利用毫秒级闪光动力学谱仪研究蜂毒不同突变体对紫膜质子泵功能的影响。实验采用对硝基苯酚（ｐ－ｎｉｔｒｏｐｈｅｎｏｌ）作为ＰＨ敏感染料来研究紫膜蛋白－菌紫质（Ｂａｃｔｅｒｉｏｒｈｏｄｏｐｓｉｎ,简称）的光反应和质子泵功能。在初步的实验中发现,在一定的温度范围内,光循环过程随着温度的升高而加快,质子泵功能却基本保持不变。另外,由于蜂毒突变体具有不同的插膜特性和不同的电荷量,通过比较它们对ＢＲ质子泵功能的影响,揭示了蜂毒小肽Ｃ端和Ｎ端的不同带电状态对质子泵功能所起的作用不同。实验结果有力地证实了蜂毒或其突变体与菌紫质蛋白具有直接的相互作用,这种相互作用的强弱与蜂毒突变体电荷的多少密切相关     

菌紫质研究的新进展

菌紫质(BR)是嗜盐菌紫膜中的唯一蛋白质,野生型的BR分子含有248个氨基酸残基,其中一个视黄醛通过希夫碱基连结在第216位赖氨酸上,它具有质子泵的功能．光照下,BR进行光循环,光循环又与质子泵过程相关联．菌紫质的结构和功能方面的研究已有很大进展,但其光循环途径和质子泵的机理还不太清楚．文章概述了近年来对菌紫质结构,光循环和质子泵机理研究的进展,尤其对争论较大的菌紫质光循环途径的四类模型作了较详细的介绍．     

菌紫质的Ser和Lys残基修饰对BR结构和功能的影响

用化学修饰研究了菌紫质（ＢＲ）的结构和功能的变化。用氮氧自由基分别对赖氨酸和丝氨酸进行修饰,研究结果表明在圆二色谱上（ＣＤ谱）,与天然紫膜样品比较,两种自由基分别修饰赖氨酸（Ｌｙｓ）和丝氨酸（Ｓｅｒ）残基２４小时后的ＣＤ谱中均只有负峰,分别在５９６ｎｍ和６０２ｎｍ,５３５ｎｍ的正峰已消失,７２小时后５３５ｎｍ的正峰部分地恢复,但１２０小时后均未见进一步恢复。与未修饰的紫膜相比,两种自由基修饰的紫膜在Ｒａｍａｎ光谱上观察到中间体Ｍ４１２的相对量要明显增加。本文对这二种化学修饰引起的ＢＲ结构和功能变化进行了初步讨论。     

紫膜上菌紫质(BR)分子间光协同作用研究

菌紫质（ＢＲ）光循环中Ｍ４１２产物受作用光强调制的现象完全可以用紫膜上ＢＲ三聚体内的光协同效应来解释。这种协同效应不仅与紫膜上ＢＲ呈三聚体的聚集状态有关,也与ＢＲ分子在紫膜上的晶型有序排列有关。紫膜在碱性介质中,加温至５０－６０℃时,三聚体仍然存在,但晶格结构已有破坏,此时也不存在协同效应。     

紫膜Langmuir-Blodgett膜的光电压

八十年代生物分子电子学和生物计算机研究的兴起,嗜盐菌紫膜蛋白菌紫质由于其结构稳定,当受到光照时,能发生同素异构变化,具有光色互变和光驱动质子泵功能、双稳态特性以及皮秒到毫秒级的光电响应特性,使其成为目前国内外关注的研究生物分子电子器件的理想材料之一,应用前途是发展生物分子器件和生物芯片等。由于紫膜具有不对称性而菌紫质的光驱动质子泵具有方向性,因而必须有序组装而且可以有序组装。紫膜     

pH对菌紫质分子的旋转运动和光电响应的影响

用闪光诱导瞬间二向色性方法测量了不同pH条件下的菌紫质分子在脂质囊泡中的旋转扩散运动.在人工平扳膜(BLM)系统中测量了不同pH条件下菌紫质分子的光电响应.在pH3至8.3的范围内没有明显观察到菌紫质分子在膜中旋转运动上的差别.pH低于3时,菌紫质分子旋转运动受到影响;pH高于11时,观察不到旋转扩散运动.在BLM系统中测量了pH2到pH11范围内菌紫质分子的光电响应信号,随着pH的增加,无论紫膜碎片还是单体菌紫质分子的光电响应逐渐由照光后快速正信号并快速衰减及撤光时的快速负信号并逐渐衰减变成慢的正信号.pH高于9.4时,单体菌紫质分子的光电响应信号由正变负,pH高于11时,观察不到信号.     

Pathways of the rise and decay of the M photointermediate(s) of bacteriorhodopsin

The photocycle of bacteriorhodopsin (BR) was studied at alkaline pH with a gated multichannel analyzer, in order to understand the origins of kinetic complexities in the rise and decay of the M intermediate. The results indicate that the biphasic rise and decay kinetics are unrelated to a photoreaction of the N intermediate of the BR photocycle, proposed earlier by others [Kouyama et al. (1988) Biochemistry 27, 5855-5863]. Rather, under conditions where N did not accumulate in appreciable amounts (high pH, low salt concentration), they were accounted for by conventional kinetic schemes. These contained reversible interconversions, either M in equilibrium with N in one of two parallel photocycles or L in equilibrium with as well as M in equilibrium with N in a single photocycle. Monomeric BR also showed these kinetic complications. Conditions were then created where N accumulated in a photo steady state (high pH, high salt concentration, background illumination). The apparent increase in the proportion of the slow M decay component by the background illumination could be quantitatively accounted for with the single photocycle model, by the mixing of the relaxation of the background light induced photo steady state with the inherent kinetics of the photocycle. Postulating a new M intermediate which is produced by the photoreaction of N was neither necessary nor warranted by the data. The difference spectra suggested instead that absorption of light by N generates only one intermediate, observable between 100 ns and 1 ms, which absorbs near 610 nm. Thus, the photoreaction of N resembles in some respects that of BR containing 13-cis-retinal.     

Chromophore-protein-water interactions in the L intermediate of bacteriorhodopsin: FTIR study of the photoreaction of L at 80 K.

Using FTIR spectroscopy, perturbations of several residues and internal water molecules have been detected when light transforms all-trans bacteriorhodopsin (BR) to its L intermediate having a 13-cis chromophore. Illumination of L at 80 K results in an intermediate L' absorbing around 550 nm. L' thermally converts to the original BR only at >130 K. In this study, we used the light-induced transformation of L to L' at 80 K to identify some amino acid residues and water molecules that closely interact with the chromophore and distinguish them from those residues not affected by the photoreaction. The L minus L' FTIR difference spectrum shows that the chromophore in L' is in the all-trans configuration. The perturbed states of Asp96 and Val49 and of the environment along the aliphatic part of the retinal and Lys216 seen in L are not affected by the L --> L' photoreaction. On the other hand, the environments of the Schiff base of the chromophore, of Asp115, and of water molecules close to Asp85 returned in L' to their state in which they originally had existed in BR. The water molecules that are affected by the mutations of Thr46 and Asp96 also change to a different state in the L --> L' transition, as indicated by transformation of a water O-H vibrational band at 3497 cm-1 in L into an intense peak at 3549 cm-1 in L'. Notably, this change of water bands in the L --> L' transition at 80 K is entirely different from the changes observed in the BR --> K photoreaction at the same temperature, which does not show such intense bands. These results suggest that these water molecules move closer to the Schiff base as a hydrogen bonding cluster in L and L', presumably to stabilize its protonated state during the BR to L transition. They may contribute to the structural constraints that prevent L from returning to the initial BR upon illumination at 80 K.     

Luminescence of bacteriorhodopsin in purple membranes from Halobacterium haolbium cells]

Red luminescence of purple membranes from Halobacterium halobium cells was found out, and its emission, excitation and polarization spectra were investigated. Simultaneous parallel measurements of absorption and luminescence changes in one sample brought about by the action of light were also carried out. The bands in the spectra can be attributed to a number of bacteriorhodpsin (BR) forms: BR(595,520), BR(650,575),BR(600-620), BR(700,625), BR(730,660) BR(780,695), where the number above is the position of the luminescence maxima, below--that of absorption. Proceding from the quantum yield of the luminescence (10(-3)) and of photoreaction (10(-1)) of BR, the photoisomerization rate constant of the latter was estimated (10(11) sec(-1). The temperature dependence of the luminescence quantum yield points to the existence of two or three quenching processes with different activation energies. BR phosphorescence was not observed in the region 500-1100 nm. High degree (36%) os luminescence polarization shows that there is no homogeneous energy transfer between BR molecules, or there is regularity in orientation of their dipoles. Energy migration from the bulk of carotenoids to BR was not found. However limited heterogeneous transfer between the different BR forms cannot be ruled out. The absence (or limitation) of migration indicated that there is a spatial separation of the chromophores. Data on possible participation of triplet states in the BR photoconversions are discussed.     

Genetic analysis of the exed region in mouse

The extraembryonic ectoderm development (exed) mutant phenotype was described in mice homozygous for the c(6H) deletion, a radiation-induced deletion in the tyrosinase region of mouse Chromosome 7. These mutants fail to gastrulate and die around embryonic day 8.0. Several genes including, for example, embryonic ectoderm development (eed), are deleted in the c(6H) mutants; however, the portion of the chromosome responsible for the more severe exed phenotype is localized to a 20-kb region called the "exed-critical region." To understand the genetics behind the exed phenotype, we analyzed this region in two ways. First, to determine whether the 20-kb exed-critical region alone causes the mutant phenotype, we removed it from a wild-type chromosome. The resulting mice homozygous for this deletion were viable and fertile, indicating that the 20-kb exed-critical region by itself is not sufficient to cause the phenotype when deleted. We then sequenced the 20-kb exed-critical region and no expressed exons were found. Several short matches to GenBank Expressed Sequence Tag (EST) databases were identified; however, none of these ESTs mapped to the region. Taken together, these results indicate that the exed phenotype may either be a position effect on a distal gene caused by the c(6H) breakpoint or the result of composite effects of nullizygosity of multiple genes in the deletion homozygotes.     

电极介导的菌紫质干膜微分光电流响应的整流性质

在长方形光脉冲光照下,菌紫质(bacteriorhodopsin,BR)干膜组装成夹层光电池具有微分光电流响应．在氧化铟锡(ITO)导电玻璃/BR膜/封口膜/不锈钢形成的干膜电池下可观察到整流特性,而在不锈钢/BR膜/封口膜/ITO导电玻璃形成的干膜电池下则观察不到整流特性,这说明是电极介导的整流．平衡电压测定表明：工作电极/BR膜表面与对电极/BR膜表面有不同的性质,电极的界面效应控制了BR的取向．酸与碱产生的瞬间电流极性也证实了电极整流行为的存在．这些结果将有助于了解BR膜的微分光电响应．     

A study on the fabrication of chemically modified bacteriorhodopsin film and its photochemical properties

Chemically modified bacteriorhodopsin (BR) films embedded in gelatin matrix were fabricated. It was found that chemically modified BR/gelatin film remained the original conformation of BR and possessed homogeneity. Then, the photochemical conversion from all-trans O state to 9-cis P state in the chemically modified BR film/gelatin was investigated. For comparison purpose, the same conversion in the wild-type BR (BRwt) film was also presented here. To our know, it is the first report to show that the absorption changes of the P state in the chemically modified BR film were larger than in the BRwt film upon illumination with red light at the low actinic power density.     

Influence of the Charge at D85 on the Initial Steps in the Photocycle of Bacteriorhodopsin

Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.