细菌视紫红质研究的新进展

细菌视紫红质（bR)自六十年代末发现以来,一直是膜蛋白领域研究的热点之一,bR不仅在基础理论研究方向有着深远的意义,而且也有着广泛的应用前景。对bR的结构、质子泵功能、折叠机制等研究前沿进行概述。     

细菌视紫红质的基因克隆与表达

细菌视紫红质的基因克隆与表达卢春林,汪俭,梅祺,韦钰（东南大学吴建雄实验室,南京２１０Ｏ１８）叶寅,田波（中国科学院微生物研究所,北京１０００８０）关键词细菌视紫红质基因;聚合酶链反应（ＰＣＲ）;基因克隆与表达细菌视紫红质（ｂａｅ快ｒｉｏｒｈｏｄｏＰ...     

细菌视紫红质对调制光的响应特性

细菌视紫红质在结构上与视紫红质的相似性使其具有某些视觉响应特性.用电泳法在不锈钢电极上沉积出定向紫膜薄膜,构成不锈钢/紫膜/凝胶/铜电极结构的光接收器.在调制光作用下,光接收器显示出对光强变化的微分响应特性.测量了光电压随调制频率和入射光功率的变化关系.比较和讨论了细菌视紫红质对调制光响应特性与视觉频闪及明暗感的相关性.     

细菌视紫红质的光电响应特性和机制

在ITO导电玻璃上制备定向细菌视紫红质 (BR)电泳沉积膜或LB膜组成光电池系统 ,在短脉冲激光照射下 ,测定其脉冲响应光电压 ;在间断光照射下 ,测定其对光强变化产生的微分响应信号。对脉冲光电响应和微分响应的机理及其关系进行理论分析和解释 ,认为脉冲响应是BR分子内部生色团快速光极化引起的电荷分离和希夫碱及其周围氨基酸去质子化和再质子化过程引起的质子定向运输产生的位移电流 ,是一个快反应过程 ,是微分响应的早期反应和基础。微分响应则是由于菌紫质的光驱动质子泵产生的连续质子流在光开和光关瞬间引起光电池系统充放电以及测量电路的耦合特性引起的 ,是一个慢变化过程     

细菌视紫红质的构象与介质pH依赖关系的荧光寿命研究

本文采用荧光探针的方法,通过测量与紫膜结合后的荧光探针ANS的荧光寿命随介质pH的变化,研究了介质pH对bR的构象的影响.结果表明:在pH4～11范围内ANS的荧光寿命呈U字形分布;其荧光寿命中含有快慢两种衰减成分;随着介质pH的变化;这两种成分的含量所占比例也有很大变化.说明介质pH能够引起蛋白构象的变化.     

基因突变细菌视紫红质薄膜的长寿命M态光存储

实验研究了D96N型基因突变细菌视紫红质薄膜的光存储性能,实现了用670 nm激光在BR膜上记录光学图像,用560 nm弱光读出图像,用488 nm激光擦除图像的写读擦操作.M态寿命在室温下延长到了3 min,比溶液状态下的野生细菌视紫红质M态寿命延长了5个数量级.     

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。     

细菌视紫红质分支光循环研究的新起点——蓝膜

细菌视紫红质分支光循环研究的新起点——蓝膜@张国艳$中国科学院化学研究所分子科学中心!北京100080 @李宝芳$中国科学院化学研究所分子科学中心!北京100080 @江龙$中国科学院化学研究所分子科学中心!北京100080~~     

细菌视紫红质的质子传输机理

细菌视紫红质（bR）是嗜盐菌紫膜中的唯一蛋白质成分, 具有质子泵、电荷分离和光致变色功能. bR分子中的发色团视黄醛通过质子化席夫碱以共价键与Lys216相连. bR分子受可见光照射后, 视黄醛发生从全-反到13-顺式构型的异构化, 导致席夫碱的去质子化,继之以可极化基团位置的改变. 力场的变化引起包括蛋白质三级结构在内的诸多变化, 这些变化促进并保证了质子从细胞质侧向细胞外侧的定向传输.     

细菌视紫红质光循环中间体M412的动力学初探

采用紫外可见吸收光谱技术和闪光光解技术,初步观察了细菌视紫红质(BR)分子在宽pH范围(2.1～12.3)内的特征吸收峰以及M412的相对浓度和M412的慢成分半衰期的变化,并对其结构和光循环功能进行了讨论.紫外可见吸收光谱实验结果显示:pH=5.0～10.0时,BR最大特征吸收峰值约为568 nm;pH＜5.0时,BR最大特征吸收峰发生红移;pH＞10.0时,BR最大特征吸收峰发生蓝移.闪光动力学光谱结果显示:pH为7.3～9.5时,M412的相对浓度(M0)基本稳定在0.038左右;pH＜7.3时,M0逐渐减小;pH＞9.5时,M0明显上升,在pH=11.8时达到最大值0.1355,随后又快速下降.pH为2.1～7.3时,M412的慢成分半衰期(ts1/2)值在(4.1±1.1)ms左右;pH＞7.3时,ts1/2值急剧延长到40 677.4 ms.推测在高pH条件下,BR分子的光循环有新的路径和机理.     

Photocurrent response of bacteriorhodopsin adsorbed on bimolecular lipid membranes

The photo response of bacteriorhodopsin adsorbed on a bimolecular lipid membrane has been investigated using short-circuit current measurements. The results revealed a biphasic current vs. time curve for the photocurrent at pH values of approx. 7. This phenomenon could be modified by altering either the value of the external applied electrical field or the proton concentration differences.The observed effects of the external applied voltage, pH gradient and lipophilic proton carriers enabled us to conclude that the bacteriorhodopsin can be adsorbed in two different states, which give rise to a pumping effect and a flux of protons in opposite directions.A theoretical analysis of the photocycle in relation to the electrical field which acts on the proton uptake and release is proposed. The main effect of this field is to diminish the pumping rate due to the proton motive force resulting from the creation of space-charge in the vicinity of purple membrane fragments.     

Electric field promotion of the bacteriorhodopsin BR570 to BR412 photoconversion in films of Halobacterium halobium purple membranes

The combined action of electric field (10⁵–10⁷ V · m^?1) and light (380–580 nm, 80 W · m^?2) activating the photoenergetic reaction of bacteriorhodopsin (BR) in dry films of purple membranes from Halobacterium halobium was studied. A new stimulating effect of the field on the BR₄₁₂ intermediate accumulation in the normal photochromic cycle of BR₅₇₀ has been observed. The formation of the product BR₄₁₂ is supposed to be accompanied by specific rearrangements of certain charged, polar and polarizable groups in the BR pigment-protein matrix. Such an intrinsic polarization could be promoted by an external electric field, the displacement vector of those groups being oriented in the direction of the field. The dielectric polarization properties of the purple membranes have been demonstrated by electret-thermal analysis.     

My remembrances of H.G. Khorana: exploring the mechanism of bacteriorhodopsin with site-directed mutagenesis and FTIR difference spectroscopy

H.G. Khorana’s seminal contributions to molecular biology are well-known. He also had a lesser known but still major influence on current application of advanced vibrational spectroscopic techniques such as FTIR difference spectroscopy to explore the mechanism of bacteriorhodopsin and other integral membrane proteins. In this review, I provide a personal perspective of my collaborative research and interactions with Gobind, from 1982 to 1995 when our groups published over 25 papers together which resulted in an early picture of key features of the bacteriorhodopsin proton pump mechanism. Much of this early work served as a blueprint for subsequent advances based on combining protein bioengineering and vibrational spectroscopic techniques to study integral membrane proteins.     

Applications of gray-level variation detection method to intracellular ice formation

Intracellular ice formation (IIF) is the major cause of death in cells subjected to freezing. The occurrence of intracellular ice prevents the penetration of light into the camera and makes the image dark. Therefore, the gray-level variation can reflect the IIF. However, cell deformation is accompanied with IIF, especially for larger cells. It is necessary to account this entire phenomenon together in a single method. In this paper, the normalized parameter C defined by the gray-level variation depending on the displacement was defined to reflect the gray-level change of each pixel point in the region of interest of the image. The process of IIF of onion epidermal cells and 293T cells was analyzed by this method.     

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。     

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.     

The absorbance spectrum of the brown holo-membrane and the comparison of pI values of bacteriorhodopsin solubilized from purple membrane and from brown holo-membrane

Brown holo-membrane was prepared by the addition of all-trans-retinal to brown apo-membrane which was isolated from Halobacterium halobium grown in the presence of nicotine. The effects of pH and NaCI concentration on the absorbance spectrum of the brown holo-membrane were investigated in comparison with those of the purple membrane. The λ_max of the dark-adapted brown holo-membrane shifted from 560 to 600 nm by lowering pH. The pK value which was determined as the mid-point pH for the spectral red-shift was 5.8 in the absence of NaCl. It was lowered to 4.5 and 3.4 in 0.1 and 1 M NaCl solutions, respectively. The pK value for the brown holo-membrane was larger than the corresponding value for the purple membrane in the NaCl solution. Bacteriorhodopsins present in the purple membrane and in the brown holo-membrane were solubilized in the nonionic detergent, lauryl ester of sucrose. For both solubilized bacteriorhodopsins, the pK value of spectral red-shift was about 3.1 in water, and the pI value, determined by chromatofocusing, was about 4.6 at 22°C.     

Effects of electric field on the photocycle of bacteriorhodopsin

The photoconversion of bacteriorhodopsin and the effects of an applied electric field (5 · 10⁷ V · m^?1) were studied in dry films of purple membranes from Halobacterium halobium. The electric field was found to cause at least two different effects: (1) it blocks in part the formation of the batho-bacteriorhodopsin (K), most probably due to electrically-induced dark transition of some bacteriorhodopsin molecules into the photochemically inactive form; (2) it decreases the rate of the intermediate M decay, the rise time of the M formation being unaffected by electric field. The observed phenomena may suggest a feedback control mechanism for the regulation of the bacteriorhodopsin photocycle in purple membranes.     

Light-driven proton transport of bacteriorhodopsin incorporated into long-term stable liposomes of a polymerizable sulfolipid

The chromoprotein bacteriorhodopsin from Halobacterium halobium has been incorporated into liposomes made of a fully synthetic, polymerizable lipid. Bacteriorhodopsin is found to be active in these polymer liposomes. The advantage in the use of such polymer systems concerning long-term stability in comparison with liposomes made of natural lipid is demonstrated.     

Effect of water on the structure of bacteriorhodopsin and photochemical processes in purple membranes

Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization.