细菌视紫红质 E204Q 和 I119T/T121S/A126T突变体的构建及其功能研究

应用定点突变的方法获得了细菌视紫红质 (bacteriorhodopsin , BR) 的单突变体 BR_E204Q和三突变体 BR_{I119T/T121S/A126T}. 通过功能研究发现, BR 蛋白的单突变体 BR_E204Q的 M 态寿命为 7.10 ms ,三突变体 BR_{I119T/T121S/A126T}的 M 态寿命为 8.23 ms ,均较野生型 BR 蛋白 (6.23ms) 有所延长,三突变体表现得更为显著,其 M 态延长时间可超出野生型的 32％. 同时单突变体 BR_E204Q和三突变体 BR_{I119T/T121S/A126T}的质子泵功能也均有改变,都比野生型 BR 蛋白有所下降,其中三突变体 BR_{I119T/T121S/A126T}下降得更为明显.     

BR—D96N光学薄膜的全息记录特性

BR D96N是具有显著光致变色特性的细菌视紫红质 (BR)经过基因定点突变的产物 ,与野生BR相比 ,它的M态寿命延长到了 5min ,因而表现出明显的饱和吸收特性和较低的饱和吸收光强 (0 .4mW /cm2 )。BR D96N用于全息记录时形成的是动态光栅 ,其特征量为光强而非曝光能量。实验表明 ,当再现光较弱时 ,衍射效率最大的记录光强基本对应于样品的饱和吸收光强。当记录光强一定时 ,再现光对所记录光栅具有擦除作用 ,再现光较弱时具有较高衍射效率 (1.8% ) ,但并不能得到高衍射光强 ,若要得到高衍射光强 ,存在着最佳再现光强 (80 μW /cm2 )。实验证明了在BR D96N薄膜上可以实现全息图像记录。     

细菌视紫红质膜质子(电荷)转移过程中的瞬态吸收研究

用自行设计的“连续泵浦－探测光＋脉冲泵浦光”的实验系统,研究细菌视紫红质（ＢＲ）的光循环过程;着重以Ｈｅ－Ｎｅ激光（６３２．８ｎｍ）为连续泵浦－探测光,研究在脉冲光共同作用下ＢＲ各能态的光吸收的动力学过程,及该吸收特性与两束光强度的关系     

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.     

一种同步观察喜树碱与菌根丛枝结构的方法

本文介绍一种用激光共聚焦扫描显微镜同步观察喜树碱、丛枝茵根及其结构的方法.喜树丛枝茵根经甘油浸润和明胶包埋后制成切片,再经酸性品红染色后在激光共聚焦扫描显微镜下观察.波长488mm处激发光下可获得清晰的丛枝茵根透射图像.364 nm处激发光下可获得喜树碱的荧光图像,通过两图像的叠加,在喜树丛枝菌根中得到喜树碱的定位图像.用此方法初步研究了喜树丛枝茼根的喜树碱分布.     

D97N突变对光受体蛋白古紫质4质子泵和能量转换效率的影响

古紫质4(archaerhodopsin 4,aR4)是新近发现的古生菌Halobacterium species xz515红膜上唯一的光敏视黄醛蛋白,具有和细菌视紫红质(Bacteriorhodopsin,bR)相似的质子泵功能,但在中性p H条件下,其质子释放和摄取顺序与bR相反。针对质子供体天冬氨酸97(Aspartic acid 97,D97)对aR4光循环;特别是对质子释放摄取顺序和菌株ATP生成率的影响,采用基因定点突变技术,构建了aR4的单突变体D97N,以及相对应的bR单突变体D96N。采用紫外-可见吸收光谱和闪光动力学光谱技术初步研究突变对视黄醛键合区、光反应中间态M态和O态、质子泵功能以及菌株ATP生成率的影响。结果表明,D97N突变对视黄醛紫外-可见光吸收波长没有太大影响,但造成M态衰减时间的显著延长、质子泵功能的消失及菌株ATP的生成率大幅降低。与bR中的D96作用相比,D97对aR4质子功能的影响有所不同,这可能与D97所处的一个更为疏水性的胞外侧环境有关。     

细菌视紫红质光学图像存储的灰阶特性研究

细菌视紫红质（Bacteriorhodopsin,BR）是一种具有优良光致变色特性的光敏蛋白分子,具有极好的抗疲劳性和高的光转换量子效率,可用于光学图像信息的获取和存储.文章讨论了BR分子膜在受到随空间位置变化的光强调制下,BR分子膜光吸收变化量的空间分布及其与存储图像灰度分布之间的关系;建立了BR灰度图像存储特性实验系统,并对BR-D96N薄膜存储的光学图像灰阶特性进行了实验研究,实验表明BR薄膜图像存储具有出色的灰度表现能力.     

温度对萝卜蚜繁殖特性的影响

在人工控制的5种温度条件下,研究了温度对生殖生物量的影响,用Weibull函数对前15天繁殖量进行了模拟,统计了各种温度下的繁殖寿命。结果表明:生殖起点温度为3.8958,与其发育起点温度接近;前15天繁殖量随温度降低呈落差态;高温下寿命缩短至30～35天,低温使寿命延长达85天。     

Time-resolved refractive index change during the bacteriorhodopsin photocycle

Kinetic refractive index spectroscopy has been applied to the study of the bacteriorhodopsin photocycle. A fully hydrated purple membrane film was examined in the temperature range from 10° to 40°C using 532 nm excitation (doubled Nd YAG laser) and 633 nm (He–Ne laser) testing beam. Multiexponential fitting of the data revealed five processes. Four of them are well known from kinetic optical absorption studies. The fifth process has only recently been observed in optical absorption experiments where it has a relatively small amplitude. In our refractive index experiments it has an amplitude of up to 30% of the full signal amplitude. It is characterized by an Arrhenius temperature dependence with an activation enthalpy of 40±5 kJ/mol and a decay time of about 0.8 ms at 20°C.     

用菌紫质薄膜传感器进行图像边缘检测的研究

应用菌紫质人工沉淀膜所具有的与动物视网膜类似的微分光电响应特性,以该人工膜为传感器构建了一个可检测图像边缘的原理系统,并成功地检测到了简单图像的边缘。本文的结果除进一步说明了菌紫质分子在图像技术中的应用前途外,还说明了这种分子在视觉功能模拟和人工视觉等方面的良好应用前景 。     

Evidence that the photoelectric response of bacteriorhodopsin occurs in less than 5 picoseconds

The initial photoinduced charge separation in bacteriorhodopsin is shown to occur in <5 ps. This result is obtained by measuring the photovoltage rise time in an oriented film of bacteriorhodopsin (BR). A dye laser syncronously pumped by an Argon ion cw mode locked laser is used to produce 3-ps light pulses which, after passing through a dye amplifier chain, photoexcite the BR sample. The photovoltage transient is detected by an ultra-fast Josephson junction digital sampling oscilloscope with liquid-helium-cooled input circuitry.     

Single bacteriorhodopsin molecules revealed on both surfaces of freeze- dried and heavy metal-decorated purple membranes

The flat sheets of the purple membrane from Halobacterium halobium contain only a single protein (bacteriorhodopsin) arranged in a hexagonal lattice. After freeze-drying at -80 degrees C (a method that is superior to air-drying), shadowing with tantalum/tungsten, and image processing, structural details on both surfaces are portrayed in the range of 2 nm. One surface is rough and lattice lines are clearly visible, whereas the other is smooth and the hexagonal order seems to be absent. The optical diffraction patterns, however, indicate a hexagonal lattice for both surfaces. In addition, these diffraction patterns are characteristic and easily distinguished. The orientation of the two surfaces was identified by silver decoration: partial condensation of silver on purple membranes enabled the smooth surface to be identified as the plasmatic and the rough surface as the exoplasmic surface. After image processing, the exoplasmic surface shows a triplet structure which exactly fits the projected structure determined by Unwin and Henderson (1975. Nature(Lond.). 257:28-32) at molecular resolution, whereas, on the plasmatic surface, four image details per unit cell are visible. Three of them match the arrangement of bacteriorhodopsin, whereas the fourth must be located over a lipidic array. Summarizing these results, it is possible to show the part of each single bacteriorhodopsin protein that is present in the surfaces of the purple membrane. By "shadowing" the membranes perpendicularly, we prove that these components of the surfaces are mainly portrayed by a decoration effect of the tantalum/tungsten condensate.     

The pH-dependence of photochemical intermediates of O and P in bacteriorhodopsin by continuous light

The pH-dependence of the O and P intermediates in the photocycle of bacteriorhodopsin (bR) on the intensity and duration of the exciting flash was investigated for bR glycerol suspensions and bR gelatin films. Green and red laser flashes (532 and 670 nm) were utilized to generate a photoequilibrium state of bR and O at ambient temperature, and UV-vis spectroscopy was used to determine the photoconversion for the bR suspensions and films. The maximal concentration of the O intermediate was observed to be pH-dependent and the dependency was most pronounced at a slightly alkaline pH values. The photochemical conversion from the O to P intermediate was investigated for both bR suspensions and films. The P intermediate was only found in bR gelatin film. These results indicate that bR gelatin film may be an attractive candidate for the information storage based on P intermediate. It is possible, with red light, to create photoproducts which are thermally stable at ambient temperature and that can be photochemically erased.     

Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures

Picosecond laser spectroscopic analysis was applied to determine how many intermediates existed in the primary photochemical process of trans-bacteriorhodopsin (light-adapted bacteriorhodopsin) at room temperature (18°C) and to calculate their absorption spectra. Irradiation of bacteriorhodopsin with a laser pulse (wavelength, 532 nm; pulse width, 25 ps) yielded the K intermediate (K) which was produced through a precursor, having an absorption maximum (λ_max) longer than that of K. K was stable during a picosecond time range (50–900 ps). The λ_max was located at 610 nm and the extinction coefficient (?_max) was 0.92-times that of bacteriorhodopsin. The same K intermediate was produced from bacteriorhodopsin even when it was excited with a high-energy pulse by which a saturation effect was induced. A transient difference spectrum measured at 150 ns after the excitation of bacteriorhodopsin was different in shape from that of the K intermediate, suggesting that an intermediate was formed by thermal decay of K. This intermediate, tentatively called the KL intermediate (KL), had a λ_max at 596 nm and an ?_max 0.80-times that of bacteriorhodopsin. KL decayed to the L intermediate (L) with a time constant of 2.2 μs. L has a λ_max at 543 nm and an ?_max 0.66-times that of bacteriorhodopsin.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field induced conformational changes of bacteriorhodopsin were studied in six types of dried film (randomly and electrically oriented membranes of purple as well as cation-depleted blue bacteriorhodopsin) by measuring the frequency dependence of the optical absorbance change and the dielectric dispersion and absorption. For the purple bacteriorhodopsin the optical absorbance change induced by alternating rectangular electric fields of ±300 kV/cm altered the sign twice in the frequency range from 0.001 Hz to 100 kHz (around 0.03 Hz and 100 kHz), indicating that the electric field induced conformational change in these samples consists of, at least, three steps. Similarly, it was found for the blue bacteriorhodopsin that at least two steps are involved. In accord with optical measurements, the dielectric behaviour due to alternating sinusoidal electric fields of±6kV/cm in the frequency range from 10 Hz to 10 MHz showed two broad dispersion/absorption regions, one below 1 kHz and the other around 10–100 kHz. This suggests that the conformational change of bacteriorhodopsin is also reflected by its dielectrical properties and that it is partially induced at 6 kV/cm. Including previous results obtained by analysis of the action of DC fields on purple membrane films, a model for a field-induced cyclic reaction for purple as well as blue bacteriorhodopsin is proposed. In addition it was found that there are electrical interactions among purple membrane fragments in dried films.     

Two-photon absorption of bacteriorhodopsin: formation of a red-shifted thermally stable photoproduct F620

By means of high-intensity 532 nm laser pulses, a photochemical conversion of the initial B(570) state of bacteriorhodopsin (BR) to a stable photoproduct absorbing maximally at approximately 620 nm in BR suspensions and at approximately 610 nm in BR films is induced. This state, which we named F(620), is photochemically further converted to a group of three products with maximal absorptions in the wavelength range from 340 nm to 380 nm, which show identical spectral properties to the so-called P(360) state reported in the literature. The photoconversion from B(570) to F(620) is most likely a resonant two-photon absorption induced step. The formation of F(620) and P(360) leads to a distinguished photo-induced permanent optical anisotropy in BR films. The spectral dependence of the photo-induced anisotropy and the anisotropy orientations at the educt (B(570)) and product (F(620)) wavelengths are strong indicators that F(620) is formed in a direct photochemical step from B(570). The chemical nature of the P(360) products probably is that of a retro-retinal containing BR, but the structural characteristics of the F(620) state are still unclear. The photo-induced permanent anisotropy induced by short laser pulses in BR films helps to better understand the photochemical pathways related to this transition, and it is interesting in view of potential applications as this feature is the molecular basis for permanent optical data storage using BR films.     

The photocycle of bacteriorhodopsin immobilized in poly(vinyl alcohol) film

The kinetics of photoelectric and optical signals were measured on samples containing oriented purple membranes immobilized in a poly(vinyl alcohol) film and on purple membranes introduced into a PVA-H2O mixture. The bacteriorhodopsin photocycle in the PVA-H2O mixture was complete. The only observed changes were the slowing down of the optical and electrical signals in relation to the M412-O640 and O640-bRall-trans steps. In the PVA film the O640 intermediate disappeared and a negative photoelectric signal appeared.     

Virtual retinal display system with nodal point image of a laser beam: construction example and evaluation of subjective brightness perception]

We investigated the perception of brightness for red monochromatic laser light. For this purpose, a modified virtual retinal display (VRD) was constructed. The modification involved projecting the laser beam into the eye. In our VRD, the laser beam pivots in the nodal point of the eye (badal system). The displayed image therefore does not depend on the refractive state of the eye. Brightness perception was assessed by means of psychophysical experiments. The results of these experiments indicate that perception of brightness at 652 nm increases more rapidly with increasing physical stimulus than does the perception of white light (colour temperature 2'935 K). At a wavelength of 652 nm, an optical power of 79 nW is required to produce an image subtending 2 degrees of equal brightness, as is perceived with an image of about 2'900 cd/m2 subtending 2 degrees.