菌紫质、磷脂与水的相互作用:红外光谱与吸附重量研究

应用紫膜等温吸附及红外差光谱技术研究细菌紫膜中水与蛋白、磷脂的相互作用.观察到酰胺Ⅰ、Ⅱ、Ⅲ带在R<0.05及0.08相似文献   

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.     

湿度和温度对酰化紫膜LB膜M衰减的影响

用闪光动力学光谱仪测量了酰化紫膜ＬＢ膜中Ｍ衰减速率的变化。酰化紫膜ＬＢ膜的衰减无论是悬浮液状态,还是ＬＢ膜中,均比未修饰的要慢。在温度为２０℃时,酰化紫膜ＬＢ随着相对湿度的增加,Ｍ衰减加快。在相对湿度较低时（ＲＨ３４—７５％）,变化较平缓,即Ｍ的衰减加快不明显;在相对湿度较高时（ＲＨ８４—９５％）,Ｍ衰减明显加快。温度的变化则随相对湿度不同而不同。相对湿度较低时,随着温度的升高,Ｍ衰减加快;相对湿度较高时,Ｍ衰减反而减慢。酰化紫膜悬浮液的Ｍ衰减随着温度的升高而明显加快．这说明酰化紫膜ＬＢ膜中ＢＲ水合程度可能是直接影响Ｍ衰减的因素之一。     

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.     

Triton X－100对紫膜蛋白的效应

本文采用同步辐射小角Ｘ射线散射方法研究了用非离子表面活性剂ＴｒｉｔｏｎＸ—１００处理后的嗜盐菌紫膜及其视紫红质蛋白结构的变化。实验结果表明,用不同浓度的ＴｒｉｔｏｎＸ—１００处理紫膜碎片时,紫膜及其蛋白所处的状态有着很大变化。     

病毒蛋白脂酰化及其功能

脂酰化是一种重要的蛋白翻译后修饰,主要包括棕榈酰化、豆蔻酰化、异戊烯化和糖基化磷脂酰肌醇(GPI)共价结合4种方式。不同的病毒蛋白可发生不同类型的脂酰化,其生物学功能也会发生相应改变。棕榈酰化通常能增强病毒跨膜蛋白的疏水性,调节这些蛋白的胞内运输及定位,进一步影响病毒感染过程中的膜融合、病毒颗粒装配及释放等步骤。豆蔻酰化则可调控病毒蛋白表面的正电荷强度,使病毒蛋白与脂质膜的亲和力改变,如preS1豆蔻酰化加强乙型肝炎病毒(HBV)和丁型肝炎病毒(HDV)的受体识别能力及感染性,而人类免疫缺陷病毒(HIV)Nef豆蔻酰化为病毒感染及免疫应答所必需。异戊烯化能使病毒游离的蛋白与膜结合,并介导蛋白间的相互作用,如大HDV抗原(L-HDAg)异戊烯化有利于其运输至内质网膜上,与HBV表面抗原(HBsAg)及HDV RNA共同形成HDV颗粒。此外,一些病毒蛋白与GPI通过共价结合形成复合物,GPI基团可改变感染细胞的膜结构及胞质内磷脂构成,如GPI与朊蛋白(PrP)结合导致细胞型朊蛋白(PrPc)交联或羊痒疫朊蛋白(PrPsc)聚集,与朊病毒引起的海绵样病变有关。进一步了解病毒蛋白脂酰化机制,有利于设计和开发以此为靶点的特异性抗病毒新药。     

肼类与蓝膜的相互作用

采用紫外可见光谱、动力学光谱和荧光光谱,研究了肼、甲基肼和偏二甲基肼与蓝膜的相互作用。结果表明这些肼类都可使蓝膜恢复为紫膜,并恢复紫膜原有的光循环方式。但是,中间体(M412)的衰減速率加快,这在金属离子重组紫膜中没有发生。另外,当肼加入蓝膜时,荧光光谱没有明显变化;而去掉金属离子的蓝膜,其荧光強度明显增加。     

膜脂环境对紫膜蛋白的影响

紫膜蛋白菌紫质（bacteriorhodopsin,BR）处于不同的膜脂环境中,通过吸收光谱、荧光光谱和闪光动力学光谱的测定,比较了3种不同膜脂环境对菌紫质分子结构和功能的影响。实验结果表明：天然膜脂是BR最稳定的膜脂环境,可以形成以三聚体为单位的二维六角形品格结构。二豆蔻酰脂酰胆碱（dimyristoyl phosphatidylcholine,DMPC）也是BR分子的稳定脂环境,但以单体形式存在于膜脂环境中,功能受到一定影响。经TritonX-100增溶处理的紫膜膜脂环境中BR为单体,不稳定,容易发生结构和功能的变化。     

紫膜的紫外荧光及分子内的能量转移

以可见光为作用光照射天然紫膜,紫膜蛋白被280nm紫外光激发所发射的荧光强度比对照略有降低.比较天然紫膜、漂白紫膜与菌蛋白三者的紫外荧光强度,前两者无显著变化,但菌蛋白的荧光强度比天然紫膜的荧光强度大2-3倍,表明生色团对蛋白质荧光可能有猝灭作用.用280nm波长光照射紫膜的暗适应形式,可使其转变成光适应形式.若有羟胺存在,以紫外光照射也可使紫膜漂白.光漂白的作用光谱,其紫外部分与紫膜蛋白部分的吸收光谱重合得很好.上述实验证明紫膜蛋白部分吸收的能量可以转移到生色团上,即紫膜存在分子内的能量转移     

高碱性pH诱导紫膜片层表面结构变化的原子力显微镜直接观测

报道了在高碱性pH下,紫膜中细菌视紫红质(BR)表面结构变化的直观信息.紫外可见光谱实验发现,当pH上升到12.6,BR分子上的生色团视黄醛脱落,分子完全变性;原子力显微镜实验观测到在此pH下,紫膜片层的晶格结构瓦解,BR分子在紫膜上无规则聚集,同时出现非特征“岛屿”结构和特征“岛屿”结构.     

Surface Charge Movements of Purple Membrane During Light-Dark Adaptation

The difference in the surface charge distribution between light-adapted and dark-adapted purple membranes was investigated with electric dichroism measurements from approximately pH 5 to pH 11. Purple membrane sheets in solution are oriented in a weak electric field by their permanent dipole moment, which is due to the charge distribution of the membrane surfaces and/or within the membrane. The degree of orientation of purple membrane sheets was obtained from the measurement of “electrical anisotropy” of retinal chromophore in the membranes. At about pH 7, there was no difference in the “electric anisotropy” between light- and dark-adapted purple membranes. At about pH 9, the electric anisotropy of dark-adapted purple membrane was larger than that of light-adapted purple membrane. But at around pH 6 the difference was opposite. Linear dichroism experiments did not show any change of retinal tilt angle with respect to the membrane normal between the two forms from approximately pH 5 to pH 10. This result indicates that the changes in the “electric anisotropy” are not due to the change of retinal tilt angle, but due to the change in the permanent dipole moment of the membrane. To estimate the change in surface charges from the permanent dipole moment, we investigated the difference of the permanent dipole moment between the native purple membrane and papain-treated purple membrane in which negative charges in the cytoplasmic-terminal part are removed. This estimation suggests that this light-dark difference at around pH 9 can be accounted for by a change of ~0.5 electric charge per bacteriorhodopsin (bR) molecule at either of the two surfaces of the membrane. We also found from pH electrode measurements that at about pH 8 or 9 light adaptation was accompanied by an uptake of ~0.1 protons per bR. A possible movement of protons during light-dark adaptation is discussed. The direction of the permanent dipole moment does not change with papain treatment. The permanent dipole moment in papain-treated purple membrane is estimated to be 27 ±2 debye/bR.     

Protein-chromophore interactions in bacteriorhodopsin: the effects of a change in surface potential.

The chromophore retinal is bound to bacteriorhodopsin via a protonated Schiff base linkage. The retinal binding site is reported to be buried in the transmembrane portion of the protein, distant from the membrane surfaces. When bound to bacteriorhodopsin, the absorption maximum of retinal is red-shifted from 366 nm to 568 nm producing a purple color. This color persists across a wide pH range. However, when the pH is raised above 12.0, the membranes become pink in color, while at pH values of 3.0 or below, a blue color is produced. The blue color can also be obtained by removing the divalent cations bound to the surface of the protein. In this study, bacteriorhodopsin was examined by circular dichroism and absorption spectroscopy to determine if protein conformational changes were associated with the color shifts. It was found that although the retinal chromophore can be completely removed by bleaching with hydroxylamine with no significant influence on the secondary structure of the protein, a change in the surface charge of bacteriorhodopsin results in measurable conformational change in the protein, which apparently affects the nature of the retinal binding site.     

Photochemical and chemical studies on the chromophore of bacteriorhodopsin.

The chromophore (purple complex) of bacteriorhodopsin is reduced by sodium borohydride upon illumination to RPhv with a three-peaked absorption band at 360 nm. Treatment of this reduction product with ultraviolet light or acid yields a modified product from which retro-retinyllysine can be obtained by alkaline hydrolysis. No reduction of the 412 nm complex was found. Under specific conditions the purple complex equilibrates with a photochemically active 460 nm form that can be reduced by borohydride in the dark. This reduction product RP460 behaves idential to RPHV. Reconstitution of the purple complex from chromophore-free membrane (apomembrane) and retinal occurs via intermediates. The first (lambdamax 400nm) shows a three-peaked absorption band and is reduced to RP400 without a change of the three-peaked absorption (lambdamax 360 nm). The same product is obtained from apomembrane and retinol. Detergents shift the absorption band to 330 nm in all cases. From the experiments described no participation of retro-retinal structures during the photochemical cycle can be concluded but stereospecific interaction of the retinal moiety with the protein resulting in a specific retinal conformation os omdocated by the spectral changes observed.     

Structure and thermal stability of monomeric bacteriorhodopsin in mixed phospholipid/detergent micelles

Thermal unfolding experiments on bacteriorhodopsin in mixed phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 +/- 13 A in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

血卟啉衍生物（HPD）对紫膜菌紫质（bR）的光敏化作用

研究了血卟啉衍生物（ＨＰＤ）对嗜盐菌紫膜上蛋白质菌紫质（ｂＲ）的光敏化作用,结果表明,ＨＰＤ与紫膜结合并不影响ｂＲ的光学性质及活性;但经光照射、ＨＰＤ光敏反应后,ｂＲ丧失光循环活性。进一步的探测显示ｂＲ中的视黄醛色素团及色氨酸均在光敏反应中受损,反映了除视黄醛色素团有可能直接受损外,深埋于折叠蛋白内部的部分色氨酸残基。亦可能在ＨＰＤ光敏化过程中被损伤。实验证明,单线态氧（＾１Ｏ２）的作用是ＨＰＤ光     

A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.     

Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol.

The molecular motion of retinal within the purple membrane was investigated by flash-induced absorption anisotropies with or without ethanol. In the absence of ethanol, the measured anisotropies at several wavelengths exhibited almost the same slow decay. This slow decay was attributed to only the rotation of purple membrane sheet itself in the aqueous suspension. In the presence of ethanol, however, we observed the wavelength-dependent anisotropies. The fluidity of the purple membrane, investigated with a fluorescence anisotropy method, was increased by the addition of ethanol. These facts indicated that the characteristic motion of bacteriorhodopsin is induced in perturbed purple membrane with ethanol. The data analysis was performed, taking account of the overlapping of absorption from ground-state bacteriorhodopsin and photointermediates. The results showed that the rotational motion of photointermediates within the membrane was more restricted than that of nonexcited bacteriorhodopsin. The addition of ethanol facilitated the rotation of nonexcited protein, whereas it did not significantly affect the motion of photointermediates. The restricted motion of photointermediates is probably caused by a conformational change in them, which may hinder the rotation of monomer protein and/or induce the interaction between photointermediate and neighboring proteins.     

Changes in the protonation state of bacterio-opsin during reconstitution of bacteriorhodopsin

Protonation changes of the protein occur during the reconstitution of bacteriorhodopsin from bacterio-opsin and all-trans retinal in the purple membrane of Halobacterium halobium. The protonation changes are conveniently determined from measures of the pH changes after photoisomerisation of 9-cis retinal in apomembrane preparations, which induces the reconstitution. In addition, to the omega-amino group of the lysine which is involved in the condensation of retinal and bacterio-opsin, the dissociation equilibria of at least two other amino acid residues are changed during the reconstitution. The results are consistent with a proposed model of chromophore structure in which an interaction of the Schiff's base occurs with two protonable amino acid residues.     

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.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field-induced absorption changes of bacteriorhodopsin were studied with different samples of purple membranes which were prepared as randomly oriented and electrically oriented films of purple as well as cation-depleted blue bacteriorhodopsin. The absorption changes were proportional to the square of the field strength up to 300 kV/cm. The electric field from the intracellular side to the extracellular side of the purple bacteriorhodopsin induces a spectrum change, resulting in a spectrum similar to that of the cation-depleted blue bacteriorhodopsin. When the field was removed, the purple state was regenerated. The blue state was mainly affected by an electric field in the opposite direction, suggesting a reversible interaction with the Schiff's base bond of the retinal. Since the field-induced reaction of bacteriorhodopsin was observed in the presence of a concomitant steady ion flux, it is assumed that the generation of a local diffusion potential may play an important role in these spectral reactions. Although the fragments were fixed in the dried film, electric dichroism was observed. The dichroic contribution of the total absorbance change was about 15%. The angular displacement of the retinal transition moment was calculated to be 1.5° toward the membrane normal.