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。     

缬氨霉素以及膜电位对菌紫质分子在脂质囊泡膜中运动的影响

用瞬间二向色性方法测量了菌紫质分子在DMPC脂质囊泡膜中的旋转扩散运动.观察了缬氨霉素和膜电位对菌紫质分子旋转扩散运动的影响.在低脂与蛋白比例时,缬氨霉素明显影响菌紫质分子的旋转扩散运动,在高脂与蛋白比例时,缬氨霉素对菌紫质分子的旋转扩散动的影响不明显.无论在高的或低的脂与蛋白比例下,膜电位都影响到菌紫质分子的旋转扩散运动.     

蜂毒素与紫膜的相互作用机理研究──与三种表面活性剂的比较

用吸收光谱和圆二色谱的方法研究了蜂毒素与嗜血菌紫膜的相互作用机理．通过与三种在结构和电荷上不同的表面活性剂与紫膜的作用相比较,可以年出蜂毒作为带正电荷的分子与同样带正电的表面活性剂ＤＴＡＢ在引起紫膜凝聚方面表现相同;但在对紫膜可见光区的吸收光谱和圆二色谱的影响上却与具有刚性结构的ＣＨＡＰＳ相似,表明蜂毒可在紫膜表面以一种刚性较大的构象（如α螺旋）存在,不能进入膜蛋白流水区的深层．另外,从紫膜－Ｔｒｉｔｏｎ－蜂毒混合作用体系的研究中得到如下推测：蜂毒与Ｔｒｉｔｏｎ竞争菌紫质分子周围的结合位点,可排斥位于菌紫质周围的Ｔｒｉｔｏｎ分子．表明蜂毒具有比Ｔｒｉｔｏｎ更强的与菌紫质的亲和力,从而提供了支持蜂毒分子存在与膜蛋白－菌紫质的直接相互作用的有力证据．     

冷冻干燥对紫膜菌紫质圆二色性的影响

无论是先冷冻后干燥,还是先干燥后冷冻,或只冷冻处理,或只干燥处理的干湿紫膜样品,也无论是在处理后将其悬浮在水中,或在十六烷中,或在大豆磷脂正己烷液中,紫膜中的菌紫质分子均丧失了天然紫膜水悬浮液中的圆二色谱的双相性。然而,对于先冷冻后干燥,只干燥处理的紫膜水悬浮液,这种双相性可以缓慢地恢复。该现象提示,冷冻和/或干燥处理对紫膜菌紫质的圆二色性的影响,可能主要通过影响脂区间接实现的;用环三体激子理论难以圆满解释该现象。     

紫膜菌紫质分子的光生物物理学研究

本文介绍了紫膜细菌视紫红质的微观结构,质子泵功能和光循环过程,及其菌紫质分子中发色团和蛋白质相互作用(结合位点)的几种光谱研究,最后讨论了菌紫质分子原初事件量子产率的双重性和相应的分子动力学模型。     

温度对脂质囊泡中单体菌紫质分子结构和光电响应的影响

本实验用人工双分子平板膜系统(BLM)测量了紫膜碎片和在DMPC脂质襄泡膜中的单体菌紫质分子的光电响应以及与温度的关系(处理温度17℃至31℃).温度对紫膜碎片的光电响应影响不大,但对单体菌紫质分子的光电响应有明显影响.用园二色(CD)方法相应地观察了温度对紫膜碎片和单体菌紫质分子在可见波长范围内的CD谱的影响 同样观察到温度对单体菌紫质分子的CD谱有明显影响.两者的影响很可能与脂质襄泡中DMPC的相变温度有关.     

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.     

菌紫质分子的重组对结构及运动的影响

本实验用闪光诱导的瞬间二向色性方法分别测量了用全反视黄醛重组以及用四溴荧光素标记的菌紫质分子在脂质囊泡膜中的旋转扩散运动以及吸收过程随时间的变化(寿命).用全反视黄醛重组后不影响菌紫质分子在膜中的旋转扩散运动,它们与蜂毒的相互作用也无明显差别.用四溴荧光素标记的菌紫质分子的寿命很短,不可能用于测量菌紫质分子在膜中的旋转运动.     

付里叶变换偏振红外光谱法研究紫膜LB膜内菌紫质的取向

用付里叶变换偏振红外法测量了紫膜LB膜内菌紫质的酰胺Ⅰ、Ⅱ和A带的二色性,计算了菌紫质的α-螺旋取向.实验表明,紫膜内菌紫质的α-螺旋轴同膜法线的平均夹角为13°,即与膜平面的平均夹角为77°.     

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     

温度及膜中蛋白质含量与膜粘度和膜中分子排列方向的关系

本实验用闪光诱导的瞬间二向色性方法测量了不同温度以及不同蛋白质含量下菌紫质分子在脂质囊泡膜中的旋转扩散运动.根据旋转扩散运动得到了温度和蛋白质含量与膜粘度以及分子在膜中排列方向的关系.温度和蛋白质的含量都影响膜的粘度,但并不影响蛋白质分子在膜中的排列方向.     

The role of the native lipids and lattice structure in bacteriorhodopsin protein conformation and stability as studied by temperature-dependent Fourier transform-infrared spectroscopy

We report the effect of partial delipidation and monomerization on the protein conformational changes of bacteriorhodopsin (bR) as a function of temperature. Removal of up to 75% of the lipids is known to have the lattice structure of the purple membrane, albeit as a smaller unit cell, whereas treatment by Triton monomerizes bR into micelles. The effects of these modifications on the protein secondary structure is analyzed by monitoring the protein amide I and amide II bands in the Fourier transform-infrared (FT-IR) spectra. It is found that removal of the first 75% of the lipids has only a slight effect on the secondary structure at physiological temperature, whereas monomerizing bR into micelles alters the secondary structure considerably. Upon heating, the bR monomer is found to have a very low thermal stability compared with the native bR with its melting point reduced from 97 to 65 degrees C, and the pre-melting transition in which the protein changes conformation in native bR at 80 degrees C could not be observed. Also, the N[bond]H to N[bond]D exchange of the amide II band is effectively complete at room temperature, suggesting that there are no hydrophobic regions that are protected from the aqueous medium, possibly explaining the low thermal stability of the monomer. On the other hand, 75% delipidated bR has its melting temperature close to that of the native bR and does have a pre-melting transition, although the pre-melting transition occurs at significantly higher temperature than that of the native bR (91 degrees C compared with 80 degrees C) and is still reversible. Furthermore, we have also observed that the reversibility of this pre-melting transition of both native and partially delipidated bR is time-dependent and becomes irreversible upon holding at 91 degrees C between 10 and 30 min. These results are discussed in terms of the lipid and lattice contribution to the protein thermal stability of native bR.     

Eu3+ binding to europium-regenerated bacteriorhodopsin upon delipidation and monomerization

We have studied the effect of monomerization of the purple membrane lattice, as well as removal of 75% of the lipids, on the binding properties of Eu(3+) ions. We found that delipidation and monomerization do not cause the cations to lose their binding ability to the protein. This suggests that the three most strongly bound Eu(3+) cations do not bind to the lipids, but directly bind to the protein. Furthermore, we found that delipidation actually causes a slight increase in the binding affinity. This is likely a result of reduced aggregation of europium-regenerated bacteriorhodopsin (bR) upon lipid removal causing more exposure of the binding sites to the Eu(3+) cations. These results, taken with those from our previous publication [Heyes and El-Sayed, Biophys. J. 85 (2003) 426-434], might suggest that the cations remain bound upon delipidation of bR, but have no effect on the function. This is discussed with respect to the role of cations in the function of native bR.     

Bicelle size modulates the rate of bacteriorhodopsin folding

The conformational equilibria of integral membrane proteins have proven extremely difficult to characterize within native lipid bilayers. To circumvent technical issues, investigations of the structure and stability of α‐helical membrane proteins are often carried out in mixed micelle or bicelle solvents that mimic the membrane and facilitate measurements of reversible folding. Under these conditions, the energetics of membrane protein folding are typically proportional to the mole fraction of an anionic detergent in the micelle. However, investigations of the folding and unfolding of bacteriorhodopsin (bR) surprisingly revealed that the folding rate is also highly sensitive to the bulk molar concentration of lipids and detergents. We show here that this rate enhancement coincides with changes in bicelle size and suggest this effect arises through restriction of the conformational search space during folding. In conjunction with previous mutagenic studies, these results provide additional evidence that a topological search limits the rate of bR folding. Furthermore, this finding provides insights into the manner by which micellar and bicellar environments influence the conformational stability of polytopic membrane proteins.     

用神经网络法预测膜蛋白RH和BR的二级结构

膜蛋白的结构预测目前还十分困难.本文以两个典型的膜蛋白为选材,用神经网络方法对其二级结构做了预测.预测结果与实验资料的符合率与该方法用于球蛋白时的结果一致,因此是较好的.鉴于目前还没有人将此方法用于膜蛋白的结构预测.我们拟进一步发展此方法.     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

嗜菌紫膜表面电位和质子泵效率间的关系

本文研究了中性红与紫膜结合,并用直接滴定法和测定结合量法,求得了结合于膜上之中性红本征PK和在不同离子强度时的表观PKa值.从它们的差值中计算得到紫膜表面电位.并得不同盐浓度的表面电位和质子泵效率作了比较.结果说明.阳离子对质子泵效率的影响不能完全用表面电位的变化来说明.这一结果暗示了一定的阳离子对于紫膜质子泵功能的完成有着特殊作用.     

N-溴代琥珀酰亚胺修饰菌紫质中色氨酸的光谱学研究

采用紫外-可见吸收光谱和荧光光谱方法研究了菌紫质（Bacteriorhodopsin,bR）中的8个色氨酸（Tryptophan,Trp）残基在被N-溴代琥珀酰亚胺（N-bromosuccinimide,NBS）修饰过程中的残基数目及对应的光谱变化。研究结果显示：随着NBS／bR摩尔比例增加逐渐被修饰的Trp残基有4个左右,如果NBS过量,则Trp残基的修饰个数最终可迭6～7个;伴随化学修饰出现Trp残基特征荧光峰值下降及峰位蓝移。研究结果揭示了bR中Trp残基可能的三种结构分布,对于进一步弄清bR中Trp-视黄醛（Retinal）偶联能量传递、单独Trp残基的荧光寿命和Tm残基在膜蛋白结构和功能中的作用具有积极而重要的意义。