【作者】 马德旺；

【导师】 丁建东；

【作者基本信息】 复旦大学 ， 高分子化学与物理， 2010， 博士

【摘要】 光敏蛋白是一种将光能转换成化学能或生理信号的生物大分子,在光合成和视觉感触方面具有举足轻重的作用。而细菌视紫红质(bacteriorhodopsin, BR)便是这类光敏蛋白中的典型代表。这种蛋白质被发现后,其所具有的独特性质引起了许多科学工作者的兴趣,很多科学工作者对其技术应用进行了研究,甚至提出了利用BR制造“蛋白质计算机”的构想。鉴于活性蛋白质自身难以成为可直接投入使用的材料,为了将来能够实现器件化和产品化,通常将BR包埋于某种合适的聚合物基质中,制作含有BR的复合材料。然而,合成高分子对于此类活性蛋白质的影响远未得到充分研究,这限制了含有光敏蛋白的材料制备技术的提高；此外,研究合成高分子与生物大分子相互作用本身也是具有一定普遍意义的基础研究课题。本博士论文课题从生物和材料学科交叉的角度入手,研究合成高分子聚合物对这种具有跨膜定向质子传递功能和光化学循环特性的BR和另一相关蛋白质古紫质4(archaerhodposin, AR4)的影响。在此基础上,综合运用化学手段和基因工程手段对上述两种膜蛋白进行改性,并将其与具有良好加工性能和力学性能的聚合物材料相结合,为制备高性能的活性蛋白质介导的复合材料提供新思路。本论文的主要创造性工作为：(一)首次发现两亲性嵌段共聚物对于细菌视紫红质的功能具有超过两亲性小分子和普通高分子的十分显著的影响,并予以了解释。研究了两亲性嵌段共聚物聚氧乙烯-b-聚氧丙烯-b-聚氧乙烯(PEO-PPO-PEO,商业名称Pluronic)对膜蛋白BR功能的影响。发现经过Pluronic处理后,BR保留了其光驱质子泵行为,但其光激发后的质子提取的时间和M中间态的寿命得到约3个数量级的显著延长；而加入均聚物聚氧乙烯(PEO)和小分子去垢剂却未发现如此显著的效应；通过圆二色谱仪检测发现,经过Pluronic处理后的BR仍然是以三聚体结构存在；而且经过Pluronic处理后BR的中间态寿命几乎不受水含量的影响；通过比较有无BR膜蛋白存在两种情况下的P123的临界胶束浓度,发现这种延长效应与共聚物胶束的形成具有密切的关联；而两亲性嵌段共聚物和膜蛋白的自组装和其所导致的紫膜表面所形成一层聚合物涂层可能是这一非常显著的延长效应的原因。(二)进一步发现两亲性合成高分子对于光敏膜蛋白的影响程度与蛋白质或膜脂种类有关。研究了嵌段共聚物Pluronic对膜蛋白AR4功能的影响,并与小分子去垢剂对AR4功能的影响进行比较。发现经过Pluronic处理后,AR4的M中间态寿命延长仅约一个数量级,与同样受Pluronic处理后BR的M中间态寿命延长三个数量级相比较,相差甚多。通过分析比较大分子表面活性剂Pluronic和小分子去垢剂Triton X-100分别对BR和AR4的M中间态的影响,提出其原因可能是由于紫红膜中的玉红素保护了AR4蛋白质,使得AR4蛋白质分子具有相对较高的稳定性。(三)综合运用两亲性共聚物水凝胶包埋与BR基因工程突变技术,制备了迄今为止在高水含量条件下具有最长M中间态寿命的含细菌视紫红质的功能材料。我们合成了F127-DA大单体并将其首次用于包埋BR并采用紫外光交联技术制备了复合水凝胶材料。对Pluronic大分子进行末端基丙烯酸修饰并不影响其与膜蛋白之间的自组装作用。向突变体BR-D96N溶液中加入嵌段共聚物F127-DA进行改性,并通过光交联大单体制备复合水凝胶材料,并且将膜蛋白的M中间态与基态之间的光致变色效应延长到半个小时以上。据我们所知,这是在高含水体系中最长的记录。(四)制备了光敏蛋白与合成高分子的系列复合膜,并通过合作研究证实,复合膜具备信息存储和处理的功能。将膜蛋白包埋于具有良好光学透明性和加工性能的聚合物基质中制备了复合膜材料并进行了初步的光学性能检测。我们通过基因工程技术成功去除了由菌株xz515中所提取到的野生AR4膜蛋白周围脂环境中的玉红素,优化了AR4的光致变色性能；同时获得了突变体BR-D96V,提高了BR的光学性能。我们首次初步尝试制备单聚体分散的细菌视紫红质/聚合物复合膜,采用具有良好生物相容性的聚氧乙烯-二丙烯酸酯(PEG-DA)大单体作为分散膜蛋白的基质,通过交联聚合制备膜蛋白/聚合物的新型复合材料；同时尝试以PEG-DA大单体为基质,制备了单聚体BR/聚合物复合材料；单聚体的稳定性仍待提高。我们利用包埋于聚乙烯醇(PVA)中的膜蛋白复合膜实现了简单的可视化的图案记录,初步表明该活性蛋白质与聚合物的复合材料具有光信息存储功能；还通过合作者的光学研究证明,BR-D96V/PVA复合膜可在部分信息存储和光信号处理中作为一种独到的新型功能材料。更多还原

【Abstract】 Photochromic proteins, which transform light energy into chemical or physiological signals, play a key role in photosynthesis and visual perception. A typical photochromic protein is bacteriorhodopsin (BR). Since its discovery, the unique properties of BR have attracted many scientific researchers. Various potential applications have been suggested including even the conceptual assumption of ’protein computer’. Since active proteins themselves are, due to poor processibility, hard to be applied straightforwardly as a "material", a strategy is preparation of polymer-based composite containing BR. However, the influence of synthetic polymers on retinal proteins is far away to be sufficiently explored, which limits the improvement of BR-related material techniques. On the other hand, the interactions between synthetic polymers and active biomacromolecules constitute a fundamental topic in the natural science, which deserves extensive investigation.This Ph.D thesis is focused upon examinations of effects of synthetic polymers on functions of active proteins. BR and its natural analog called archaerhodposin 4 (AR4) were employed as model proteins. Composite materials were obtained via combining polymeric technique and gene engineering technique, which shed light onto the functional polymeric materials including active biomacromolecules.The original work and results are summarized as follows:(1) It has been unexpectedly found that amphiphilic macromolecules influence the function of BR much more significantly than either normal polymers or small molecular amphiphiles, and an interpretation has been further put forward. Effects of a macromolecular amphiphile poly(ethylene oxide)-b-poly (propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) on functions of BR were examined. After incubation of BR in Pluronic solutions, BR maintained its function of a light-driven proton pump; however, the rate of proton uptake and lifetime of the M intermediate of BR upon illumination were, under appropriate conditions, prolonged for about three orders of magnitude compared with that of native BR, even at neutral pH. Addition of homopolymer PEO or small molecular detergent Triton X-100 didn’t result in so significant effects. BR molecules were still in a trimer state after treatment by the copolymers. And the functions of BR/P123 assemblies were not significantly influenced by water content. Determination of critical micelle concentration of P123 with and without BR reveals that this prolongation is closely related to formation of micelles. And formation of a local polymer coating due to self assembly of the copolymer and protein molecules might be responsible for this very significant prolongation effect, which is beneficial for some potential application of BR as information materials.(2) We further found that the effects of amphiphilic synthetic polymers on functions of retinal proteins were dependent upon kinds of proteins or surrounding lipids. Effects of Pluronic copolymers PEO-PPO-PEO on functions of AR4 were also examined. After treatment by Pluronic, lifetime of the M intermediate of AR4 was only one order of magnitude longer than that without Pluronic, and that is quite different with the results from BR treated by Pluronic. Comparison of effects of various detergents on AR4 and BR revealed that carotenoids existing in native claret membrane might protect AR4 from interacting with Pluronics.(3) The composite hydrogel with so far longest photochromic response of BR under high water content was achieved by combining addition of amphiphilic synthetic polymers and modification of proteins by gene engineering. Macromonomer F127-diacrylate was synthesized and first used for encapsulating BR to fabricate composite hydrogel (F127 is a Pluronic amphiphile.) The subtle end modification of Pluronic has little influence on assembly of copolymer and membrane proteins. After photopolymerization of the macromonomer in mixture of mutation BR-D96N and F127-diacrylate, a composite hydrogel material was formed and the photochromic response was prolonged to over half an hour, which is the longest record under high water conditions to the best of our knowledge.(4) A series of composite films of membrane proteins and synthetic polymers were fabricated, and the feasibility of information storage and processing of the resultant films was confirmed via collaboration with physicists. The carotenoid in the native lipid environment of H.sp.xz515 was successfully removed via gene engineering, and the contrast between M410 intermediate and ground state was greatly enhanced. A mutation BR-D96V with improved optical properties was also obtained. Polymeric composite encapsulating retinal protein monomers was first tried to be prepared. A new composite material was fabricated by employing macromonomer poly(ethylene oxide)-diacrylate (PEO-DA) with good biocompatibility as matrixes to encapsulate membrane proteins in either the trimer state or the monomer state, although the improvement of stability of monomers (non-aggregated proteins) is still called for. Composite films were also made by embedding the recombinant AR4 and mutation BR-D96V into poly(vinyl alcohol) (PVA). A simple pattern recording in the composite films was also realized, which demonstrates its ability for optical information storage. Investigation from our collaborator further confirmed that our BR-D96V/PVA film was available for information storage and processing.更多还原