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分子自组装是自然界进化过程中普遍存在的现象，核酸、蛋白质、细胞甚至生命的形成都是通过自组装实现的，而生物体内的大多数自组装活动均是在各种各样的限域环境中进行的，如细胞、脂质膜、酶囊、囊泡、胶束生物有机骨架等。短肽分子本质上是一种低分子量的生物活性蛋白，因其内在的生物相容性、生物降解性以及低免疫原等特点成为当下的研究热点。作为一种自下而上构建超分子纳米材料的结构基元，短肽分子的组装基础是各种分子间协同的相互作用力，这些作用力可通过一些常用的动力学参数进行调制，包括pH、温度、金属离子、酶以及小分子等。基于此，我们从仿生学的角度出发，通过改变共价键、非共价键以及组装环境，以温和的生物刺激来触发分子的自组装活动，并通过模拟生物体内的受限环境来探究限域空间对短肽自组装的影响。首先，设计了一系列Fmoc修饰的二肽及氨基酸来探索Thermolysin的催化作用。实验和计算模拟结果表明，氨基酸的疏水性和序列与Thermolysin催化反应的结合能力和催化效率有显著的相关性。根据不同的底物设计，Thermolysin可以催化反应向水解或缩合方向发展。利用Thermolysin的双向催化作用设计了一个两步反应，完成了Fmoc-YL-COOH向Fmoc-YY-NH2的转变，并实现了凝胶-溶胶-凝胶的过渡。其次，为了模拟自然界中酶触发的分子自组装，我们将Thermolysin催化的短肽缩合反应用于AAO模板的限域空间中。在整个过程中，我们选择具有不同空间结构和孔径的AAO模板来探究它们对短肽自组装的影响。同时，我们还通过不同序列的短肽来揭示分子自身对限域空间内自组装的影响。此外，选用几种亲疏水性不同的Fmoc-氨基酸，探究L-NH2对Fmoc-氨基酸组装的影响。研究发现，L-NH2与Fmoc-氨基酸之间通过形成新的氢键来改变组装体形貌以及二级结构，从一种无规则的形态转变成了有规则形貌的晶体结构，完成了固-固相的转变。最后，选用不同孔径的毛细管作为限域空间来研究L-NH2调控下Fmoc-氨基酸的自组装，并通过毛细管壁的吸附作用使短肽在其中形成类似细胞外基质的组装体，从而促进类似于管状结构的细胞层形成。;Molecular self-assembly is a common phenomenon in the evolution of nature. The formation of nucleic acids, proteins, cells and even life are achieved by self-assembly, and most self-assembly activities in living organisms take place in a variety of confined environments, such as cells, lipid membranes, enzyme capsules, vesicles, micelle biological organic framework and so on. The short peptide molecule is essentially a low molecular weight bioactive protein, which has become a current research focus due to its inherent biocompatibility, biodegradability and low immunogenicity. As a building block for the “bottom-up” fabrication of supramolecular nanomaterials, the basis of the assembly of short peptide molecules is the synergy of various intermolecular interaction forces, which can be modulated by some commonly used kinetic parameters, including pH, temperature, metal ions, enzymes and small organic molecules. Based on this, from the perspective of bionics, we change the covalent bond, non-covalent bond, and assembly environment, trigger molecular self-assembly activities with mild biological stimulation, and explore the influence of the confined space on the self-assembly of short peptides by simulating the localized environment in organisms.First, a series of Fmoc-modified dipeptides and amino acids were designed to explore the catalytic effect of Thermolysin. The results from experiments and computational simulation showed that the hydrophobicity and sequence of amino acids had significant correlation with thermolysin catalytic reactions including binding capacity and catalytic efficiency. Depending on the substrate design, Thermolysin could catalyze the reaction towards hydrolysis or condensation. Taking advantage of the bidirectional catalytic actions of thermolysin, a two-step reaction was designed to modify the sequence of Fmoc-dipeptide from Fmoc-YL-COOH to Fmoc-YY-NH2 with gel-sol-gel transition.Second, in order to to mimic the self-assembly of molecules triggered by enzyme in nature, Thermolysin-catalyzed short peptide condensation reactions was applied to the confined space of AAO templates. During the whole process, different spatial structure and pore size of AAO templates were chosen to investigate their effects on the self-assembly of short peptides. Meanwhile, short peptides with various sequences were also tested to reveal the influence of molecule-self on the self-assembly in confined environment.In addition, several Fmoc-amino acids with different hydrophobic properties were selected to investigate the effect of L-NH2 on the assembly of Fmoc-amino acid. The study showed that L-NH2 and Fmoc-amino acids formed new hydrogen bonds to change the morphology and secondary structure of the assembly, and transformed from an irregular morphology to crystalline structure with regular morphology accompanying solid-solid phase transformation.At last, the capillaries with different pore diameters were used as confined space to study the self-assembly of Fmoc-amino acids under the control of L-NH2, and the short peptides formed assembly similar to the extracellular matrix through the adsorption of the capillary wall, thus promoting the formation of the cell layer similar to tubular structure.
|王眉月. 酶及小分子诱导的短肽自组装行为在限域空间的研究和应用[D]. 中国科学院大学,2020.|
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