人工设计蛋白质及其定向进化突变体的实验分析

发布时间：2018-05-09 06:31

本文选题：蛋白质设计 + 蛋白质折叠　；参考：《中国科学技术大学》2016年博士论文

【摘要】：蛋白质从头设计最基本的目标是让设计的氨基酸序列折叠成预期的三维结构。这一思路能够让我们更好地理解一级结构即氨基酸序列是怎么决定蛋白质三级结构从而决定其功能的。现阶段尽管蛋白质设计已有多个实验验证成功的例子,但其应用仍然受到诸多的限制。其瓶颈是其成功率不理想,追根溯源是对于蛋白质折叠的理解不够深入,设计方法不够成熟。其中,完全基于结构分析对于设计结果进行实验验证的方法成本高、周期长,导致对理论方法的验证缺乏系统性,可靠的反馈信息较少等等,限制了设计方法的改进。为克服这一困难,本文应用了一种把目的蛋白质结构稳定性与抗生素抗性偶联在一起的高效实验体系,实现了对设计蛋白折叠性能的高效检验。本文进一步应用该体系对初始折叠特性不佳的设计序列进行定向进化,发现这些设计结果实际上只包含小的设计序列错误,可通过定向进化导入的点突变得以纠正。本文使用溶液核磁共振或X-射线晶体衍射技术解析了三个人工蛋白质的三维结构,其一为直接设计的结果,另两个为经定向进化后的突变体。这三个实测结构均和相应设计模板高度一致。人工设计蛋白质和相应天然模板蛋白的高分辨率晶体结构比较揭示了若干对决定三维结构可能具有关键作用的序列位点,本文进一步通过定位点突变对这些位点的结构效应进行了分析。同时,还使用圆二色谱、差热扫描量热分析研究了人工蛋白质的高温变性过程。这些设计蛋白质在表现出高热稳定性的同时,其去折叠过程缺乏协同性。在这一点上,我们获得的人工设计蛋白与大多数天然蛋白质不同, 然而与文献报道的大部分人工设计蛋白类似。尽管其原因有待进一步研究,但至少证明折叠协同性并非形成正确、稳定的三维结构所必须的。在此基础上,我们选择了多个高度规则的空间结构分别作为理论设计的模板,以对理论设计成功率进行较全面的鉴定。核磁共振谱图表明,第一轮设计结果中约80%的设计蛋白能够折叠成稳定的空间结构。与此同时,约50%的设计蛋白质溶解度低。基于这些实验结果,我们改进了设计方法,并进行了第二轮设计。核磁共振谱图表明,该论设计蛋白在形成稳定三维结构的同时,溶解度显著改善。
[Abstract]:The basic goal of protein ab initio design is to fold the amino acid sequence into the desired three-dimensional structure. This idea allows us to better understand how the primary structure, the amino acid sequence, determines the tertiary structure and functions of proteins. At present, although there have been many successful examples of protein design, its application is still limited. The bottleneck is that the success rate is not ideal, the source of tracing is that the understanding of protein folding is not deep enough, and the design method is not mature enough. Among them, the design method based on structural analysis has high cost and long period, which leads to the lack of systematic verification of theoretical methods, less reliable feedback, and so on, which limits the improvement of design methods. In order to overcome this difficulty, an efficient experimental system combining the structural stability of the target protein with antibiotic resistance was used to test the folding performance of the designed protein. In this paper, we further apply this system to the directional evolution of design sequences with poor initial folding characteristics. It is found that these design results contain only a small number of design sequence errors and can be corrected by point mutation introduced by directional evolution. In this paper, three dimensional structures of three artificial proteins have been analyzed by solution nuclear magnetic resonance (NMR) or X-ray crystal diffraction technique. One is the result of direct design and the other two are directional evolution mutants. The measured structures are highly consistent with the corresponding design templates. The high resolution crystal structure comparison of artificially designed proteins and corresponding natural template proteins reveals a number of sequence sites that may play a key role in determining three-dimensional structures. The structural effects of these loci were further analyzed by locus mutation. At the same time, circular dichroism and differential scanning calorimetry (DSC) were used to study the denaturation of artificial proteins at high temperature. These design proteins exhibit high thermal stability and lack of synergy in the unfolding process. At this point, the artificial design proteins we obtained are different from most natural proteins, but similar to most of the artificial design proteins reported in the literature. Although the reasons need to be further studied, it is at least proved that folding synergy is not necessary for the formation of a correct and stable three-dimensional structure. On this basis, we select a number of highly regular spatial structures as the template of theoretical design, in order to evaluate the success rate of theoretical design. NMR results show that about 80% of the designed proteins can be folded into a stable spatial structure in the first round design. At the same time, about 50% of the designed proteins have low solubility. Based on these experimental results, we improve the design method and carry out the second round design. Nuclear magnetic resonance spectroscopy (NMR) showed that the solubility of the designed protein was significantly improved while the stable three-dimensional structure was formed.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q51

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