本文選題：蛋白質(zhì)設計 + 蛋白質(zhì)折疊　； 參考：《中國科學技術大學》2016年博士論文

【摘要】：蛋白質(zhì)從頭設計最基本的目標是讓設計的氨基酸序列折疊成預期的三維結(jié)構。這一思路能夠讓我們更好地理解一級結(jié)構即氨基酸序列是怎么決定蛋白質(zhì)三級結(jié)構從而決定其功能的�，F(xiàn)階段盡管蛋白質(zhì)設計已有多個實驗驗證成功的例子,但其應用仍然受到諸多的限制。其瓶頸是其成功率不理想,追根溯源是對于蛋白質(zhì)折疊的理解不夠深入,設計方法不夠成熟。其中,完全基于結(jié)構分析對于設計結(jié)果進行實驗驗證的方法成本高、周期長,導致對理論方法的驗證缺乏系統(tǒng)性,可靠的反饋信息較少等等,限制了設計方法的改進。為克服這一困難,本文應用了一種把目的蛋白質(zhì)結(jié)構穩(wěn)定性與抗生素抗性偶聯(lián)在一起的高效實驗體系,實現(xiàn)了對設計蛋白折疊性能的高效檢驗。本文進一步應用該體系對初始折疊特性不佳的設計序列進行定向進化,發(fā)現(xiàn)這些設計結(jié)果實際上只包含小的設計序列錯誤,可通過定向進化導入的點突變得以糾正。本文使用溶液核磁共振或X-射線晶體衍射技術解析了三個人工蛋白質(zhì)的三維結(jié)構,其一為直接設計的結(jié)果,另兩個為經(jīng)定向進化后的突變體。這三個實測結(jié)構均和相應設計模板高度一致。人工設計蛋白質(zhì)和相應天然模板蛋白的高分辨率晶體結(jié)構比較揭示了若干對決定三維結(jié)構可能具有關鍵作用的序列位點,本文進一步通過定位點突變對這些位點的結(jié)構效應進行了分析。同時,還使用圓二色譜、差熱掃描量熱分析研究了人工蛋白質(zhì)的高溫變性過程。這些設計蛋白質(zhì)在表現(xiàn)出高熱穩(wěn)定性的同時,其去折疊過程缺乏協(xié)同性。在這一點上,我們獲得的人工設計蛋白與大多數(shù)天然蛋白質(zhì)不同, 然而與文獻報道的大部分人工設計蛋白類似。盡管其原因有待進一步研究,但至少證明折疊協(xié)同性并非形成正確、穩(wěn)定的三維結(jié)構所必須的。在此基礎上,我們選擇了多個高度規(guī)則的空間結(jié)構分別作為理論設計的模板,以對理論設計成功率進行較全面的鑒定。核磁共振譜圖表明,第一輪設計結(jié)果中約80%的設計蛋白能夠折疊成穩(wěn)定的空間結(jié)構。與此同時,約50%的設計蛋白質(zhì)溶解度低。基于這些實驗結(jié)果,我們改進了設計方法,并進行了第二輪設計。核磁共振譜圖表明,該論設計蛋白在形成穩(wěn)定三維結(jié)構的同時,溶解度顯著改善。
[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.
【學位授予單位】：中國科學技術大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q51

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