本文選題：蛋白質(zhì)設(shè)計(jì) + 蛋白質(zhì)折疊��； 參考：《中國科學(xué)技術(shù)大學(xué)》2016年博士論文

【摘要】：蛋白質(zhì)從頭設(shè)計(jì)最基本的目標(biāo)是讓設(shè)計(jì)的氨基酸序列折疊成預(yù)期的三維結(jié)構(gòu)。這一思路能夠讓我們更好地理解一級結(jié)構(gòu)即氨基酸序列是怎么決定蛋白質(zhì)三級結(jié)構(gòu)從而決定其功能的。現(xiàn)階段盡管蛋白質(zhì)設(shè)計(jì)已有多個實(shí)驗(yàn)驗(yàn)證成功的例子,但其應(yīng)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(shè)計(jì)方法不夠成熟。其中,完全基于結(jié)構(gòu)分析對于設(shè)計(jì)結(jié)果進(jìn)行實(shí)驗(yàn)驗(yàn)證的方法成本高、周期長,導(dǎo)致對理論方法的驗(yàn)證缺乏系統(tǒng)性,可靠的反饋信息較少等等,限制了設(shè)計(jì)方法的改進(jìn)。為克服這一困難,本文應(yīng)用了一種把目的蛋白質(zhì)結(jié)構(gòu)穩(wěn)定性與抗生素抗性偶聯(lián)在一起的高效實(shí)驗(yàn)體系,實(shí)現(xiàn)了對設(shè)計(jì)蛋白折疊性能的高效檢驗(yàn)。本文進(jìn)一步應(yīng)用該體系對初始折疊特性不佳的設(shè)計(jì)序列進(jìn)行定向進(jìn)化,發(fā)現(xiàn)這些設(shè)計(jì)結(jié)果實(shí)際上只包含小的設(shè)計(jì)序列錯誤,可通過定向進(jìn)化導(dǎo)入的點(diǎn)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(shù)解析了三個人工蛋白質(zhì)的三維結(jié)構(gòu),其一為直接設(shè)計(jì)的結(jié)果,另兩個為經(jīng)定向進(jìn)化后的突變體。這三個實(shí)測結(jié)構(gòu)均和相應(yīng)設(shè)計(jì)模板高度一致。人工設(shè)計(jì)蛋白質(zhì)和相應(yīng)天然模板蛋白的高分辨率晶體結(jié)構(gòu)比較揭示了若干對決定三維結(jié)構(gòu)可能具有關(guān)鍵作用的序列位點(diǎn),本文進(jìn)一步通過定位點(diǎn)突變對這些位點(diǎn)的結(jié)構(gòu)效應(yīng)進(jìn)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(shè)計(jì)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(diǎn)上,我們獲得的人工設(shè)計(jì)蛋白與大多數(shù)天然蛋白質(zhì)不同, 然而與文獻(xiàn)報(bào)道的大部分人工設(shè)計(jì)蛋白類似。盡管其原因有待進(jìn)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(gòu)所必須的。在此基礎(chǔ)上,我們選擇了多個高度規(guī)則的空間結(jié)構(gòu)分別作為理論設(shè)計(jì)的模板,以對理論設(shè)計(jì)成功率進(jìn)行較全面的鑒定。核磁共振譜圖表明,第一輪設(shè)計(jì)結(jié)果中約80%的設(shè)計(jì)蛋白能夠折疊成穩(wěn)定的空間結(jié)構(gòu)。與此同時,約50%的設(shè)計(jì)蛋白質(zhì)溶解度低�；谶@些實(shí)驗(yàn)結(jié)果,我們改進(jìn)了設(shè)計(jì)方法,并進(jìn)行了第二輪設(shè)計(jì)。核磁共振譜圖表明,該論設(shè)計(jì)蛋白在形成穩(wěn)定三維結(jié)構(gòu)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(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q51

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