《蛋白質(zhì)分子設(shè)計(jì)》ppt課件 (2)-全文預(yù)覽
【正文】 Data Bank)已收集數(shù)以萬(wàn)計(jì)個(gè)蛋白質(zhì)晶體結(jié)構(gòu)，但是通常蛋白質(zhì)序列的數(shù)目比蛋白質(zhì)三維結(jié)構(gòu)的數(shù)目大 100倍。 ? 計(jì)算機(jī)模擬技術(shù)在蛋白質(zhì)設(shè)計(jì)循環(huán)中占有重要位置。由于基因工程的發(fā)展，真核基因表達(dá)技術(shù)的發(fā)展使動(dòng)物蛋白質(zhì)與植物蛋白質(zhì)的數(shù)目迅速增長(zhǎng)，又增加了新的生物活性物質(zhì)源 。 蛋白質(zhì)設(shè)計(jì)在許多方面取得的顯著進(jìn)展 Protein engineering: by which we mean mutating the gene of an existing protein in an attempt to alter its function in a predictable way Protein Design: which has the more ambitious goal of designing de novo a protein to fulfill a desired function 第一節(jié) 基于天然蛋白質(zhì)結(jié)構(gòu)的分子設(shè)計(jì) 一、概述 蛋白質(zhì)結(jié)構(gòu)與功能的關(guān)系的認(rèn)識(shí)對(duì)蛋白質(zhì)設(shè)計(jì)是至關(guān)重要的，蛋白質(zhì)的結(jié)構(gòu)涉及一級(jí)結(jié)構(gòu) (序列 )及三維結(jié)構(gòu)。第二章 蛋白質(zhì)分子設(shè)計(jì) 第一節(jié) : 基于蛋白質(zhì)天然結(jié)構(gòu)的分子設(shè)計(jì) 第二節(jié) : 全新蛋白質(zhì)分子設(shè)計(jì) 第三節(jié) : 計(jì)算蛋白質(zhì)分子設(shè)計(jì) 蛋白質(zhì)分子設(shè)計(jì) ? 蛋白質(zhì)分子設(shè)計(jì)是一門(mén)新興的研究領(lǐng)域，其本身在不斷地發(fā)展，其內(nèi)容也在不斷地更新。 蛋白質(zhì)設(shè)計(jì)是多學(xué)科的交叉領(lǐng)域 。由于真菌與細(xì)胞相對(duì)容易處理，因此它們是一個(gè)生物活性物質(zhì)源。一些新技術(shù)，如 PCR及自動(dòng)化技術(shù)的發(fā)展使各種類型的基因工程變得快速、容易。 蛋白質(zhì)三維結(jié)構(gòu)知識(shí)的必要性 蛋白質(zhì)三維結(jié)構(gòu)知識(shí)對(duì)于蛋白質(zhì)工程是絕對(duì)必要的。 設(shè)計(jì)目標(biāo)及解決辦法 ? 蛋白質(zhì)結(jié)構(gòu)與功能的關(guān)系對(duì)于蛋白質(zhì)工程及蛋白質(zhì)分子設(shè)計(jì)都是至關(guān)重要的。所謂內(nèi)核是指蛋白質(zhì)在進(jìn)化中保守的內(nèi)部區(qū)域。 ④ 疏水及親水基團(tuán)需要合理地分布在溶劑可及表面及不可及表面。 ⑧ 結(jié)構(gòu)及功能的專一性。 ? 最大量的定位突變是在體外利用重組 DNA技術(shù)或 PCR方法。 ? Mathews及其合作者在溶菌酶內(nèi)核中替換多至 10個(gè)殘基。根據(jù)所希望的結(jié)構(gòu)及功能設(shè)計(jì)蛋白質(zhì)或多肽的氨基酸序列。 ? 為達(dá)到這些目的可以采用兩條不同的途徑：反向?qū)崿F(xiàn)蛋白質(zhì)與工程底物的契合，改變功能；從頭設(shè)計(jì)功能蛋白質(zhì)。 二、蛋白質(zhì)的功能設(shè)計(jì) 1）通過(guò)反向 Mimicking天然蛋白質(zhì)設(shè)計(jì)新功能 2）鍵合及催化的從頭設(shè)計(jì) 3）在全新蛋白質(zhì)中引入結(jié)合位點(diǎn) 4）催化活性蛋白質(zhì)的設(shè)計(jì) 5）膜蛋白及離子通道的設(shè)計(jì) 6）新材料的設(shè)計(jì) Introduction to Structural Biology: prediction, engineering, and design of protein structures Proteins can be made more stable by engineering The factors that are important to protein stability can be revealed by doing protein engineering studies. An example: T4 lysozyme (from the work done by Brian Mathews, Univ. of Oregon). T4 lysozyme (a) Is a 164aa polypeptide chain that folds into two domains: The Nterminal domain is of ?+? type, and the Cterminal domain prises 7 short ? helices. (b) Has no disulfide bonds (c) Has two Cys residues, Cys54 and Cys97 (that are far apart in the folded structure) T4 lysozyme (contd.) Tm (the melting temperature) Tm (the melting temperature): the temperature at which 50% of the enzyme is inactivated (or more rigorously, 50% of the enzyme is unfolded) during reversible heat denaturation. The higher Tm, the more stable the protein. WT T4 lysozyme’s Tm: 176。C (a)Oxidized mutants are more stable than WT. (b) Reduced mutants are less stable than WT. (c) The longer the loop between the cysteine residues of the mutants with single disulfide bonds, the larger was the effect on stability. (d) The mutational effects were additive. Triple mutants: ++11 ?22176。 this strongly indicates that the effect on Tm of Ala82Pro is indeed due to entropy changes. Stabilizing the dipoles of ? helices increases stability The helix dipole concept: the positive charge is at the Nterminus of the helix, and the negative charge is at the Cterminus of the helix. Thus, negative ions are usually bound to the Nterminal end of the helix. Results, in T4 lysozyme, Ser38Asp (Tm increase of 2176。 Interestingly, these segments form units of secondary structure. A model based on the Xray structure of one of the native enzymes Additional material on Eisenberg’s 3D profile method Ref: Science (1991), 253:164170 Schematic description of the construction of a 3D structure profile Schematic description of the construction of
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