蛋白質(zhì)分子設(shè)計(jì)ppt課件(2)-在線瀏覽
2025-03-07 18:10本頁(yè)面
【正文】 白質(zhì)在進(jìn)化中保守的內(nèi)部區(qū)域。 ②所有蛋白質(zhì)內(nèi)部都是密堆積 (很少有空穴大到可以結(jié)合一個(gè)水分子或惰性氣體 )，并且沒(méi)有重疊。 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。 ⑥ 對(duì)于金屬蛋白，圍繞金屬中心的第二殼層中的相互作用是重要的。 ⑧ 結(jié)構(gòu)及功能的專一性。實(shí)踐表明這是蛋白質(zhì)設(shè)計(jì)最困難的問(wèn)題。 ? 最大量的定位突變是在體外利用重組 DNA技術(shù)或 PCR方法。 ? Fersht等替換了 Barnase的所有內(nèi)核殘基。 ? Mathews及其合作者在溶菌酶內(nèi)核中替換多至 10個(gè)殘基。 ? 這些結(jié)果說(shuō)明不同的氨基序列具有相近的設(shè)計(jì)的結(jié)構(gòu)。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。 一、 蛋白質(zhì)結(jié)構(gòu)的從頭設(shè)計(jì) 1) 二級(jí)結(jié)構(gòu)模塊單元的自組裝 2) 配體誘導(dǎo)組裝 3) 通過(guò)共價(jià)交叉連接實(shí)現(xiàn)肽的自組裝： SS；DAB 4) 在合成模板上的肽自組裝 5) 線性多肽折疊為球狀結(jié)構(gòu) 6) 基于組合庫(kù)的全新蛋白質(zhì)設(shè)計(jì) 二、蛋白質(zhì)功能的全新設(shè)計(jì) ? 蛋白質(zhì)設(shè)計(jì)的目標(biāo)是產(chǎn)生既能折疊為預(yù)想的結(jié)構(gòu)又具有有趣和有用的功能。 ? 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)。這個(gè)循環(huán)已經(jīng)在蛋白質(zhì)的合理設(shè)計(jì)中得到了許多重要進(jìn)展。 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 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。 C) Ala82Pro (Tm increase of 2176。 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。 C) Stabilizing the dipoles of ? helices increases stability (contd.) Stabilizing the dipoles of ? helices increases stability (contd.) Mutations that fill cavities in hydrophobic cores do not stabilize T4 lysozyme, although they may stabilize other proteins The elimination of a cavity of the size of one –CH2 group in the protein interior stabilizes the enzyme by about 1 kcal/mol. Results: in T4 lysozyme, Leu23Phe amp。 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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