《蛋白質(zhì)分子設(shè)計》ppt課件 (2)-文庫吧
2025-01-03 18:10 本頁面
【正文】 頭設(shè)計 ? 取得的進(jìn)展：血紅素結(jié)合蛋白、 氧化還原活性蛋白質(zhì)、 DNA結(jié)合蛋白及基于蛋白質(zhì)的高分子材料。 一、 蛋白質(zhì)結(jié)構(gòu)的從頭設(shè)計 1) 二級結(jié)構(gòu)模塊單元的自組裝 2) 配體誘導(dǎo)組裝 3) 通過共價交叉連接實現(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) 基于組合庫的全新蛋白質(zhì)設(shè)計 二、蛋白質(zhì)功能的全新設(shè)計 ? 蛋白質(zhì)設(shè)計的目標(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。功能設(shè)計主要涉及鍵合及催化。 ? 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)計功能蛋白質(zhì)。 蛋白質(zhì)的功能設(shè)計 １．通過反向擬合天然蛋白質(zhì)設(shè)計新的功能 ２．鍵合及催化的從頭設(shè)計 ３．在全新蛋白質(zhì)中引入結(jié)合位點 ４．催化活性蛋白質(zhì)的設(shè)計 ５．膜蛋白及離子通道的設(shè)計 ６．新材料的設(shè)計 第三節(jié) 計算蛋白質(zhì)設(shè)計 ? 蛋白質(zhì)設(shè)計是一個理論與實驗之間的循環(huán)。這個循環(huán)已經(jīng)在蛋白質(zhì)的合理設(shè)計中得到了許多重要進(jìn)展。 ? 計算蛋白質(zhì)設(shè)計包括能量表達(dá)、能量優(yōu)化、側(cè)鏈構(gòu)象的離散化、殘基分類 (內(nèi)核、表面、邊界 )、功能位點設(shè)計、專一性、穩(wěn)定性及序列空間的穩(wěn)健性預(yù)測等方面內(nèi)容。 二、蛋白質(zhì)的功能設(shè)計 1）通過反向 Mimicking天然蛋白質(zhì)設(shè)計新功能 2）鍵合及催化的從頭設(shè)計 3）在全新蛋白質(zhì)中引入結(jié)合位點 4）催化活性蛋白質(zhì)的設(shè)計 5）膜蛋白及離子通道的設(shè)計 6）新材料的設(shè)計 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 Thermodynamic parameters obtained from DSC measurements DSC: Differential Scanning Calorimetry 差示掃描量熱法 Determination of ?H and ?S from DSC Cp T Thermal denaturation of a protein Three methods to engineer a more thermostable protein than wildtype T4 lysozyme (1) reducing the difference in entropy between folded and unfolded protein. (in practice, reducing the number of conformations in the unfolded state) (2) stabilizing the ? helices. (3) increasing the number of hydrophobic interactions in the interior core. Disulfide bridges increase protein stability One way to reduce the number of unfolded conformations is to introduce a disulfide bridge. The geometry of the CH2SSCH2 bridge in proteins is confined to rather narrow conformational limits. Thus, deviations from this geometry will introduce strains into the folded structure. So, pairs of ideal positions needed to be sought in order to acmodate disulfide bridges. Disulfide bridges increase protein stability (contd.) Three candidate disulfide bridges remained after this long filtering process: 397, 9164, 21142 (by the way, Cys54 was mutated to Thr to avoid the formation of incorrect disulfide bonds during folding). Disulfide bridges increase protein stability (contd.) 176。c 176。c 11176。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 m
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