蛋白質(zhì)分子設計ppt課件(2)-免費閱讀
【正文】 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。 二、蛋白質(zhì)的功能設計 1）通過反向 Mimicking天然蛋白質(zhì)設計新功能 2）鍵合及催化的從頭設計 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)的設計 5）膜蛋白及離子通道的設計 6）新材料的設計 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。根據(jù)所希望的結(jié)構(gòu)及功能設計蛋白質(zhì)或多肽的氨基酸序列。 ? 最大量的定位突變是在體外利用重組 DNA技術或 PCR方法。 ④ 疏水及親水基團需要合理地分布在溶劑可及表面及不可及表面。 設計目標及解決辦法 ? 蛋白質(zhì)結(jié)構(gòu)與功能的關系對于蛋白質(zhì)工程及蛋白質(zhì)分子設計都是至關重要的。一些新技術，如 PCR及自動化技術的發(fā)展使各種類型的基因工程變得快速、容易。 蛋白質(zhì)設計是多學科的交叉領域 。 蛋白質(zhì)設計在許多方面取得的顯著進展 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)的分子設計 一、概述 蛋白質(zhì)結(jié)構(gòu)與功能的關系的認識對蛋白質(zhì)設計是至關重要的，蛋白質(zhì)的結(jié)構(gòu)涉及一級結(jié)構(gòu) (序列 )及三維結(jié)構(gòu)。 ? 計算機模擬技術在蛋白質(zhì)設計循環(huán)中占有重要位置。如果我們想改變蛋白質(zhì)的性質(zhì)，必須改變蛋白質(zhì)的序列。 ⑤ 在金屬蛋白中，配位殘基的替換要滿足金屬配位幾何。 ? 突變的加和性原則 突變蛋白質(zhì)結(jié)構(gòu)的評估 ? 溶解性 ? 熱力學分析 ? Ｘ射線晶體學及ＮＭＲ譜 ? 園二色散方法 ? 單克隆抗體探測構(gòu)象變化 結(jié)構(gòu)與功能的容忍度 ? 蛋白質(zhì)結(jié)構(gòu)及功能對殘基的替換有一定的容忍度，即結(jié)構(gòu)與功能關系有一定的穩(wěn)健度。 蛋白質(zhì)的全新設計 內(nèi)容 ? 蛋白質(zhì)結(jié)構(gòu)的從頭設計 ? 蛋白質(zhì)功能的從頭設計 ? 取得的進展：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。 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 stra
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