蛋白質(zhì)結構與功能的nmr研究-資料下載頁
2025-08-11 14:41本頁面
【正文】 W和 V66W 突變體 (G88W1 V66W110)的折疊研究 G88W110 V66W110 SNase110 SNase110(1110殘基 )， SNase121(1121基 )， SNase135 (1135殘基 ) 片段的折疊路徑研究 Two Folding pathways： ? Thick arrow: Folding pathway energetically more favorable for SNase fragments ? Thin arrow: Folding pathway disadvantage energetically for SNase fragments SNase的 β與 ?亞結構域片段 (SNase121與 SNase ?3)復合體的溶液三維結構和相互作用機制的研究 SNase121在復合體中的 HSQC譜 SNase121的 HSQC譜 可以看到 SNase121在復合體中的譜峰分布與 SNase的 HSQC譜近。 SNase?3的構象 : 在復合體中的 HSQC譜 在水溶液中的 HSQC譜 可以看到 SNase ?3在復合物中已具有一定的構象。 SNase*－ DNA復合物的 NMR研究 在復合物中 SNase*的 HSQC譜 SNase的 HSQC譜 3. 凋亡相關蛋白PDCD5的研究 通過化學位移和弛豫 參數(shù)對難以解析結構 的蛋白質(zhì) (凋亡相關 蛋白 PDCD5)進行結 構特征鑒定 N端 核心結構 C端 4. Ssh10b與核酸相互作用溫度依賴性的結構基礎 Ssh10b在核磁譜圖上呈現(xiàn)兩種象，比例 與溫度相關．譜圖分析表明兩種構象是 L61P62順反異構造成的 L61P62 反式 順式 在新藥發(fā)現(xiàn)中的 NMR 應用 An important technique in support of structurebased drug design ? provide information on the nature of molecular interactions, ? identify weakbinding pounds ? to aid development of the druglike inhibitors for use as lead pounds in drug discovery. NMR進行新藥研究的策略 檢測蛋白質(zhì)： 1. 選擇性地同位素標記某種類型的氨基酸殘基 。 2. ‘segmental labelling’: discrete segments of the polypeptide chain are uniformly labelled。 3. Chemicalshift mapping or Differential chemicalshift mapping 檢測底物小分子： Based on T2 and T1enhancement: the large differences in the rates of rotational and translational motions of a small molecule in the free state relative to when it is bound to a macromolecule 新的脈沖方法： SEATROSY: selects solventexposed amide protons in uniformly deuterated 15Nlabelled proteins dissolved in H2O by magization transfer from H2O. 二氫葉酸還原酶 (DHPR) Met ε13CH3 resonance region a. Chemicalshift mapping 方法： 綠色表示沒有底物存在，藍色表示存在 底物類似物 pyridine2,6dicarboxylate (PDC)． b. Differential chemicalshift mapping方法： 藍色表示結合 PDC，紅色表示結合 4chloroPDC. SEATROSY脈沖方法 Conventional [15N,1H]HSQC [15N,1H]TROSY SEATROSY selects solventexposed 1HN Methods based on T2 and T1enhancement 1. T2 (小分子 ) T2 (蛋白質(zhì) ) the resonance lines in the NMR spectra of small molecules are much narrower than those in the spectra of macromolecules. 2. T1 (小分子 ) transferred NOEs: small positive NOEs for free small ligand。 negative NOEs for bound ligand. SAR (structure–activity relationship) by NMR SAR by NMR : use of protein chemicalshift changes to screen for low affinity ligands, and the collection of structural information to direct a linkedfragment approach to enhancing binding affinities. a,b. Small molecules that bind to two distinct sites on the target protein. A second screen of close analogues to the hit(s) can be used to optimize the affinity for the sites. c. Knowledge of the positions of the two sites in the protein is then used to guide the synthesis of pounds in which the two smallmolecule fragments are linked (it is possible to obtain inhibitors that bind very strongly). df. Example of the design and discovery of a potent stromelysin. Thanks!
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