【作者】 李兵；

【导师】 罗永章；

【作者基本信息】 清华大学 ， 生物学， 2004， 硕士

【摘要】 内皮抑素(endostatin)是一种潜在的新生血管形成抑制剂,对多种肿瘤模型均具有较强的抑制效果, 且无耐药性和毒副作用。其独特的结果特点在于它的二级结构含有大量的β折叠,而只有极少量的α螺旋;它还包含有两对巢式二硫键。因此我们选择endostatin作为一种模型蛋白来研究这一类蛋白的折叠机理。我们用核磁共振(NMR)、内源荧光(intrinsic fluorescence)、圆二色相(CD)等手段来监测endostatin的酸变性过程;用尿素变性来测量endostatin在中性条件和酸性条件下的稳定性。我们的结果显示:Endostatin是一个特别耐酸的蛋白质,在pH 3才开始变性,在pH 1.6完全变性。在pH 3.5,虽然endostatin仍处于天然状态,但是其稳定性已经降低。5% TFE使天然状态的endostatin更不稳定,然而却使endostatin更耐酸;而金属锌离子的结合使天然状态的endostatin更稳定,也使endostatin更耐酸。Endostatin的酸变性是缓慢的、可逆的。Endostatin在pH 1.6的完全变性需要大约90分钟,而完全变性的endostatin在稀释到pH 6.0的时候能很快恢复其天然结构。在pH 1.6,当endostatin完全变性后,0~30% TFE能线性增加变性endostatin的α螺旋二级结构,这表明endostatin有内在的α螺旋结构倾向性。我们的发现对于更有效地复性endostatin有一定的指导意义。Endostatin的耐酸性质可能使其能有效地抵抗体内的酸性环境如溶酶体(lysosome)以增加其体内稳定性。更多还原

【Abstract】 Endostatin is a potent angiogenesis inhibitor. The structure of endostatin is unique in that its secondary structure is mainly irregular loops, β-sheets, and contains only a small fraction of α-helices, with two pairs of disulfide bonds in a nested pattern. We choose human endostatin as a model system to study the folding mechanism of this kind. Nuclear magnetic resonance (NMR), intrinsic Trp emission fluorescence, and circular dichroism (CD) were used to monitor the unfolding process of endostatin upon acid titration. Urea-induced unfolding was used to measure the stability of endostatin under different conditions. Our results show that endostatin is acid very resistant, it still remains some native structure even at pH 2 as evidenced by 1H-NMR. Stability measurement of endostatin suggests that endostatin is still in native structure at pH 3.5 despite of the decreased stability. Trifluoroethanol (TFE) destabilizes native endostatin while makes endostatin even more acid resistant in low pH region; while zinc binding makes native endostatin stable, and more acid resistant. Acid-induced unfolding of endostatin is reversible, although it requires a long time to reach equilibrium below pH 3. Surprisingly, the α-helical content of endostatin is increased when it is unfolded at pH 1.6, and the α-helical content of the polypeptide chain of unfolded endostatin increases linearly on TFE concentration in the range of 0 ~ 30%. This observation indicates that the polypeptide chain of unfolded endostatin has an intrinsic α-helical propensity. Our discoveries may provide clues for refolding endostatin more efficiently. The acid-resistance property of endostatin may have biological significance in that it cannot be easily digested by proteases in acidic environment such as lysosome in the cell.更多还原