Acid-Induced Unfolding Mechanism of Recombinant Human Endostatin
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- 作者：Bing Li ; Xiaoyu Wu ; Hao Zhou ; Qianjie Chen ; and Yongzhang Luo
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：March 9, 2004
- 年：2004
- 卷：43
- 期：9
- 页码：2550 - 2557
- 全文大小：127K
- 年卷期：v.43,no.9(March 9, 2004)
- ISSN：1520-4995
文摘
Endostatin is a potent angiogenesis inhibitor. The structure of endostatin is unique in that itssecondary structure is mainly irregular loops and 
-sheets and contains only a small fraction of 
-heliceswith two pairs of disulfide bonds in a nested pattern. We choose human endostatin as a model system tostudy the folding mechanism of this kind. Nuclear magnetic resonance (NMR), tryptophan emissionfluorescence, and circular dichroism (CD) were used to monitor the unfolding process of endostatin uponacid titration. Urea-induced unfolding was used to measure the stability of endostatin under differentconditions. Our results show that endostatin is very acid-resistant; some native structure still remainseven at pH 2 as evidenced by 1H NMR. Trifluoroethanol (TFE) destabilizes native endostatin, while itmakes endostatin even more acid-resistant in the low pH region. Stability measurement of endostatinsuggests that endostatin is still in native structure at pH 3.5 despite the decreased stability. Acid-inducedunfolding of endostatin is reversible, although it requires a long time to reach equilibrium below pH 3.Surprisingly, the -helical content of endostatin is increased when it is unfolded at pH 1.6, and the -helicalcontent of the polypeptide chain of unfolded endostatin increases linearly with TFE concentration in therange of 0-30%. This observation indicates that the polypeptide chain of unfolded endostatin has anintrinsic -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 easilydigested by proteases in an acidic environment such as in a lysosome in the cell.
-sheets and contains only a small fraction of -heliceswith two pairs of disulfide bonds in a nested pattern. We choose human endostatin as a model system tostudy the folding mechanism of this kind. Nuclear magnetic resonance (NMR), tryptophan emissionfluorescence, and circular dichroism (CD) were used to monitor the unfolding process of endostatin uponacid titration. Urea-induced unfolding was used to measure the stability of endostatin under differentconditions. Our results show that endostatin is very acid-resistant; some native structure still remainseven at pH 2 as evidenced by 1H NMR. Trifluoroethanol (TFE) destabilizes native endostatin, while itmakes endostatin even more acid-resistant in the low pH region. Stability measurement of endostatinsuggests that endostatin is still in native structure at pH 3.5 despite the decreased stability. Acid-inducedunfolding of endostatin is reversible, although it requires a long time to reach equilibrium below pH 3.Surprisingly, the -helical content of endostatin is increased when it is unfolded at pH 1.6, and the -helicalcontent of the polypeptide chain of unfolded endostatin increases linearly with TFE concentration in therange of 0-30%. This observation indicates that the polypeptide chain of unfolded endostatin has anintrinsic -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 easilydigested by proteases in an acidic environment such as in a lysosome in the cell.