| 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. |
