Acid-induced unfolding mechanism of recombinant human endostatin.
Endostatin is a potent angiogenesis inhibitor. The structure of endostatin is unique in that its secondary structure is mainly irregular loops and beta-sheets and contains only a small fraction of alpha-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), tryptophan 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 very acid-resistant; some native structure still remains even at pH 2 as evidenced by (1)H NMR. Trifluoroethanol (TFE) destabilizes native endostatin, while it makes endostatin even more acid-resistant in the low pH region. Stability measurement of endostatin suggests that endostatin is still in native structure at pH 3.5 despite the decreased stability. Acid-induced unfolding of endostatin is reversible, although it requires a long time to reach equilibrium below pH 3. Surprisingly, the alpha-helical content of endostatin is increased when it is unfolded at pH 1.6, and the alpha-helical content of the polypeptide chain of unfolded endostatin increases linearly with TFE concentration in the range of 0-30%. This observation indicates that the polypeptide chain of unfolded endostatin has an intrinsic alpha-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 an acidic environment such as in a lysosome in the cell.
Department of Biological Sciences and Biotechnology, MOE laboratory of Protein Science, Tsinghua University, Beijing 100084, PR China.
Bing
B
Li
Xiaoyu
X
Wu
Hao
H
Zhou
Qianjie
Q
Chen
Yongzhang
Y
Luo
D019342:Acetic Acid / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D043169:Endostatins / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry; D019346:Sodium Acetate / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors; D014270:Trifluoroethanol / Q000737:chemistry
D043169:Endostatins; D011994:Recombinant Proteins; D019346:Sodium Acetate; D014270:Trifluoroethanol; D019342:Acetic Acid