Protein folding by the effects of macromolecular crowding
Protein folding by the effects of macromolecular crowding
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DOI：
10.1110/ps.03288104
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发表时间：
2004-01-01
期刊：
影响因子：
8
通讯作者：
Yomo, T
中科院分区：
文献类型：
--
作者：
Tokuriki, N;Kinjo, M;Yomo, T
利用核糖核酸酶A的非折叠态研究了大分子拥挤对大分子紧密性和蛋白质折叠的影响。通过圆二色谱法、荧光相关光谱法和NMR光谱法在聚乙二醇（PEG）或Ficoll作为拥挤剂的存在下测量蛋白质折叠和紧凑的程度。通过加入35%PEG 20000或Ficoll 70，RNase A在pH 3.0的2.4 M尿素溶液中的未折叠状态在构象和致密性上变得天然。此外，以荧光标记的聚乙二醇为测试分子，采用荧光相关光谱法研究了大分子拥挤对惰性大分子致密性的影响。随着PEG 20000或Ficoll 70浓度的增加，荧光标记的PEG的尺寸显著减小。这些结果表明，在高浓度的大分子的存在下，大分子是有利的紧凑的构象，并表明拥挤的环境的折叠和稳定的球状蛋白质的重要性。此外，不同大小的背景分子对大分子拥挤的影响的大小进行了研究。RNase A和标记的PEG没有变得紧凑，并通过加入PEG 200而具有折叠构象。还讨论了化学势对测试分子与测试分子和背景分子的相对尺寸的压实的影响。
Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.