Effects of macromolecular crowding on alkaline phosphatase unfolding, conformation and stability.
The interior of the cell is tightly packed with various biological macromolecules, which affects physiological processes, especially protein folding process. To explore how macromolecular crowding may influence protein folding process, alkaline phosphatase (ALP) was chosen as a model protein, and the unfolding process of ALP induced by GdnHCl was studied in the presence of crowding agents such as PEG 4000, Dextran 70 and Ficoll 70. The effect of macromolecular crowding on the denatured state of ALP was directly probed by measuring enzyme activities, fluorescence spectroscopy and circular dichroism. From the results of circular dichroism, GdnHCl induced a biphasic change, suggesting that a three-state unfolding mechanism was involved in the denaturation process irrespective of the absence or presence of crowding agents. It was also found that crowding agents had a little impact on the unfolding process of ALP. The results of phase diagrams also demonstrated that the unfolding process of ALP induced by GdnHCl was three-state mechanism. Moreover, the results of fluorescence spectra demonstrated that with the increase of GdnHCl concentration, the structure of protein had changed, but existence of crowding agents can make protein structure more stable. Our results can provide valuable information for understanding the protein folding in vivo.
Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
Jiajia
J
Jia
School of Life Sciences, Tianjin University, Tianjin 300072, PR China. Electronic address: pengxin5403@tju.edu.cn.
Xin
X
Peng
Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China; State Key Laboratory of Chemical Engineering, Tianjin University, Tianjin 300072, PR China; Collaborative Innovation Center of Chemistry Science and Engineering (Tianjin), Tianjin 300072, PR China; Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin, 300072, PR China. Electronic address: qiwei@tju.edu.cn.
Wei
W
Qi
Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China; State Key Laboratory of Chemical Engineering, Tianjin University, Tianjin 300072, PR China; Collaborative Innovation Center of Chemistry Science and Engineering (Tianjin), Tianjin 300072, PR China; Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin, 300072, PR China.
Rongxin
R
Su
Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China; State Key Laboratory of Chemical Engineering, Tianjin University, Tianjin 300072, PR China.
Zhimin
Z
He
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability; D019791:Guanidine / Q000494:pharmacology; D011487:Protein Conformation; D058767:Protein Unfolding / Q000187:drug effects
D000469:Alkaline Phosphatase; D019791:Guanidine
Alkaline phosphatase; Macromolecular crowding; Unfolding