The stability and the metal ions binding properties of mutant A85M of CopC.
In this work, the mutant A85M of CopC was obtained. The stability of mutant A85M of CopC and the binding properties of metal ions were clarified through various spectroscopic techniques. The binding capacity of A85M to metal ions was measured by fluorescence spectroscopy and UV differential absorbance. The results suggested that Cu(2+) can bind with A85M in 1:1 form, and the constant of A85M was nearly the same as that of CopC. Ag(+) can occupy the Cu(+) binding site located at C-terminal, and the binding constant was (2.64±0.48)×10(6)L/mol. Hg(2+) not only can occupy the Cu(+) binding site located at C-terminal, but also can occupy the Cu(2+) binding site located at N-terminal. The stability of A85M was measured by chemical unfolding experiment. The intermediate was observed in the unfolding pathway of A85M-Cu(2+) induced by urea. In addition, the interaction of SDS with A85M also can result in the formation of the intermediate. The effect of metal ions on the stability of intermediate suggested that the C terminal region of intermediate was unfolded and the N terminal region suffered few effects. Compared with CopC, the stability of A85M was decreased. The main reason was the lower stability of N terminal region. The results of molecular dynamic simulation suggested that when the alanine at 85 site was mutated to methionine, the hydrophobic almost unchanged, but the distance between the phenylalanine at 25 site and tryptophan at 83 site increased because of the spatial effect. And it made the stacking interaction of aromatic rings decreased, which was the main reason for the decreasing stability of N terminal region for A85M.
Taiyuan Normal University Department of Chemistry, Taiyuan 030012, China.
Zhen
Z
Song
Taiyuan Normal University Department of Chemistry, Taiyuan 030012, China; Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China.
Jinlong
J
Dong
Taiyuan Normal University Department of Chemistry, Taiyuan 030012, China.
Wen
W
Yuan
Taiyuan Normal University Department of Chemistry, Taiyuan 030012, China; Humic Acid Engineering and Technology Research Center of Shanxi Province, Taiyuan 030012, China.
Caifeng
C
Zhang
Taiyuan Normal University Department of Chemistry, Taiyuan 030012, China.
Yuehong
Y
Ren
Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China. Electronic address: yangbs@sxu.edu.cn.
Binsheng
B
Yang
D001419:Bacteria / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions / Q000737:chemistry; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D001426:Bacterial Proteins; C071265:CopC protein, Bacteria; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins
Conformation stability; Intermediate; Metal ions