Effect Of Several Molecular On Copper Regulation Of Copper Chaperone (CopC)

The copper chaperone Cop C found in Gram-negative bacteria in California tomatoes from a high concentration of copper,it consists of 102 amino acid residues,and its molecular weight of approximately 10 k Da.The NMR structural data indicates that Cop C combined with Cu²⁺ and Cu⁺features a Greek barrel-like structure formed by 9 ?-sheets,two of which are parallel and the rest are anti-parallel.The protein contains a hydrophobic core,and the only tryptophan 83 and tyrosine 79 residues are located in the hydrophobic barrel.Cop C has a high affinity for both Cu⁺ and Cu²⁺.The binding site of Cu²⁺ is located at the N-terminus of Cop C,with two histidine residues,one glutamic acid and one aspartic acid residue,which are His1,His91,Glu27 and Asp89,respectively.The other end of the barrel is a Cu⁺binding site with four methionine residues,Met40,Met43,Met46 and Met51.Cop C is involved in the regulation of copper as a redox switch,and the interaction of Cop C with small molecules will affect the regulation of copper by Cop C.Based on the interaction between HSSC?SSC,salicyl sulfamate-anion?,L1,curcumin and Cop C.In this paper,the study focused on the interaction between doxorubicin?ADM?and Cop C,and the effect of some small molecular on Cop C as a redox switch.Firstly,this paper studied the interaction between CopC and ADM.Fluorescence spectroscopy,UV-vis spectroscopy and isothermal titration calorimetry?ITC?analysis showed that Cop C and ADM were bound by 1:1,and the conditional stability constant was about 105 L/mol.Infrared spectroscopy,CD spectroscopy and urea-induced unfolding experiments showed that binding of doxorubicin resulted in conformational change of Cop C,accompanied by a decrease in ?-sheet and an increase in random coiling,and the stability of Cop C was reduced.Computational docking studies showed that the main force between ADM and Cop C was hydrogen bonding and hydrophobic interaction,and the binding site of ADM is located at the N-terminus of the protein.Secondly,the interaction of Cu²⁺ with ADM?ADM-Cop C was studied.Fluorescence spectra,UV-vis spectra,ITC and fluorescence lifetime analysis indicated that Cu²⁺ and ADM were combined by 1:2,and the cumulative conditional stability constant K[Cu?II?-ADM] is 1.90×109 L2/mol2;kinetics and thermodynamic experiments showed that Cu²⁺,ADM and Cop C can form a ternary complex Cop C-Cu²⁺-ADM,and the formation of complex greatly reduced the reduction rate of Cu²⁺,the combination of ADM,affects the role of Cop C as a redox switch.Finally,the effects of ADM,HSSC,L1 and curcumin on Cop C copper regulation were studied.The UV reduction kinetics indicated that the main factors affecting the regulation of Cop C copper by small molecules were Cu²⁺ reduction and Cu⁺ migration.HSSC binds to C-terminal of Cop C,L1 and ADM bind to N-terminal of Cop C,the binding will change the conformation of Cop C,expand the exit and inlet of Cu⁺ migration channel in Cop C,and make migration of Cu⁺ easy,and curcumin which bind to outside the middle of the Cop C hydrophobic bucket did not change the migration pathway of Cu⁺,and did not affect the migration rate of Cu⁺;By constructing an amino acid mutation in the center of the hydrophobic barrel,the alanine at position 85 is mutated to methionine,forming the Cu⁺ weak binding position in the barrel,making migration of Cu⁺ easier,which indicates that the reduction pathway of Cop C-Cu²⁺ may be an internal pathway.