| Trifluxazine(TFP)is mostly used in cell signaling and neurological researches.Because of its ability to bind to calmodulin,it is widely used as a blocker of calmodulin,and then to observe the activity of downstream calmodulin-dependent proteins.TFP is a mature drug for the treatment of mental illness in medicine,and has the advantage of less cardiovascular damage.Centrin and calmodulin have similar structures,both contain four EF hand domains,and can coordinate with four Ca2+.After binding to Ca2+,the protein conformation changes from a closed state to an open state,and the hydrophobic region is exposed,making it easy to bind other signaling proteins.Centrin plays an extremely important role in the cell life cycle,involving all aspects of cell division,movement,and maintaining normal physiological activity.The most important thing is that centrin plays an irreplaceable role in the process of nucleotide excision and repair.In view of the similarity between central protein and calmodulin,this paper selects the Euplotes octocarinatus centrin as the research object,and deeply understands the binding of TFP and central protein and the effects on central protein function,we hope to provide a theoretical basis for subsequent disease prevention and control.First,the relevant properties of TFP were studied by using fluorescence spectroscopy,ultraviolet-visible absorption spectrum and other methods.The results show that the polarity,temperature,and p H of the solvent all has a significant effect on the spectral properties of TFP,while the salt concentration has almost no effect on the spectral properties of TFP;the TFP concentration affects resonance light scattering spectrum.The change of the map means the change of TFP structure and morphology,that is,TFP will exist in different forms under different experimental conditions.In order to reduce the generation of by-products,the reaction conditions must be strictly controlled during the experiment.Next,the interaction of trifluproxazine(TFP)with the N-terminus(apo N-Eo Cen)and the C-terminus(apo C-Eo Cen)was studied using fluorescence spectroscopy,isothermal titration calorimetry,circular dichroism,resonance light scattering and other methods.The results showed that in p H=7.4 Hepes,10 mmol/L Hepes buffer,TFP was able to bind to apo N-Eo Cen at a ratio of 1:1 and apo C-Eo Cen at a ratio of 2:1,and the conditional binding constants were 103 L/mol and 107 L2/mol2,respectively.The combination of TFP with protein leads to a decrease in the protein ? helix content,and at the same time inhibits the protein's aggregation function,nucleotide excision repair function,and Tb3+ sensitized fluorescence.The explanation for these phenomena is due to the change in protein conformation,leading to these functions inhibition or loss.Finally,the interaction between TFP and WT-Eo Cen was studied using fluorescence spectroscopy,isothermal titration calorimetry,infrared spectrum,and polyacrylamide gel electrophoresis.The results show that TFP binds to WT-Eo Cen at a 3:1 binding ratio,and the conditional binding constant is 1012 L3/mol3,which is slightly increased compared to the cumulative binding constant between the semimolecules,this indicating the existence of domains between WT-Eo Cen Synergistic effect,but the effect is weak;TFP combined with WT-Eo Cen leads to a decrease in ? helix content.It is this conformational change that inhibits WT-Eo Cen functions,such as aggregation function,nucleotide excision repair function,binding to target peptide function and Tb3+-sensitized fluorescence function.The effect of TFP on WT-Eo Cen is similar to that on semi-molecules.By comparison,it was found that the effect of the semi-molecular aggregation was simply added and compared with the effect on WT-Eo Cen,and the results were consistent,indicating the synergistic effect between the WT-Eo Cen N-terminal domain and the C-terminal domain is weak... |
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