Study On The Conformational Changes Of Knotted Protein 1O6D Under Denatured Conditions

Knotted protein is a protein with high-level topological structure,which plays an important role in a variety of cellular physiological activities(such as DNA methylation,protein enzymatic hydrolysis,etc.).Studies have shown that this high-level topological structure plays a very important role in the structural stability and normal functioning of proteins.Therefore,the formation mechanism of this topological structure has also attracted widespread attention.At present,there are still many problems in the research on the folding mechanism of knotted proteins,such as whether the knotted structure exists after denaturation,and how the conformation changes during denaturation and unfolding.This thesis takes the knot protein1O6 D as the research object,and uses genetic engineering and time-resolved fluorescence techniques to study the conformational changes of knot protein 1O6 D under denaturing conditions.The main research contents of the thesis are as follows:(1)Using circular PCR site-directed mutagenesis technology,a series of mutants of knot protein 1O6 D were successfully constructed and transformed into E.coli expression system for expression.The recombinant protein was purified by chromatography and verified by electrophoresis.Its molecular weight is close to the theoretical value of 19 k Da and the sample purity is greater than 95%.The CD spectrum analysis of the mutants showed that each mutant has a similar secondary structure to the wild-type protein.The results of the chemical denaturation titration experiment of guanidine hydrochloride showed that the D50% of the four mutants were 3.75 M,3.99 M,3.53 M,and 3.33 M,respectively,which was slightly lower than the 4.05 M of the wild-type protein.Since most of the mutation sites are located inside the knotted structure and at the nodes,this also indirectly proves that the amino acid sites of the knotted structure have an important influence on the stability of the knotted protein.(2)The mutant protein was labeled with 1,5-IAEDANS fluorescent dye to form a FRET system(Tryptophan-1,5-IAEDANS)inside the protein molecule.Using time-resolved fluorescence technology,under different denaturation conditions of guanidine hydrochloride,The FRET efficiency and distance changes between the donor and the acceptor in each mutant protein were characterized.The results showed that with the increase of denaturant concentration,the FRET efficiency of the F84W-76 C mutant first remained unchanged and then decreased,indicating that the distance between the two points was first unchanged and then increased;F120W-76 C mutant FRET efficiency as the denaturant concentration increases,it first increases and then decreases,which indicates that the distance between the two points first decreases and then increases.In the same way,the distances between different labeling sites in Y106W-76 C,F127W-76 C,F84W-33 C,F120W-33 C,and F127W-33 C mutants all showed a gradually increasing trend with the increase of denaturant concentration.According to the changes in the distance between the labeled sites of each mutant during the denaturation process,we speculate that when the low concentration of guanidine hydrochloride is denatured,the secondary structure of the protein is not fully opened,the knotted structure becomes loose and slides toward the CTD,and the CTD peptide chain gradually Go inside the structure.As the denaturant concentration increases,the secondary structure of the protein is completely opened,and the knotted structure gradually becomes larger and looser,and continues to slide toward the CTD,showing a tendency to open from the CTD.(3)Use time-resolved fluorescence anisotropy technology to characterize the fluorescence anisotropy of the tryptophan sites of each mutant under different denaturing conditions of guanidine hydrochloride,so as to investigate the environment and space of each tryptophan site during the denaturation process Changes in restrictions.The research results show that the decay time of Trp84 and Trp106 located inside the knotted structure under non-denaturing conditions are 10.96 ns and 8.77 ns,respectively,while the decay time of Trp120 and Trp127 located outside the structure is significantly shortened to 2.30 ns and 2.08,respectively.ns.This indicates that there is a significant difference in the rotation rate of Trp inside and outside the knotted structure.Under the denaturation condition of 1-2 M low concentration of guanidine hydrochloride,the fluorescence anisotropy decay time of Trp120 and Trp127 of tryptophan increased to 5.56 ns and 5.81 ns,which is close to the decay time of Trp84 and Trp106 located inside the structure,showing Similar to the rotation state,it is speculated that Trp120 and Trp127 gradually enter the knotted structure with the protein denaturation process.When the denaturation concentration of guanidine hydrochloride increased to 3 M,the tryptophan decay time at different sites gradually shortened,and the decay time of Trp84 was the most significant,which was presumably caused by the gradual movement of Trp84 to the outside of the knotted structure.When the high concentration of guanidine hydrochloride is denatured,due to the complete denaturation of the secondary structure,the knotted structure is looser,and the fluorescence anisotropy decay time of tryptophan at 4 sites is significantly shortened.This is consistent with the results of the above FRET experiment,and further verified our speculation about the conformational changes of the knotted protein under denaturing conditions.The research in this thesis provides a theoretical basis for further elucidating the unfolding mechanism of knotting protein 1O6 D in the denaturation process,as well as for disease treatment and drug design targeting knotting protein.