The Study Of The Unfolding Process Of Knotted Protein 1O6D

The classical protein folding theory believes that complex topological structures are generally not formed in proteins,but with the development of computer simulation technology,NMR,and X-Ray technology,more and more knotted proteins are continuously discovered.Studies have shown that knotting structures not only play an important role in the structure and function of proteins,but also have important biological effects on the treatment of genetic diseases and the regulation of physiological activities.However,the folding mechanism of knotted proteins and how the structure of knots affects the folding process of proteins remain to be uncover.In this dissertation,the thermodynamics and kinetics of the knotted protein 1O6D in the unfolding process was studied to reveal the folding mechanism of knotted proteins.?1?High-purity 1O6D wild-type protein was prepared in E.coli expression system and characterized.SDS-PAGE and mass spectrometry analysis showed that the relative molecular weight of the prepared 1O6D protein was 19112.12 Da,which was consistent with the theoretical molecular weight.The result of circular dichroism spectroscopy showed that the1O6D wild-type protein showed a distinct?-helix structure,which is consistent with the literature reports.Chemical denaturation titration showed that the half denaturation concentration?D50%?of the wild-type 1O6D protein was 4.05 mol/L GdnCl.Temperature denaturation experiments showed that the wild-type 1O6D protein is of high thermal stability,and its secondary structure at 80°C is similar to that at room temperature.Kinetic experiments show that the unfolding of 1O6D involves both slow and fast processes.?2?Five 1O6D tryptophan insertion mutants were successfully constructed using PCR technology.The SDS-PAGE and mass spectrometry experiments confirmed that the actual relative molecular weight of the mutants were consistent with its theoretical value.Circular dichroism experiments showed that the secondary structures of the mutants were almost identical to the wild type.Chemical titration experiments showed that D50%of mutants were lower than wild-type 1O6D.By calculating?G₀ of each mutant,the wild-type 1O6D structure was found to be the most stable,while the stability of F120W and F84W mutants was poor.Temperature denaturation titration studies found that the thermal stability of F68W and Y106W ware similar to that of wild-type,while the thermal stability of F84W and F120W ware significantly reduced,which is consistent with the results of chemical denaturation titration,indicating that these two mutation sites are of great importance to maintain the structural stability of the protein.?3?The mutant proteins were labeled with the fluorescent dye IAEDANS.The time-resolved fluorescence resonance energy transfer?TR FRET?technique was used to detect the change of the tryptophan fluorescence lifetime of the mutants under different concentrations of GdnCl.As a result,it was found that the variation of amino acid distances among the five 1O6D mutants during the denaturation process is:the distance of the mutants of F84W and Y106W in knotted core has an increase and then a decrease between the donor and acceptor in the denaturation process;the distance between donor and acceptor in mutants of F68W,F120W and F127W during denaturation was gradually increased.It can be seen that there is indeed a significant conformational change in the knotting structure during the denaturation process,but further detailed experiments or molecular dynamics simulations are needed to verify the detailed dynamic change process of the knotting structure.