Study On The Impact Of Chaperonin GroEL-GroES On Bacteriorhodopsin Folding And Membrane Integration

Cell homeostasis is crucial for the survival of cels and even whole organisms.This requires the entire process of protein folding,degradation,and transport to maintain homeostasis.However,a family of proteins that caled molecular chaperones regulates this protein homeostasis.Molecular chaperones act as regulatory factor in the process of protein homeostasis,which can prevent protein aggregation,promote proper protein folding and regulate proper protein degradation under physiological and stimulatory conditions.However,most of these studies are directed at water-soluble proteins,the mechanism of molecular chaperone system-assisted membrane protein folding is rarely studied at present.In this thesis,we used the seven-transmembrane protein bacteriorhodopsin(BR)as the substrate protein to study the mechanism of action of the chaperonin GroEL-GroES when BR was folded in the DDM micelles.At the same time,a preliminary study was carried out on the GroEL-GroES mediated denatured BR insertion and the folding process in simulated biofilms.In the second chapter of the study,the expression and purification of the substrate protein BR were carried out successfully,and a large number of BRs were successfully prepared,which laid the material basis for subsequent folding studies.The chaperonin GroEL and its comolecular chaperones GroES and SR1 were prepared for follow-up experiments,which is the experimental basis for this study.In the third chapter,BR was used as the substrate membrane protein and based on the UVVis absorption spectrum of retinal,we studied the effects of different combinations of GroEL,GroES,and ATP on the kinetics of denatured BR folding,and compared with the mutant SR1.Based on the UV-Vis absorption spectrum,the influence of different combinations of GroEL,GroES and ATP on the structure of the folded BR was also studied;based on the absorption spectrum or fluorescence spectrum,we studied the different combinations of GroEL(SR1),GroES,ATP on the water soluble protein.—Enhanced Green Fluorescent Protein(EGFP)—the effect of folding Kinetics;Based on BR intrinsic fluorescence,the effect of GroEL,GroES,and ATP on BR folding kinetics was studied that using a technique of stop-flow.Based on the results of the relevant experimental data and analysis,we found that the binding affinity of the denatured BR to the chaperone GroEL is higher than the native BR.GroEL has a significant effect on refolding of BR,but the presence of ATP is necessary to increase the rate and yield of GroEL-mediated BR folding.On the contrary,GroES has an adverse effect on its folding yield.Finally,in the fourth chapter,we prepared E.coli inverted vesicles.Based on the UV-Vis absorption spectra of retinal in BR,the effect of different combinations of GroEL,GroES and ATP on the process kinetics of denatured BR was studied,which compared with the role of GroEL mutant SR1.We found that GroEL-GroES can also mediate the membrane integration of BR,however,the synergy between ATP and GroES proved to be necessary not only for the release of high-affinity denatured BR from GroEL,but also for misfolding.This is consistent with the observed results of BR insertion rate enhancement and maximized folding rate when using the complete GroEL-GroES system.The results we obtained support that the repeated annealing mechanism previously proposed for the soluble protein GroEL-GroES effect,in which GroEL can perform repeated unfolding and release of BR,thus providing an opportunity for BR to correctly fold or membrane integration.