Study On GroEL-GroES-assisted Bacteriorhodopsin Folding

Proteins are widely involved in various life activities of organisms,but nascent peptide chains must be folded into specific structures to function,and misfolding of proteins is an important cause of many diseases.Molecular chaperones often play an important role in the folding of many proteins.E.coli-derived chaperone GroEL and its cofactor GroES are a very important class of molecular chaperones.Many studies have shown that the molecular chaperone GroEL-GroES can assist in the folding of many water-soluble proteins.Recently,our research group showed that the molecular chaperone GroEL-GroES can also assist in the folding of membrane proteins,which has a significant effect on the production and activity of membrane proteins.This finding is of great significance for the synthesis of membrane proteins.It is still unclear how GroEL-GroES assists in the folding of membrane proteins,so it is difficult to further regulate this folding process.In this paper,the process of folding the GroELGroES assisted membrane protein was studied by using the bacteriorhodopsin with seven transmembrane alpha helix structures.It is expected to provide an experimental basis for regulating the folding of membrane proteins while elucidating this folding process.In this paper,we used single-molecule fluorescence technology to investigate the mechanism of action of GroEL-GroES in bacteriorhodopsin folding,including its effect on the dynamic changes of molecular conformation in bacterial rhodopsin folding/unfolding.In Chapter 2,based on the previous work of the laboratory,we successfully prepared two double-site mutants of bacteriorhodopsin BR(0C-V130C)and apobacteriorhodopsin BO(0C-249C).The fluorescent proteins AF488 and AF647 were fluorescently double-labeled with the mutant protein,and the results of the characterization of the labeled protein samples indicated that the double labeling was successful.The proteins such as GroEL and GroES were also successfully prepared.The next step was to study the effects of GroEL-GroES on the folding of bacteriorhodopsin by single-molecule fluorescence resonance energy transfer(smFRET)and fluorescence correlation spectroscopy(FCS).In Chapter 3,we explored the kinetics and thermodynamics of different combinations of GroEL/GroES/ATP combined with denatured BR(0C-V130C)and natural BR(0C-V130C).It was found that ATP acts as an activator and can greatly promote hydrophobic binding of GroEL to the denatured substrate protein BR,thereby promoting refolding of denatured proteins.At the same time,we believe that ATP,GroES,and ATP-GroES can have different effects on the structure of GroEL,respectively,resulting in changes in the affinity of GroEL for specific substrates.Thermodynamic experiments show that the affinity of denatured BR to the molecular chaperone GroEL is higher than that of native BR.In Chapter 4,we used fluorescence correlation spectroscopy to study the spontaneous renaturation of BR molecules,doubling under the help of GroEL,and BR renaturation mediated by GroEL-GroES.It is found that the denatured BR molecules are prone to aggregation when they are spontaneously renatured,which is not conducive to their folding into the correct natural state.The molecular chaperone GroEL binds to the denatured BR which is easy to aggregate and evacuates the aggregated BR molecule.When GroEL-GroES is present at the same time,the effect of the evacuation of the BR molecules in the aggregated state is more obvious.By binding to the BR folding intermediate,the non-native BR aggregation is reduced,thereby facilitating the folding of the substrate protein into its natural state.At the same time,the dynamic changes of molecular conformation in the folding/unfolding of BO mutants were characterized by single-molecule fluorescence resonance energy transfer technique.It was found that there were many different sets in the unfolding process of BO molecules.The spontaneous refolding process of BO molecules is slow and the renaturation yield is low.During GroEL-mediated BO renaturation,the rate of refolding increased,but the rate of refolding was very low.The cooperation of the cofactor GroES and the activator ATP can further aggravate the aggregated BO,thereby maximally increasing the folding rate and folding efficiency of the denaturing BO,regenerating the GroEL activity,and promoting the refolding of the degenerate BO.