The Role Of Chaperonin GroEL-GroES In The Folding Of Seven-transmembrane Proteins

GroEL-GroES system is the best characterized member of molecular chaperone family,and plays very critical roles in E.coli.They are required for the correct folding,assembly,and translocation of newly translated polypeptide chains.However,most of our knowledge on chaperone assisted folding was based on the folding of water soluble substrate proteins.The role of GroEL-GroES in the folding of newly translated membrane protein chains,especially G protein-coupled receptors(GPCRs),is still unclear.GPCRs play pivotal roles in various physiological,neurological and immunological processes,and constitute the most frequently addressed targets for current pharmaceutical therapies.It is of great scientific significance in the field of both biology and medicine to reveal the action mechanism of GroEL-GroES in the expression and folding of membrane protein,and to elucidate the relationship between the folding and function of GPCRs.In this study,we adopted the cell-free system to express the target GPCRs and investigate the effects of GroEL-GroES on the folding dynamics,structural stability,biological activity and expression level of newly translated GPCRs,also elaborated the mechanism of GroEL-GroES assisted-folding of nascent GPCRs polypeptides chains.Furthermore,the mechanism of chaperonin-assisted folding of membrane proteins was verified with bacteriorhodopsin(BR)which is a classical model of seven transmembrane proteins.Finally,based on the typical helical structures of GPCRs,the seven transmembrane peptides from CXCR4 were used to further study the binding and dissociation of GPCRs with GroEL.In chapter 2,the in vitro folding of newly translated human CC chemokine receptor type 5(CCR5)has been studied in cell-free system where the effects of GroEL-GroES on the translation and folding of nascent CCR5 were investigated directly.The freshly synthesized CCR5 can spontaneously fold into its biologically active state but this process was slow and inefficient.In comparision,on addition of GroEL-GroES,the folding rate,structural stability,functional expression level and ligand binding ability of the nascent CCR5 was significantly enhanced with ~36-fold,~3.5-fold,~1.3-fold and ~1-fold,respectively.Moreover,GroEL was partially effective on its own,but for maximum efficiency both the GroEL and its GroES lid were necessary.According to these results,we speculated and built the mechanism model of GroEL-GroES assisted-folding of GPCRs.In chapter 3,we analyzed synergies of GroEL-GroES and cell-free expression for the production of functionally folded C-X-C chemokine GPCR type 4(CXCR4)to further explore the application potential and versatility of GroEL-GroES in the functional expression of GPCRs.GroEL-GroES added was found to significantly enhance the productive folding of newly synthesized CXCR4,by increasing both the rate(~30-fold)and the yield(~1.4-fold)of folding over its spontaneous behavior.Meanwhile,the structural stability of CXCR4 was also improved with supplied GroEL-GroES,as was the soluble expression of biologically active CXCR4 with a ~1.4-fold increase.The essential chaperonin GroEL was shown to be partially effective on its own,but for maximum efficiency both GroEL and its co-chaperonin GroES were necessary.The method reported here should prove generally useful for cell-free production of large amounts of natively folded GPCRs.Meanwhile,we verified the action mode of GroEL-GroES in the folding of GPCRs with bacteriorhodopsin(BR)which is the ideal pattern protein of GPCRs due to the similar structure with GPCRs.We found that BR binds asymmetrically to the double-ring GroEL,and the denatured state exhibited a much higher affinity for the chaperonin than the native state.ATP is necessary to significantly enhance both the rate and yield of the GroEL-mediated folding,in contrast to the adverse effect of GroES on the folding yield.GroEL might perform repeated unfolding and release of BR,thus offering additional opportunities for timely folding.In chapter 4,the transmembrane peptides were used as mimics of exposed hydrophobic residues of GPCRs,which are crucial for GroEL recognition and assisted folding.By this reduction approach,complications like the kinetic competition between CXCR4 folding and binding to GroEL could be circumvented.Each of the CXCR4-derived peptides exhibited high affinity for GroEL with a binding stoichiometry near seven.It is found that the peptides interact with the paired ? helices in the apical domain of the chaperonin similar to the binding of SBP(strongly binding peptide: SWMTTPWGFLHP).Complementary binding study with a single-ring version of GroEL indicates that each of the two chaperonin rings is competent for accommodating all the seven CXCR4 peptides bound to GroEL under saturation condition.The binding kinetics of CXCR4 peptides with GroEL was also examined.In addtion,we found that ATP alone,or in combination of GroES evidently promoted the release of the peptide substrates from the chaperonin.These results obtained by the reduced approach of employing CXCR4 hydrophobic peptides would be beneficial to understand the thermodynamic and kinetic nature of GroEL-GPCRs interaction which is the central molecular event in the assisted folding process.