The Folding Mechanism Of Chemokine CCL11 And Its Cocrystallization With CCR3

The eotaxin-1(CCL11)belongs to the ? chemokines or CC chemokines family,which has a chemotactic function to immune cells.The chemokine receptor CCR3 is a member of family A GPCR embeded in the leukocytes cell surface as membrane proteins.CCL11 plays a crucial role in asthma and other allergic inflammation by binding to specific sites of the chemokine reporter CCR3.It has been supposed that blocking the CCR3 signal transduction pathway could cure or release related diesease.Only a few systematic studies for the folding mechanism exist so far.In addition,there has not determinated for the crystal structure of chemokine receptor CCR3.In this thesis,the thermodynamic stability and the folding mechanism of CCL11 were systematically investigated.Alao biologically active chemokine receptor CCR3 was prepared by using both the prokaryotic and the mammalian expression systems,and the crystallization conditions of CCR3 and CCL11 complexes were also carried out using high throughput screening.In the thermodynamic stability part,two CCL11 mutants each with the removal of a pair of disulfide bond were prepared.it was shown that the mutant CCL11 was completely denatured in 4.5 mol/L guanidine hydrochloride and the D50=2.20 mol/L and ?G=3.39 kcal.mol-1 On the other hand,the mutants stay in the denaturized states during the entire equilibrium titration and CD spectrum measurements,indicates that both pairs of disulfide bonds are important in maintaining the CCL11 structural stability.CCL11 cannot be refolded in the temperature denaturation experiments,and a Tm value of 325.1 K was obtained by fitting the fluorescence curves.Folding kinetics experiments indicate that a folding intermediate exists during the denaturing with guanidine hydrochloride concentration in the range 0-3.5M.This three-state pathway is competing with the two-state direct conversion,which is supported by our further analysis of the Chevron plots,where the conversion rate constants can be evaluated by fitting the Gibbs free energy change between different states The intermediate state was not observed in measurements with guanidine hydrochloride concentrations in the range 3.5-8M,of which experimental data agrees well with the two-state model.In CCR3/CCL11 crystallization conditions screening parts,at fist,the expression vectors with MBP fusion label is used to expres the fusion protein MBP-CCR3 and the biological activity of MBP-CCR3 is vertified by the way of binding to CCL11,the result shown that MBPCCR3 has a high yield(2mg/L),however,binding assay in vitro shown a low binding affinity between MBP-CCR3 and CCL11.So the other two expression vectors in which including SUMO3 and GST two different fusion label ware designed in the prokaryotic expression system,and then the expression conditions were optimized,the results show that the high expression system(1.2mg/L)taking GST fusion label were selected.The fusion protein expressed by taking GST fusion label expression system were detected beding located in the cell membrane,and form of fusion protein of taking GST fusion label was aggregation.For the mammalian expression system,we detected all the same content as well as prokaryotic expression system.The results show that CCR3 expressed by mammalian cell has a high product(milligram grade),existing a dimer and monomer and could form complex with CCL11.Lastly,the crystal condition of CCR3 and CCL11 complex were screened.The result shown that there were no growing crystals for CCR3 and CCL11 complex except crystals of CCL11.The crystallization reagent condition of CCL11 was 30%v/v polyethytene glycol 400,0.1M sodium acetate,0.2M Calcium acetate,p H=4.5.