dnak蛋白质的纯化及其生物化学性质析-食品科学专业毕业论文.docxVIP

ABSTRACTHeat ABSTRACT Heat Shock Protein 70kDa，Hsp70s，are a class of highly conseⅣed m01ecular chaperone WIlich are essential for maintaining cellular protein homeostasis in orgaIlisms ranging f．rom bacteria to m猢a1． DnaK，a major Hsp70 in E cD，f，has been widely used as a paradigm for studying Hsp70s．nle latest s01Ved c呵stal灿cture of Dn水in complex wi也ATP shows t11ere are two m01ecules of DnaK—AIP in me asymmetric u11it．Interestingly，谢m seVeral hydrogen bonds t11e interface between tlle two molecules of DnaK are 1a玛e witll good sul．|’ace complementaIi够，suggesting functional importallce of mis crystallograpKc dimer． In ttlis study，crosslim【ing expe加ent were done based on the Dn址oAI’P strIlcture and confinned that DnaK fo蛐s specific dimer in an ArP—dependent maIlner．Tb understalld the physiological如nction of the dimer，、ve mutated five residues on the dimer interface．The growtll test加1，如D showed that f01lr mutations，R56A，T301A，N537A and D540A，result in loss of chaperone actiVi吼indicating me如nctionaJ imporCallce of t11e dimer．111en a11 above mlltant DnaK protein were expressed in E cD，f and purified witll HisTrap column aIld Hislhp Q coh】mn．FurtheImore we did nuorescence anisotropy peptide substrate binding assay．Dissociation constaJlts(＆)were detemined by fitting aJlisotropy data using PⅪSM sof．eware．The reSults didn’t show appreciable defect in substrate binding compared wim WT DnaK．Anomer nyptophaIl nuorescence assay were did to test whetller me disrupted dimer fomation could innuence ATP—induced a110Steric coupling．Compared wim 6 i1111 blue s11iR of WT DnaK，a11 t11e dimer mutants sho、ved similar锄ount of blue s11in， suggesting吐le ATP-induced anosteric coupling is la玛ely intact． In summary，this study suggests tllat DnaK foms transient dimer upon ATP binding and mis diIIler s协lcture is imponant to its m01ecular chaperone actiVi哆．Disrupting dimer fo衄ation reduced协e chaperone activi哆．Sufprisingly，neitller me peptide binding abili哆 nor tlle A1’P．induced a110steric coupling