Probing the Functional Mechanism of Escherichia coli GroEL Using Circular Permutation 英文参考文献.docVIP

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Probing the Functional Mechanism of Escherichia coli GroEL Using Circular Permutation 英文参考文献.doc

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Probing the Functional Mechanism of Escherichia coli GroEL Using Circular Permutation 英文参考文献

ProbingtheFunctionalMechanismofEscherichiacoli GroELUsingCircularPermutation TomohiroMizobata1,3*,TatsuyaUemura3,KazuhiroIsaji1,TakumaHirayama2,KunihiroHongo1,3, YasushiKawata1,3 1DepartmentofChemistryandBiotechnology,GraduateSchoolofEngineering,TottoriUniversity,Tottori,Japan,2DepartmentofBiotechnology,FacultyofEngineering, TottoriUniversity,Tottori,Japan,3DepartmentofBiomedicalScience,InstituteofRegenerativeMedicineandBiofunction,GraduateSchoolofMedicalScience,Tottori University,Tottori,Japan Abstract Background: The Escherichia colichaperonin GroEL subunit consists of three domains linked via two hingeregions, and eachdomainisresponsibleforaspecificroleinthefunctionalmechanism.Here,wehaveusedcircularpermutationtostudy thestructuralandfunctionalcharacteristicsoftheGroELsubunit. Methodology/Principal Findings: Three soluble, partially active mutants with polypeptide ends relocated into various positionsoftheapicaldomainofGroELwereisolatedandstudied.Thebasic functionalhallmarks ofGroEL(ATPaseand chaperoningactivities)wereretainedinallthreemutants.Certainfunctionalcharacteristics,suchasbasalATPaseactivity andATPaseinhibitionbythecochaperoninGroES,differedinthemutantswhileatthesametime,theabilitytofacilitatethe refolding of rhodanese was roughly equal. Stopped-flow fluorescence experiments using a fluorescent variant of the circularlypermutedGroELCP376revealedthataspecifickinetictransitionthatreflectsmovementsoftheapicaldomainwas missing in this mutant. This mutant also displayed several characteristics that suggested that the apical domains were behavinginanuncoordinatedfashion. Conclusions/Significance: The loss of apical domain coordination and a concomitant decrease in functional ability highlights the importance of certain conformational signals that are relayed through domain interlinks in GroEL. We proposethatcircularpermutationisaveryversatiletooltoprobechaperoninstructureandfunction. Citation: Mizobata T, Uemura T, Isaji K, Hirayama T, Hongo K, et al