Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP 英文参考文献.docVIP

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Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP 英文参考文献.doc

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Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP 英文参考文献

DistinctEffectsofGuanidineThiocyanateonthe StructureofSuperfolderGFP OlesyaV.Stepanenko1,OlgaV.Stepanenko1,IrinaM.Kuznetsova1,DariaM.Shcherbakova2, VladislavV.Verkhusha2,KonstantinK.Turoverov1* 1LaboratoryofStructuralDynamics,StabilityandFoldingofProteins,InstituteofCytologyoftheRussianAcademyofSciences,St.Petersburg,Russia,2Departmentof AnatomyandStructuralBiology,AlbertEinsteinCollegeofMedicine,NewYork,NewYork,UnitedStatesofAmerica Abstract Havingahighfoldingefficiencyandalowtendencytoaggregate,thesuperfolderGFP(sfGFP)offersauniqueopportunity tostudythefoldingofproteinsthathaveab-barreltopology.Here,westudiedtheunfolding–refoldingofsfGFPthatwas induced by guanidine thiocyanate (GTC), which is a stronger denaturing agent than GdnHCl or urea. Structural perturbations of sfGFP were studied by spectroscopic methods (absorbance, fluorescence, and circular dichroism), by acrylamide quenching of tryptophan and green chromophore fluorescence, and by size-exclusion chromatography. Low concentrationsofGTC(upto0.1M)inducesubtlechangesinthesfGFPstructure.Thepronouncedchangesinthevisible absorption spectrum of sfGFP which are accompanied by a dramatic decrease in tryptophan and green chromophore fluorescence was recorded in the range 0–0.7M GNC. These alterations of sfGFP characteristics that erroneously can be mixed up with appearance of intermediate state in fact have pure spectroscopic but not structural nature. Higher concentrationsofGTC(from0.7to1.7M),induceadisruptionofthesfGFPstructure,thatismanifestedinsimultaneous changes of all of the detected parameters. Full recovery of native properties of denaturated sfGFP was observed after denaturant removal. The refolding of sfGFP passes through the accumulation of the off-pathway intermediate state, in whichinneralpha-helixandhencegreenchromophoreandTrp57arestillnottunedupto(properlyintegratedinto)the alreadyformedb-barrelscaffoldofprotein.Incorporationofthechromophoreintheb-barrelinthepathwayofrefolding andrestorationofits