F？rster Resonance Energy Transfer Measurements of Ryanodine Receptor Type 1 Structure Using a Novel Site-Specific Labeling Method 英文参考文献.docVIP

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F？rster Resonance Energy Transfer Measurements of Ryanodine Receptor Type 1 Structure Using a Novel Site-Specific Labeling Method 英文参考文献.doc

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F？rster Resonance Energy Transfer Measurements of Ryanodine Receptor Type 1 Structure Using a Novel Site-Specific Labeling Method 英文参考文献

Fo¨rsterResonanceEnergyTransferMeasurementsof RyanodineReceptorType1StructureUsingaNovel Site-SpecificLabelingMethod JamesD.Fessenden* BostonBiomedicalResearchInstitute,Watertown,Massachusetts,UnitedStatesofAmerica Abstract Background:WhilethestaticstructureoftheintracellularCa2+releasechannel,theryanodinereceptortype1(RyR1)has been determined using cryo electron microscopy, relatively little is known concerning changes in RyR1 structure that accompany channel gating. Fo¨rster resonance energy transfer (FRET) methods can resolve small changes in protein structure although FRET measurements of RyR1 are hampered by an inability to site-specifically label the protein with fluorescentprobes. Methodology/PrincipalFindings:Anovelsite-specificlabelingmethodispresentedthattargetsaFRETacceptor,Cy3NTA to10-residuehistidine(His)tagsengineeredintoRyR1.Cy3NTA,comprisedofthefluorescentdyeCy3,coupledtotwoNi / 2+ nitrilotriaceticacidmoieties,wassynthesizedandfunctionallytestedforbindingtoHis-taggedgreenfluorescentprotein (GFP).GFPfluorescenceemissionandCy3NTAabsorbancespectraoverlappedsignificantly,indicatingthatFRETcouldoccur (Fo¨rsterdistance=6.3nm).Cy3NTAboundtoHis10-taggedGFP,quenchingitsfluorescenceby88%.GFPwasthenfusedto theN-terminusofRyR1andHis10tagswereplacedeitherattheN-terminusofthefusedGFPorbetweenGFPandRyR1. Cy3NTAreducedfluorescenceofthesefusionproteinsby75%andthisquenchingcouldbereversedbyphotobleaching Cy3,thusconfirmingGFP-RyR1quenchingviaFRET.AHis10tagwasthenplacedataminoacidposition1861andFRETwas measuredfromGFPlocatedateithertheN-terminusoratposition618toCy3NTAboundtothisHistag.WhileminimalFRET was detected between GFP at position 1 and Cy3NTA at position 1861, 53% energy transfer was detected from GFP at position618toCy3NTAatposition1861,thusindicatingthatthesesitesareincloseproximitytoeachother. Conclusions/Significance:Thesefindingsillustratethepotentialofthissite-specificlabelingsystemforuseinfutureFRET- basedexperimentstoelucidatenovelaspectsofRyR