Oct. 13, 2005GW Canters, Leiden Univ1. Oct. 13, 2005GW Canters, Leiden Univ2 Förster Resonance Energy Transfer principle The efficiency E of the resonance.

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Presentation transcript:

Oct. 13, 2005GW Canters, Leiden Univ1

Oct. 13, 2005GW Canters, Leiden Univ2 Förster Resonance Energy Transfer principle The efficiency E of the resonance energy transfer from a fluorescent donor to an acceptor molecule is E = R 0 6 /(R 6 +R 0 6 ), R – donor-acceptor distance, R 0 – F ö rster radius R 0 6 ~  2 J( ),  2 – orientation factor, J( ) - spectral overlap between the donor fluorescence and acceptor absorption J( ) Sonya Kuznetsova, MetProt Grp 2005

Oct. 13, 2005GW Canters, Leiden Univ3 FRET as a molecular ruler: R 0 R R 0 is constant and determined by the spectral properties of the donor and the acceptor. The donor-acceptor distance R is changing and can be determined from the fluorescence intensity. donor: attached label ; acceptor: attached quencher R R Sonya Kuznetsova, MetProt Grp 2005

Oct. 13, 2005GW Canters, Leiden Univ4 3(2002)908

Oct. 13, 2005GW Canters, Leiden Univ5 D A emissionexcitation fluorescence F D excitation energy transfer fluorescence F DA A D emissionexcitation Oxidation Reduction

Oct. 13, 2005GW Canters, Leiden Univ SO O 3 S  N O O N N O N

Oct. 13, 2005GW Canters, Leiden Univ7 + Ferricyanide + DTT ChemPhysChem (2005), 6: J. Biol. Inorg.Chem (2005)

Oct. 13, 2005GW Canters, Leiden Univ8 Single Molecules: Wide Field Set- up Stokes- shift Ralf Schmauder, 2005

Oct. 13, 2005GW Canters, Leiden Univ9 Single Molecules: Raw Data  m Ralf Schmauder, 2005

Oct. 13, 2005GW Canters, Leiden Univ10 Single Molecules: brightness oxidizedreduced ChemPhysChem (2005), 6:

Oct. 13, 2005GW Canters, Leiden Univ11 Control: Zn-Azurin ChemPhysChem (2005), 6:

Oct. 13, 2005GW Canters, Leiden Univ12 Close up: h*  h*  1 fl = l ChemPhysChem (2005), 6:

Oct. 13, 2005GW Canters, Leiden Univ13 The raw data: chemistry time fluorescence DetectorLaser ChemPhysChem (2005), 6:

Oct. 13, 2005GW Canters, Leiden Univ14 Azurin Data: Cu ChemPhysChem (2005), 6:

Oct. 13, 2005GW Canters, Leiden Univ15 Azurin Data: Zn ChemPhysChem (2005), 6:

Oct. 13, 2005GW Canters, Leiden Univ16

Oct. 13, 2005GW Canters, Leiden Univ17 Cholesterol oxidase Science 282(1998)1877

Oct. 13, 2005GW Canters, Leiden Univ18 C.antarctica Lipase B Angew. Chem. 44(2005)560