single-molecule fluorescence spectroscopy 5 dynamics of a single molecule by FRET michael börsch 18/07/2003
topics theory of fluorescence resonance energy transfer solvent effects and fluorescence quenching conformational dynamcis of single ribozymes ( X. Zhuang, Harvard) a rotary molecular motor @ work: single F o F 1 -ATP synthase during ATP hydrolysis and ATP synthesis
fluorescence resonance energy transfer : FRET (1) distance dependence on nanometer scale: 1-10 intensity traces
fluorescence resonance energy transfer : FRET (2) non-radiative
fluorescence resonance energy transfer : FRET (3) distance dependence and Förster radius R 0
fluorescence resonance energy transfer : FRET (4) spectral overlap (depends on λ):
fluorescence resonance energy transfer : FRET (5) orientational factor κ 2 freely rotating: κ 2 = 2/3
fluorescence resonance energy transfer : FRET (6) transfer efficiency and distance measurement
fluorescence resonance energy transfer : FRET (7) fluorophore interaction: energy, symmetry, distance
fluorescence resonance energy transfer : FRET (8) Donor Acceptor Table 1.4 R 0 values for Alexa Fluor dyes* Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 555 Alexa Fluor 568 Alexa Fluor 594 Alexa Fluor 647 Alexa Fluor 488 NA 64 70 62 60 56 Alexa Fluor 546 NA 70 71 74 Alexa Fluor 555 NA 47 51 Alexa Fluor 568 NA 82 Alexa Fluor 594 NA 85 *R 0 values in angstroms (Å) represent the distance at which fluorescence resonance energy transfer from the donor dye to the acceptor dye is 50% efficient. Values were calculated from spectroscopic data as outlined (Fluorescence Resonance Energy Transfer (FRET)). NA = Not applicable.
fluorescence resonance energy transfer : FRET (9)
fluorescence resonance energy transfer : FRET (10)
fluorescence resonance energy transfer : FRET (11)
why single-molecule FRET? conformational dynamics in biomacromolecules www.courses.fas.harvard.edu/ ~chem163/lectures/ 'Frontiers in Molecular Biophysics' lectures by Xiaowei Zhuang
single-molecule FRET imaging TIRFM with CCD
solvent effect and FRET the FRET efficiency depends on the donor fluorescence spectrum ( spectral overlap J) gated spectra
quenching and FRET (1) the FRET efficiency depends on quantum yields
quenching and FRET (2) O 2 quenching of perylene concentration dependece of quenching: modified Stern-Volmer plot reveals the number of quenchable fluorophores.
confocal single-molecule FRET - a molecular motor @ work - rotation in F o F 1 -ATP synthase biochemistry ATP mechanical work signalling
F o F 1 -ATP synthase from E. coli structure function two motors ADP + P i ATP @ F 1 3x per 360 rotation rotor stator 8 nm B. Böttcher (2000) JMB 296, 449 proton translocation @ F o 10 14x per 360 rotation
F 1 motor @ ATP hydrolysis substeps during ATP hydrolysis (Kinosita & Yoshida groups, nature 2001) Mvrdbqr7.mov high [ATP] M1qa0p1o.mov low [ATP]
How F o F 1 might synthesize ATP animation from W. Junge @ Universität Osnabrück F o driven by [H + ] continuous rotation of the c-ring stepwise rotation of γ and ε (3 steps) induced conformational changes at β (3) site 1: release of ATP site 2: formation of ATP site 3: waiting for ADP,P i
rhodamine B sulfonic acid fluoride! β-lysine-4, non-rotating subunit Rotation monitored by Fluorescence Resonance Energy Transfer Cy5 FRET ( published 2003 in PROC. SPIE 4962 )
FRET efficiency and rotation two fluorophores spectral overlap const. orientation κ 2? distance changes distinct steps (3) sequential transitions rigid protein domains long observation times 3 F 1 catalysis (ATP hydrolysis): 1 2 γ rotates stepwise in 120 (90 + 30 ) and counterclockwise 1 2 3 1
F 1 : 2-step FRET labelling 1 : Cy5@γ-Cys-106 2 : RhB@β-Lys-4 RhB Cy5 Cy5
Selectivity of subunit labelling by SDS-PAGE of F 1 and UV-VIS RhB RhB + Cy5 Cy5 absorbance 3 2 1 normalized absorbance F 1 -βrhb-γcy5 F 1 -βrhb F 1 -γcy5 0 200 300 400 500 600 700 800 wavelength / nm
Single-molecule detection in solution confocal set-up focussed laser spot inside solution, (open volume, diffusing molecules) pinhole (100 µm) eliminates out-of-focus FL two detectors count photons in two channels (FRET) photon bursts during transit
Set-up Nov-2000 laser : 532 nm, 50 mw, cw objectives : 60x, n.a. 1.2, W 40x, n.a.1.15, W PIFOC two SPAD 1: 545-610 nm 2: LP 665 nm PMS300 for MCS
1,2 F 1 FRET labelling. 3,4 Reconstituting F o. 5 Reassembling F o F 1 1 2 RhB RhB 5 Cy5 Cy5 Cy5 3 4 5
Observation times of proteoliposomes by FCS (fluorescence correlation spectroscopy) g (2) (τ), norm. 1.2 1.0 0.8 0.6 0.4 0.2 Rhodamine B F 1 -βrhb F o F 1 -βrhb in liposomes 0.0 10-3 10-2 10-1 10 0 10 1 10 2 10 3 10 4 correlation time τ c / ms
Identification of a single F o F 1 P = I ac / (I ac +I do ) counts per ms proximity factor
Opposite direction of γ rotation : the '80-degree problem' of γ stop positions hydrolysis 86% A1=M A2=L A3=H counter-clockwise rotation synthesis 84% A3=H A2=L A1=M clockwise rotation ATP synthesis: virtual γ stops ccw shifted by 80? observed order was L-M-H substeps: catalysis 90 waiting states 0 =120
Proc. SPIE 4962, pp. 11-21 beta-4 gamma-106 FRET approach with single F o F 1 reconstituted into liposomes during ATP hydrolysis and synthesis Institute of Physical Chemistry (University of Freiburg) Prof. P. Gräber Manuel Diez Boris Zimmermann Stefan Steigmiller Matthias Trost (GBF Braunschweig) Paola Turina (University of Bologna) 3 rd Institute of Physics (University of Stuttgart) Prof. J. Wrachtrup Nawid Zarrabi Michael Börsch (www.m-boersch.org)
reference for the single-molecule FRET approach F o F 1 -ATP synthase @ ATP synthesis PDF version of papers: www.m-boersch.org/ email: m.boersch@physik.uni-stuttgart.de... let's go back to work :-)
people & projects 1. Manuel Diez: rotation γ vs. b 2 2. Boris Zimmermann: regulatory function of ε 3. Stefan Steigmiller: ATP binding and mode of catalysis 4. Monika Düser: rotary motions in F o 5. Nawid Zarrabi: rotation of γ vs b 2 with immobilized F o F 1 6. Mark Mozer: proton translocation of single F o F 1 7. Jörg Breitbarth: coincidence analysis of ATP binding 8. N.N.: ATP synthase powered (nano)devices...
thank you.