Molekulare Maschinen bei der Arbeit: Untersuchungen von Proteindynamik mit Einzelmolekül-FRET

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1 Molekulare Maschinen bei der Arbeit: Untersuchungen von Proteindynamik mit Einzelmolekül-FRET Dagmar Klostermeier Biophysikalische Chemie Biozentrum, Uni Basel

2 Outline 1. Fluorescence, FRET, and single molecules 1.1. Fluorescence 1.2. Conformational changes and FRET 1.3. Confocal microscopy 1.4. Total internal reflection microscopy 1.5. Information obtained in smfret experiments 2. Helicases: RNA unwinding 2.1. The RNA helicase YxiN 2.2. Donor-acceptor labeling 2.3. Single molecule FRET results 3. DNA topoisomerases

3 Green fluorescent protein (GFP) Aequorea victoria GFP

4 Jablonski diagram E IC S 2 IC Internal conversion (10-12 s) ISC Inter-system crossing (10-8 s) VR vibron. Relaxation (10-12 s) VR S 1 ISC T 1 V n V 0 S 0 ΔE = h ν = h c / λ Absorption Fluorescence (τ) Phosphorescence (τ) s 10-9 s s

5 conformational changes DNA polymerase β apo enzyme DNA nucleotide complex

6 Two fluorescent dyes: FRET Fluorophor 1 Donor Fluorophor 2 Acceptor abs fluo abs fluo λ (nm)

7 E Jablonski diagram: Energy transfer IC S 2 k T = 1 τ D R r VR k T S 1 S 1 V n V n V 0 S 0 V 0 S 0 Donor Acceptor

8 Two fluorescent dyes: FRET E Dipolar coupling Transfer efficiency E 6 kt R0 E = = 6 k + k + k R + r T F S 6 0 S 1 Förster distance R κ R = φ n D 4 J abs fluo Orientation factor κ 2 κ 2 = (cosθ Overlap integral J 4 J = F ( λ ) ε ( λ ) λ dλ D DA 3 cosθ A D cosθ A ) 2 S 0 donor acceptor

9 orientation factor κ 2 D κ 2 =4 θ DA θ D r φ θa θ A κ 2 =1 A κ 2 =0 κ 2 = (cosθ DA 3 cosθ D cosθ A ) 2

10 Calculating distances from FRET efficiencies R ln10 φ κ = F π N n D 4 D A 0 ( λ) ε ( λ) λ dλ Validation of the orientation factor approximation Fluorescence anisotropy decays r(t) = β + t/ φ1 t/ φ 1 e β2 e 2 θ local global flexibility Ordnungsparameter S S = β 1 θ = cos 1 β2 + β 2 ( S 1)

11 Förster distances Donor Acceptor R 0 (nm) Naphtalene Dansyl Pyrene Cy3 Fluorescein Terbium Europium Tryptophan Dansyl Fluorescein Coumarin Cy5 TMR Rhodamine Cy5 Nitrobenzoyl Dansyl IAEDANS Tyr-NO 2 Pyrene Heme

12 Fluorescence resonance energy transfer (FRET) as a molecular ruler E = R R r FRET efficiency in % R distance (nm)

13 FRET as a molecular ruler fluorescence fluorescence λ (nm) λ (nm) E = 1 I I DA D = 6 R0 R + r 6 0 6

14 Why single molecules?

15 I II III II I t I III II

16 confocal microscopy 1 fl = l

17 confocal microscopy Si-chip electrons per photon

18 single molecule FRET

19 Total internal reflection (TIR) microscopy donor Prism Objective CCD camera z z acceptor Charge proportional to integrated illumination

20 experimental set-up confocal APD d TIR L F d beamsplitter APD a L F a beamsplitter L 2 pinhole L 1 F a L F d CCD

21 Surface immobilization Streptavidin / Biotin

22 Surface immobilization nucleic acids streptavidin proteins biotin-lipid proteins/ nucleic acids biotin-bsa biotin-peg biotin-d/a-rna biotin-d/a-protein

23 Einzelne Moleküle im Fokus Donor-Fluoreszenz I D gross Abstand klein 900 photons / ms Ereignisse Akzeptor-Fluoreszenz I A t (s) FRET-Effizienz FRET-Effizienz E FRET = I D IA + I A

24 Towards corrected FRET efficiencies Determination of correction parameters E FRET = ( 1+ βγδ) ( 1+ βγδ) I I + ( γ + γδ) ( I βi ) A I α + γδ 1+ βγδ A α + γδ I 1+ βγδ D D D A I D, I A : background-corrected donor and acceptor fluorescence intensities α: cross-talk donor into acceptor channel β: cross-talk acceptor into donor channel γ: sensitivity ratio for donor and acceptor fluorescence δ: direct excitation of the donor E FRET B. Theissen, A.R. Karow, J. Köhler, A. Gubaev, D. Klostermeier, PNAS 2008

25 Generation of FRET histograms smfret in solution smfret on surfaces donor 3 1 acceptor # of events E FRET

26 Dynamic information: FRET traces and dwell time histograms smfret in solution dwell time E FRET photons smfret on surfaces t (ms) donor acceptor # of events k conf dwell time (ms)

27 Outline 1. Fluorescence, FRET, and single molecules 1.1. Fluorescence 1.2. Conformational changes and FRET 1.3. Confocal microscopy 1.4. Total internal reflection microscopy 1.5. Information obtained in smfret experiments 2. Helicases: RNA unwinding 2.1. The RNA helicase YxiN 2.2. Donor-acceptor labeling 2.3. Single molecule FRET results 3. DNA topoisomerases

28 ie offene Konformation der DEAD Box Helikasen

29 SF2 helicase modules DEAD box helicases Reverse gyrase Chromatin remodelling enzymes Type I restriction enzymes

30 YxiN : Ribosomenbiogenese YxiN (3-367) YxiN ( ) Wang et al. (2006) RNA 12,

31 YxiN : preparation for smfret experiments Activity assays: Nucleotide binding RNA binding (RNA-stimulated) ATPase activity RNA unwinding 1. exchange intrinsic cysteines to other aa 2. verify wild-type like activity 3. introduce cysteines for labeling in desired positions 4. verify wild-type like activity 5. test reactivity of cysteines 6. optimize labeling 7. verify wild-type like activity of labeled protein

32 Von einzelnen Molekülen zum Mechanismus Liganden Mutationen FRET-Effizienz mapping Biochemie k obs Ereignisse Ereignisse FRET-Effizienz [RNA]

33 FRET histogram YxiN S108C S229C

34 FRET histograms: YxiN in the absence of ligands B. Theissen, A.R. Karow, J. Köhler, A. Gubaev, D. Klostermeier, PNAS 2008

35 YxiN: the open conformation B. Theissen, A.R. Karow, J. Köhler, A. Gubaev, D. Klostermeier, PNAS 2008

36 eschlossene Konformation mit RNA und ADPNP Ereignisse Ereignisse - Nukleotid ADPNP RNA 0.50 FRET-Effizienz FRET-Effizienz B. Theissen, A.R. Karow, J. Köhler, A. Gubaev, D. Klostermeier, PNAS 2008

37 YxiN: the closed the conformation B. Theissen, A.R. Karow, J. Köhler, A. Gubaev, D. Klostermeier, PNAS 2008

38 Direkte Beobachtung eines katalytischen Zyklus FRET-Effizienz Zeit (ms) B. Theissen, A.R. Karow, J. Köhler, A. Gubaev, D. Klostermeier, PNAS 2008

39 Open and closed conformations of YxiN mjdead Vasa B. Theissen, A.R. Karow, J. Köhler, A. Gubaev, D. Klostermeier, PNAS 2008

40 Konformationen im Nukleotidzyklus Zustand vor der Hydrolyse ADP BeF x ADP? Hydrolyse Zustand nach der Hydrolyse ADP MgF x P i Öffnen Freisetzung von RNA? R. Aregger & D. Klostermeier, Biochemistry 2009

41 Verschiedene Klassen von Mutanten ATPase (s -1 ) ss ds wt mut - RNA-stimulierte ATPase - RNA Entwindung RNA (nm) ss ds wt mut ATPase (s -1 ) RNA-stimulierte ATPase - RNA Entwindung Kooperativität RNA (nm) A.R. Karow & D. Klostermeier, NAR 2009

42 Verschiedene Klassen von Mutanten Ereignisse Ereignisse Ereignisse FRET-Effizienz +ADPNP +RNA FRET-Effizienz +ATP +RNA FRET-Effizienz - RNA-stimulierte ATPase - RNA Entwindung RNA-stimulierte ATPase - RNA Entwindung Kooperativität A.R. Karow & D. Klostermeier, NAR 2009

43 Konformation und RNA Entwindung Wildtyp ATPasedefizient unvollständiges Schliessen unwinding Konformation der RNA? - Zweiter Entwindungsschritt? - M. Hilbert, A.R. Karow & D. Klostermeier Biol. Chem A.R. Karow & D. Klostermeier, NAR 2009

44 Mechanismus der ATP-abhängigen RNA Entwindung durch DEAD Box Proteine ATPase-def. unvollständiges Schliessen ADPÿMgF x ADPÿAlF x ADPNP unvollständiges Schliessen ADPÿBeF x M. Hilbert, A.R. Karow & D. Klostermeier, Biological Chemistry 2009

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