© D. Goodsell

1. Single Molecule Spectroscopy of Protein Folding Dynamics Ben Schuler © D. Goodsell EMBO Practical Course, September 23-28, 2009

2. Deciding how to fold Pathways (the “old view”) • structurally defined folding intermediates • well-defined “path” to the native state • “chemical” picture for large, multi-domain proteins Landscapes (the “new view”) • stresses ensemble character  statistical mechanics, microscopic states • alternative routes to native state • elementary properties of the protein folding reaction • most suitable models: small two-state proteins from Dobson, 2000

3. Förster Resonance Energy Transfer (FRET) Unfolded state collapse unfolded folded Acceptor  Acceptor Donor Donor

4. History of FRET Since 1934

5. Confocal single molecule fluorescence detection

6. Probing protein folding with single molecule FRET Single molecule FRET: two-state folding and unfolded state collapse Distance distributions in the unfolded state Unfolded statedynamics Kramers-type descriptions of protein folding dynamics: Socci, Onuchic & Wolynes, J. Chem. Phys. 1996; Klimov & Thirumalai, Phys. Rev. Lett., 1997 Deniz et al., PNAS 2000 Schuler et al., Nature 2002 Lipman et al., Science 2003 Kuzmenkina et al., PNAS 2005 Laurence et al., PNAS 2005 Magg et al., JMB 2006 Tezuka et al., Biophys J 2006 Sherman & Haran, PNAS 2006 Möglich et al., PNAS 2006 Huang et al., PNAS 2007 Hoffmann et al., PNAS 2007 Merchant et al., PNAS 2007 Nettels et al., PNAS 2007Hofmann et al., JMB 2008 The holy grail: microscopic distribution of folding paths Figure adapted from Dinner et al., 2000

7. Distance distributions from FRET efficiencies where with lp: persistence lengthn: number of peptide bondsl: peptide segment length (3.8 Å) • but: direct information about P(r) lost because of ms-averaging

8. Distance distributions from fluorescence lifetimes D A subpopulation-specific fluorescence intensity decays

9. Distance distributions in the unfolded state of CspTm transfer efficiencyhistograms • collapse is largely uniform • close to random Gaussian chaineven when collapsed lifetime distributions Hoffmann, Kane, Nettels, Hertzog, Baumgärtel, Lengefeld, Reichardt, Seckler, Bakajin & Schuler (2007) Proc Natl Acad Sci USA 104, 105-110.

10. Probing protein folding with single molecule FRET Distance distributions in the unfolded state Unfolded statedynamics Kramers-type descriptions of protein folding dynamics: Socci, Onuchic & Wolynes, J. Chem. Phys. 1996; Klimov & Thirumalai, Phys. Rev. Lett., 1997 Figure adapted from Dinner et al., 2000

11. tDD = 43 ns 4 M GdmCl Dynamics from single molecule photon statistics photon bunching G(t) photon antibunching 0 t (ns) • chain dynamics are very rapid (~“Zimm time“) • fundamental property of completely unfolded proteins • dynamics slow down when chain collapses HanburyBrown &Twiss • physical model?

12. Combining distance distributions and dynamics Diffusive motion in a potential of mean force for a Gaussian chain Photophysics log r r only free parameter Nettels, Gopich, Hoffmann & Schuler (2007) Proc Natl Acad Sci USA 104, 2655-2660.

13. Unfolded state dynamics and collapse tDD (raw data) tDD = 43 ns (viscosity corrected) 4 M GdmCl • collapse slows down chain dynamics (inreasing internal friction/roughness) Nettels, Gopich, Hoffmann & Schuler (2007) Proc Natl Acad Sci USA 104, 2655-2660.

14. The free energy surface of unfolded Csp “speed limit”/ preexponential factor: ~1/0.4 µs collapsed Csp: ~20% -structure content of N (SRCD) diffusive unfolded state dynamics~50 ns = collapse time(Onsager!) roughness of the free energy surface increases upon collapse (~1.3 kT) unfolded Csp: Gaussian chain, even upon collapse (Zwanzig, 1988)

15. Temperature-induced unfolded-state collapse 1. GdmCl dissociation: Makhatadze& Privalov1992 2. Intrachain interactions: • unfolded chain collapses with increasing temperature both via dissociation of denaturant and by increasing intramolecular interactions Nettels et al., submitted

16. Rhodanese Folding and Aggregation native denatured 7M GdmCl refolded + 500 nM unlabeled rhodanese Hillger, F., Nettels, D., Dorsch, S., & Schuler, B. (2007) J. Fluoresc. 17, 759-765.

17. Rhodanese-chaperone interactions DA rapid chain dynamics DA D-only GroEL/rhodaneserotation no distance dynamics! Hillger, Hänni, Nettels, Geister, Grandin, Textor & Schuler (2008) Angew Chem Int Ed 47, 6184-88

18. Conclusions • Intramolecular distance distributions and dynamics from nanoseconds to seconds can be obtained from single molecule FRET • Unfolded state collapse of Csp results in slowed chain dynamics • Unfolded proteins compact with increasing temperature • Charge repulsion can dominate unfolded state dimensions in intrinsically disordered proteins • Single molecule FRET allows the investigation of protein aggregation and the influence of cellular factors on protein folding mechanisms

19. University of Zurich Institute of Biochemistry Daniel NettelsArmin Hoffmann Frank Hillger Hagen HofmannDominik Hänni Sonja Geister www.bioc.uzh.ch/schuler NIH Laboratory of Chemical Physics Irina Gopich Attila Szabo University ofPotsdamPhysical Biochemistry Klaus GastBen Heinz Frank Küster René Wuttke Luc Reymond Jennifer Clark Bengt Wunderlich Andrea Soranno UC Santa BarbaraDepartment of Physics Everett Lipman Shawn Pfeil University ofCambridgeDepartment of Chemistry Robert Best