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

Landscapes (the “new view”) Pathways (the “old view”) structurally defined folding intermediates well-defined “path” to the native state “chemical” picture for large, multi- domain proteins 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 Deciding how to fold

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

Since 1934 History of FRET

Confocal single molecule fluorescence detection

Probing protein folding with single molecule FRET Figure adapted from Dinner et al., 2000 Distance distributions in the unfolded state Single molecule FRET: two-state folding and unfolded state collapse 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 2007 Hofmann et al., JMB 2008 Socci, Onuchic & Wolynes, J. Chem. Phys. 1996; Klimov & Thirumalai, Phys. Rev. Lett., 1997 Unfolded state dynamics Kramers-type descriptions of protein folding dynamics: The holy grail: microscopic distribution of folding paths

where withl p : persistence length n: number of peptide bonds l: peptide segment length (3.8 Å) Distance distributions from FRET efficiencies  but: direct information about P(r) lost because of ms-averaging

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

Distance distributions in the unfolded state of CspTm  collapse is largely uniform  close to random Gaussian chain even when collapsed Hoffmann, Kane, Nettels, Hertzog, Baumgärtel, Lengefeld, Reichardt, Seckler, Bakajin & Schuler (2007) Proc Natl Acad Sci USA 104, transfer efficiency histograms lifetime distributions

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

0  (ns) Hanbury Brown & Twiss photon bunching photon antibunching G()G()  DD = 43 ns 4 M GdmCl Dynamics from single molecule photon statistics  physical model?  chain dynamics are very rapid (~“Zimm time“)  fundamental property of completely unfolded proteins  dynamics slow down when chain collapses

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

 DD = 43 ns 4 M GdmCl  DD (raw data) (viscosity corrected)  collapse slows down chain dynamics (inreasing internal friction/roughness) Unfolded state dynamics and collapse Nettels, Gopich, Hoffmann & Schuler (2007) Proc Natl Acad Sci USA 104,

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

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

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

Rhodanese-chaperone interactions rapid chain dynamics GroEL/ rhodanese rotation no distance dynamics! DADAD-only DADA Hillger, Hänni, Nettels, Geister, Grandin, Textor & Schuler (2008) Angew Chem Int Ed 47,

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

University of Zurich Institute of Biochemistry Daniel Nettels Armin Hoffmann Frank Hillger Hagen Hofmann Dominik Hänni Sonja Geister NIH Laboratory of Chemical Physics Irina Gopich Attila Szabo UC Santa Barbara Department of Physics Everett Lipman Shawn Pfeil Frank Küster René Wuttke Luc Reymond Jennifer Clark Bengt Wunderlich Andrea Soranno University of Cambridge Department of Chemistry Robert Best University of Potsdam Physical Biochemistry Klaus Gast Ben Heinz