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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

Donor-acceptor distance r

Tra

nsfe

r ef

ficie

ncy

E

0.0

0.5

1.0

R0

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

1

2B

G

k Tf

r

k e

Deniz et al., PNAS 2000Schuler et al., Nature 2002Lipman et al., Science 2003Kuzmenkina et al., PNAS 2005Laurence et al., PNAS 2005Magg et al., JMB 2006Tezuka et al., Biophys J 2006Sherman & Haran, PNAS 2006Möglich et al., PNAS 2006Huang et al., PNAS 2007Hoffmann et al., PNAS 2007Merchant et al., PNAS 2007Nettels et al., PNAS 2007Hofmann et al., JMB 2008

Socci, Onuchic & Wolynes, J. Chem. Phys. 1996;Klimov & Thirumalai, Phys. Rev. Lett., 1997

Unfolded statedynamics

Kramers-type descriptions of protein folding dynamics:

The holy grail: microscopic distribution of folding paths

where

with lp: persistence lengthn: number of peptide bondsl: 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, 105-110.

transfer efficiencyhistograms

lifetime distributions

Probing protein folding with single molecule FRET

Figure adapted from Dinner et al., 2000

Distance distributions in the unfolded state

1

2B

G

k Tf

r

k e

Socci, Onuchic & Wolynes, J. Chem. Phys. 1996;Klimov & Thirumalai, Phys. Rev. Lett., 1997

Unfolded statedynamics

Kramers-type descriptions of protein folding dynamics:

0 (ns)

HanburyBrown &Twiss

photon bunching

photon antibunching

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

( ) ln( ( ))eqG r kT p r

( )eqp r ( )G r

( , ) 1( ) ( ) ( , )

( )eqeq

r tD p r r r t

t r r p r

0

pI K p *

*

* *

( , )

( , )

( , )

( , )

( , )

DA

D A

DA

D A

p r t

p r t

p r t

p r t

r t

p

Diffusive motion in a potentialof mean forcefor 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, 2655-2660.

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, 2655-2660.

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& Privalov1992

1. GdmCl dissociation:

2. Intrachain interactions:

Nettels et al., submitted

Rhodanese Folding and Aggregation

native

denatured7M GdmCl

refolded + 500 nMunlabeledrhodanese

Hillger, F., Nettels, D., Dorsch, S., & Schuler, B. (2007) J. Fluoresc. 17, 759-765.

Rhodanese-chaperone interactions

rapid chain dynamics

GroEL/rhodaneserotation

no distance dynamics!

DA D-only

DA

Hillger, Hänni, Nettels, Geister, Grandin, Textor & Schuler (2008) Angew Chem Int Ed 47, 6184-88

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 ZurichInstitute 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

UC Santa Barbara Department of Physics

Everett LipmanShawn Pfeil

Frank Küster René WuttkeLuc ReymondJennifer ClarkBengt WunderlichAndrea Soranno

University of Cambridge Department of Chemistry

Robert Best

University of Potsdam Physical Biochemistry

Klaus GastBen Heinz