Transcript Slide 1

Single Molecule Spectroscopy
of Protein Folding Dynamics
© D. Goodsell
Ben Schuler
EMBO Practical Course, September 23-28, 2009
Deciding how to fold
Pathways (the “old view”)
• structurally defined folding
intermediates
• well-defined “path” to the native
state
• “chemical” picture for large, multidomain 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 twostate proteins
from Dobson, 2000
Förster
Resonance
Energy Transfer (FRET)
Unfolded
state collapse
unfolded
folded
Acceptor

Acceptor
Donor
Donor
Transfer efficiency E
1.0
0.5
0.0
R0
Donor-acceptor distance r
History of FRET
Since 1934
Confocal single molecule fluorescence
detection
Probing protein folding with single molecule
FRET
Unfolded state
dynamics
Distance distributions
in the unfolded state
Single molecule FRET:
two-state folding and
unfolded state collapse
Kramers-type
descriptions of
protein folding
dynamics:
Socci, Onuchic &
Wolynes,
J. Chem. Phys. 1996;
Klimov & Thirumalai,
Phys. Rev. Lett., 1997
The holy grail:
microscopic distribution
of folding paths
Figure adapted from Dinner et al., 2000
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
Distance distributions from FRET efficiencies
where
with
lp: persistence length
n: number of peptide bonds
l: peptide segment length (3.8 Å)
 but: direct information about P(r) lost because of ms-averaging
Distance distributions from fluorescence
lifetimes
D
A
subpopulation-specific
fluorescence intensity decays
Distance distributions in the unfolded state of
CspTm
transfer efficiency
histograms
 collapse is largely uniform
lifetime
distributions
 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.
Probing protein folding with single molecule
FRET
Unfolded state
dynamics
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
Distance distributions
in the unfolded state
G(t)
Dynamics from single molecule photon
statistics
photon
bunching
4 M GdmCl
tDD = 43 ns
photon
antibunching
0
t (ns)




chain dynamics are very rapid (~“Zimm time“)
Hanbury
fundamental property of completely unfolded proteins Brown &
Twiss
dynamics slow down when chain collapses
physical model?
Combining distance distributions and dynamics
Diffusive motion in a potential
of mean force G(r )   kT ln( peq (r ))
for a Gaussian chain
Photophysics
peq (r )
G (r )
log
r
r

p(r , t )  

1
  D peq (r )
I  K 0 ( r )  p( r , t )
t
r peq (r )
 r

only free parameter
 pDA (r , t ) 
 p (r , t ) 

p(r , t )   D*A
 pDA* (r , t ) 


p
(
r
,
t
)
 D*A*

Nettels, Gopich, Hoffmann & Schuler (2007) Proc Natl Acad Sci USA 104, 2655-2660.
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.
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!)
unfolded Csp:
Gaussian chain,
even upon collapse
roughness of the
free energy surface
increases upon
collapse (~1.3 kT)
(Zwanzig, 1988)
Temperature-induced unfolded-state collapse
1. GdmCl dissociation:
Makhatadze
& Privalov
1992
2. Intrachain interactions:
 unfolded chain collapses with increasing
temperature both via dissociation of denaturant
and by increasing intramolecular interactions
Nettels et al., submitted
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.
Rhodanese-chaperone interactions
DA
DA
rapid
chain
dynamics
D-only
GroEL/
rhodanese
rotation
no
distance
dynamics!
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
Zurich
Institute of Biochemistry
Frank
Küster
Daniel Nettels René Wuttke
Armin HoffmannLuc Reymond
Frank Hillger Jennifer Clark
Hagen HofmannBengt
Dominik Hänni Wunderlich
Sonja Geister Andrea
Soranno
www.bioc.uzh.ch/schuler
NIH
Laboratory of Chemical
Physics
University of
Potsdam
Physical Biochemistry
Irina Gopich
Attila Szabo
UC Santa
Barbara
Klaus Gast
Ben Heinz
University of
Cambridge
Department of Physics
Department of Chemistry
Everett Lipman
Shawn Pfeil
Robert Best