Transcript HHMI meeting, FOLDING

University of Orange Free State
Bloemfontain, South Africa
September 5, 2007
Protein folding. A theoretical view
Alexei Finkelstein
Institute of Protein Research,
Russian Academy of Sciences,
Pushchino, Moscow Region, Russia
U
N
TWO protein folding problems:
1) How does protein structure fold? √
2) How to predict protein structure
from the chain’s a. a. sequence?
BASIC FACTS:
Protein chains has unique sequence
& unique 3D structure
Protein chain can fold
spontaneously
(RNase, Anfinsen, 1961;
RNase, Merrifield, 1969)
Folding time:
in vivo: Biosynthesis +Folding < 10–20 min
in vitro: from microseconds to hours
BASIC FACTS:
Protein chains has unique sequence
& unique 3D structure
Protein chain can fold
spontaneously
(RNase, Anfinsen, 1961;
RNase, Merrifield, 1969)
Folding time:
in vivo: Biosynthesis +Folding < 10–20 min
in vitro: from microseconds to hours
For:
Water-soluble
single-domain proteins;
or separate domains
How CAN protein fold in a “bio-reasonable” time?
Levinthal paradox (1968):
Native protein structure
refolds from various starts,
i.e., it behaves as
thermodynamically stable.
U
N
RANDOM
Random exhaustive enumeration
Special pathway?
Folding intermediates?
HOW CAN it be found within seconds - among
zillions of the others?
Is “Levinthal paradox” a paradox at all?
Is “Levinthal paradox” a paradox at all?
L-dimensional
“Golf course”
Is “Levinthal paradox” a paradox at all?
L-dimensional
“Golf course”
…any tilt of energy surface solves this “paradox”… (?)
L-dimensional
“Funnel”
Zwanzig, 1992;
Bicout & Szabo, 2000
L-dimensional “folding funnel”?
Cunning simplicity of a “funnel”
(without phase separation) folding
U
E
L-
Resistance of entropy at T>0
E
N
All-or-none transition
for 1-domain proteins
(in thermodynamics: Privalov,1974;
in kinetics: Segava, Sugihara,1984)
- NO simultaneous explanation to
(I) “all-or-none” transition
(II) folding within non-astron. time
at mid-transition
ST
~L
Funnel helps, but ONLY when
N is much more stable than U !!
A special pathway?
Phillips (1965) hypothesis:
folding nucleus is formed by the N-end of the nascent protein
chain, and the remaining chain wraps around it.
for single-domain proteins: NO:
Goldenberg & Creighton, 1983:
circular permutants:
N-end has no special role in the in vitro folding.
A special pathway?
Phillips (1965) hypothesis:
folding nucleus is formed by the N-end of the nascent protein
chain, and the remaining chain wraps around it.
for single-domain proteins: NO:
Goldenberg & Creighton, 1983:
circular permutants:
N-end has no special role in the in vitro folding.
However, for many-domain proteins:
Folding from N-end domain,  domain after domain
DO NOT CONFUSE N-END DRIVEN FOLDING WITHIN DOMAIN
(which seems to be absent)
and
N-DOMAIN DRIVEN FOLDING IN MANY-DOMAIN PROTEIN
(which is observed indeed)
Folding intermediates?
HYPOTHESIS:
Stages in the mechanism of self-organization of protein molecules
O.B.Ptitsyn, Dokl. Akad. Nauk SSSR. 1973; 210:1213-1215.
NOW:
pre-molten
globule
NOW:
MOLTEN
GLOBULE
e
PROTEIN
FOLDING:
current picture
(Dobson, 2003)
TRUE: FOLDING
Hierarchic (stepwise) folding
avoids many “bad” pathways
with observable
(accumulating in experiment)
U
pre-MG
intermediates
MG
= MG
N
INDEED,
NO exhaustive enumeration
when N is much more stable then U
U
N
TRUE: FOLDING
Hierarchic (stepwise) folding
avoids many “bad” pathways
with observable
(accumulating in experiment)
U
pre-MG
intermediates
MG
= MG
N
INDEED,
NO exhaustive enumeration
when N is much more stable then U
U
Special pathway N
Folding intermediates they help, but ONLY when N is much more stable than U !!
Cunning simplicity of
BUT ALSO: FOLDING
hierarchic folding
WITHOUT ANY observable
intermediates  as applied to resolve
the Levinthal paradox
U
N
All-or-none transition
for 1-domain proteins
(in thermodynamics: Privalov,1974;
in kinetics: Segava, Sugihara,1984)
U
N
NO hierarchic folding –
NO “special pathways”,
NO explanation of
non-astron. folding time at
“all-or-none” transition,
especially close to mid-transition
How CAN protein fold in a “bio-reasonable” time?
Levinthal paradox (1968):
Native protein structure
refolds from various starts,
i.e., it behaves as if
thermodynamically stable.
U
N
RANDOM
HOW can it be found within seconds - among
zillions of the others?
SEARCH TIME AT
Special pathway?
MID-TRANSITION= ???
Folding intermediates?
“Funnel”?
Can help…, but ONLY when N is much more stable then U …
Kinetics vs. stability:
Native protein structure:
That, which folds most rapidly?
That, which is the most stable?
Practical questions:
What to predict?
What to design?
Kinetics vs. stability:
Native protein structure:
That, which folds most rapidly?
That, which is the most stable?
Practical questions:
What to predict?
What to design?
(railway? airport?)
Kinetics vs. stability:
Native protein structure:
That, which folds most rapidly?
That, which is the most stable? √
Practical questions:
What to predict?
What to design?
However:
Is there a contradiction between the “foldable”
structure and the “most stable” structure?!
NO!
Computer experiments (Shakhnovich et al, 1993-96);
general theory (Finkelstein et al., 1995-97) √
Nucleation: Folding with phase separation
L
1
folding interm.
Nucleation occurs at the
“all-or-none” transition
(N and U states are observed only):
U
N
Nucleation results from the “energy gap”
Energy landscape
gap
The “energy gap” is: - necessary for unique protein structure
- necessary for fool-proof protein action
- necessary for direct UN transition
- necessary for fast folding
Nucleation: Folding with phase separation
L
1
folding interm.
Nucleation: Folding with phase separation
“Detailed Balance”: at given conditions, folding pathway = unfolding pathway
L
1
folding interm. = unfolding interm.
Nucleation: Folding with phase separation
“Detailed Balance”: at given conditions, folding pathway = unfolding pathway
L
1
folding interm. = unfolding interm.
folding pathway = unfolding pathway at mid-transition  TtrS = H
folding pathway  unfolding pathway close to mid-transition  TS  90%H
“close to”  T  90%Ttr
indeed:  T  300oK,
 Ttr  330oK
Nucleation: Folding with phase separation
“Detailed Balance”: at given conditions, folding pathway = unfolding pathway
L
1
folding interm. = unfolding interm.
 F # ~ L2/3  surface tension
a) micro-; b) loops
[from melting] [from Flory]
Ln(kf ) ~ F #/RT ~ (1/2  3/2) L2/3
↓
↓
loops
At mid-transition
intermediates
do not matter…
Corr. = 0.7
Any stable fold is automatically a focus of rapid folding
pathways. No “special pathway” is needed.
ΔFN ↓
U
N
↓
↓
↓
ΔFN ↓
↓
When globules (N & M) become more stable than U:
a
ΔFN ↓

GAP 

b
1) Acceleration:
lnkf  -1/2FN/RT
2) Large gap  large
acceleration before
“rollover” caused by
intermediates M
at “bio-conditions”
b
↓
↓
↓
ΔFN ↓
↓

GAP 

a
α-helices decrease
effective chain length.
THIS HELPS TO FOLD!
In water
α-HELICES
ARE
PREDICTED
FROM THE
AMINO ACID
SEQUENCE
Corr. = 0.84
Ivankov D.N., Finkelstein A.V. (2004) Prediction of protein folding rates from the amino-acid
sequence-predicted secondary structure. - Proc. Natl. Acad. Sci. USA, 101:8942-8944.
2) One still cannot predict protein structure from the a. a.
sequence without homologues… WHY??
choice of one structure out of two
DOES NOT require too precise estimate of interactions
GAP
choice of one structure out of zillions
REQUIRES very precise estimate of interactions
GAP
University of Orange Free State
Bloemfontain, South Africa
September 4, 2007
Protein folding. A theoretical view
Alexei Finkelstein
Institute of Protein Research,
Russian Academy of Sciences,
Pushchino, Moscow Region, Russia
Gratitude to: D.A. Dolgikh, R.I. Gilmanshin, A.E. Dyuysekina, V.N. Uversky,
E.N. Baryshnikova, B.S. Melnik, V.A. Balobanov, N.S. Katina,
N.A. Rodionova, R.F. Latypov, O.I Razgulyaev, E.I. Shakhnovich,
A.M. Gutin, A.Ya. Badretdinov, O.V. Galzitskaya, S.O. Garbuzynskiy,
D.N.Ivankov, N.S. Bogatyreva, V.E. Bychkova, G.V. Semisotnov
The Russian Acad. Sci. Program “Mol. & Cell Biology”,
The Russian Foundation for Basic Research,
ISSEP, HFSPO, CRDF, INTAS,
The Howard Hughes Medical Institute
Consider thermodynamic
mid-transition U ↔ N.
M: all unstable
L
#
Detailed Balance:
at given conditions,
folding pathway = unfolding pathway
unstable
semi-folded
U: stable
N: stable
Consider sequential folding (with phase separation)
? HOW FAST the most stable state is achieved ?
ESTIMATE free energy barrier F
Experiment:
Rearrangement of 1 residue takes 1-10 ns
#

1
ns
F # ~ L2/3
HOW FAST the most stable state isL achieved?
free energy barrier 
  F # ~ L2/3  surface tension
F (U)
=
F (N)
a) micro-;
b) loops
compact folded nucleus: ~1/2 of the chain
micro:
F #  L2/3 [/4];  2RT0 [experiment]
loops:
F # ≤ L2/31/2[3/2RTln(L1/3)]e-N/(100)
[Flory]
[knots]
F # ~ (1/2  3/2) L2/3
micro
loops
Any stable fold is automatically a focus of rapid folding
pathways. No “special pathway” is needed.
1
ns