ANRV343-BB37-14 ARI 9 April 2008 11:34

The Protein Folding

Problem
Ken A. Dill,
1,2
S. Banu Ozkan,
3
M. Scott Shell,
4
and Thomas R. Weikl
5
1
Department of Pharmaceutical Chemistry,
2
Graduate Group in Biophysics,
University of California, San Francisco, California 94143;
email: [email protected]
3
Department of Physics, Arizona State University, Tempe, Arizona 85287;
email: [email protected]
4
Department of Chemical Engineering, University of California, Santa Barbara,
California 93106; email: [email protected]
5
Max Planck Institute of Colloids and Interfaces, Department of Theory and
Bio-Systems, 14424 Potsdam, Germany; email: [email protected]
Annu. Rev. Biophys. 2008. 37:289316
The Annual Review of Biophysics is online at
biophys.annualreviews.org
This articles doi:
10.1146/annurev.biophys.37.092707.153558
Copyright c 2008 by Annual Reviews.
All rights reserved
1936-122X/08/0609-0289$20.00
Key Words
structure prediction, funnel energy landscapes, CASP, folding
code, folding kinetics
Abstract
The protein folding problem consists of three closely related puz-
zles: (a) What is the folding code? (b) What is the foldingmechanism?
(c) Can we predict the native structure of a protein from its amino
acid sequence? Once regarded as a grand challenge, protein fold-
ing has seen great progress in recent years. Now, foldable proteins
and nonbiological polymers are being designed routinely and mov-
ing toward successful applications. The structures of small proteins
are now often well predicted by computer methods. And, there is
now a testable explanation for how a protein can fold so quickly: A
protein solves its large global optimization problem as a series of
smaller local optimization problems, growing and assembling the
native structure from peptide fragments, local structures rst.
289
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Contents
INTRODUCTION. . . . . . . . . . . . . . . . . 290
THE FOLDING CODE: WHAT
BALANCE OF FORCES
ENCODES NATIVE
STRUCTURES? . . . . . . . . . . . . . . . . 290
Annsens Thermodynamic
Hypothesis . . . . . . . . . . . . . . . . . . . . 290
One Dominant Driving Force
or Many Small Ones? . . . . . . . . . . 291
Designing New Proteins and
Nonbiological Foldamers . . . . . . 292
COMPUTATIONAL PROTEIN
STRUCTURE PREDICTION
IS INCREASINGLY
SUCCESSFUL . . . . . . . . . . . . . . . . . . 293
CASP: A Community-Wide
Experiment . . . . . . . . . . . . . . . . . . . 293
ARE THERE MECHANISMS OF
PROTEIN FOLDING?. . . . . . . . . . 294
There Have Been Big Advances in
Experimental and Theoretical
Methods . . . . . . . . . . . . . . . . . . . . . . 294
The PSB Plot: Folding Speed
Correlates with the Localness
of Contacts in the Native
Structure. . . . . . . . . . . . . . . . . . . . . . 295
Proteins Fold on Funnel-Shaped
Energy Landscapes . . . . . . . . . . . . 296
The Zipping and Assembly
Hypothesis for the Folding
Routes . . . . . . . . . . . . . . . . . . . . . . . . 300
PHYSICS-BASED MODELING OF
FOLDING AND STRUCTURE
PREDICTION . . . . . . . . . . . . . . . . . . 301
SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . 303
INTRODUCTION
The proteinfoldingproblemis the questionof
how a proteins amino acid sequence dictates
its three-dimensional atomic structure. The
notion of a folding problem rst emerged
around 1960, with the appearance of the rst
atomic-resolution protein structures. Some
formof internal crystalline regularity was pre-
viously expected(117), and-helices hadbeen
anticipated by Linus Pauling and colleagues
(180, 181), but the rst protein structuresof
the globinshad helices that were packed
together in unexpected irregular ways. Since
then, the protein folding problem has come
to be regarded as three different problems:
(a) the folding code: the thermodynamic
question of what balance of interatomic
forces dictates the structure of the protein,
for a given amino acid sequence; (b) protein
structure prediction: the computational
problem of how to predict a proteins native
structure from its amino acid sequence; and
(c) the folding process: the kinetics question
of what routes or pathways some proteins
use to fold so quickly. We focus here only
on soluble proteins and not on brous or
membrane proteins.
THE FOLDING CODE: WHAT
BALANCE OF FORCES ENCODES
NATIVE STRUCTURES?
Annsens Thermodynamic
Hypothesis
A major milestone in protein science was
the thermodynamic hypothesis of Christian
Annsen and colleagues (3, 92). From his
now-famous experiments on ribonuclease,
Annsen postulated that the native structure
of a protein is the thermodynamically stable
structure; it depends only on the amino acid
sequence and on the conditions of solution,
and not on the kinetic folding route. It be-
came widely appreciated that the native struc-
ture does not depend on whether the protein
was synthesized biologically on a ribosome or
with the help of chaperone molecules, or if,
instead, the protein was simply refolded as
an isolated molecule in a test tube. [There
are rare exceptions, however, such as insulin,
-lytic protease (203), and the serpins (227),
in which the biologically active form is ki-
netically trapped.] Two powerful conclusions
followed from Annsens work. First, it en-
abled the large research enterprise of in vitro
290 Dill et al.
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ANRV343-BB37-14 ARI 9 April 2008 11:34
protein folding that has come to understand
native structures by experiments inside test
tubes rather than inside cells. Second, the An-
nsen principle implies a sort of division of
labor: Evolution can act to change an amino
acidsequence, but the folding equilibriumand
kinetics of a given sequence are then matters
of physical chemistry.
One Dominant Driving Force
or Many Small Ones?
Prior to the mid-1980s, the protein folding
code was seen a sum of many different small
interactionssuch as hydrogen bonds, ion
pairs, van der Waals attractions, and water-
mediated hydrophobic interactions. A key
idea was that the primary sequence encoded
secondary structures, which then encoded
tertiary structures (4). However, through
statistical mechanical modeling, a different
viewemerged in the 1980s, namely, that there
is a dominant component to the folding code,
that it is the hydrophobic interaction, that the
folding code is distributed both locally and
nonlocally inthe sequence, andthat a proteins
secondary structure is as much a consequence
of the tertiary structure as a cause of it
(48, 49).
Because native proteins are only 5
10 kcal/mol more stable than their denatured
states, it is clear that no type of intermolec-
ular force can be neglected in folding and
structure prediction (238). Although it re-
mains challenging to separate in a clean and
rigorous way some types of interactions from
others, here are some of the main observa-
tions. Folding is not likely to be dominated by
electrostatic interactions among charged side
chains because most proteins have relatively
fewcharged residues; they are concentrated in
high-dielectric regions on the protein surface.
Protein stabilities tend to be independent of
pH (near neutral) and salt concentration, and
charge mutations typically leadtosmall effects
on structure and stability. Hydrogen-bonding
interactions are important, because essentially
all possible hydrogen-bonding interactions
are generally satisedinnative structures. Hy-
drogen bonds among backbone amide and
carbonyl groups are key components of all
secondary structures, and studies of mutations
in different solvents estimate their strengths
to be around 14 kcal/mol (21, 72) or stronger
(5, 46). Similarly, tight packing in proteins im-
plies that van der Waals interactions are im-
portant (28).
However, the question of the folding code
is whether there is a dominant factor that
explains why any two proteins, for example,
lysozyme and ribonuclease, have different na-
tive structures. This code must be written in
the side chains, not in the backbone hydrogen
bonding, because it is through the side chains
that one protein differs from another. There
is considerable evidence that hydrophobic in-
teractions must play a major role in protein
folding. (a) Proteins have hydrophobic cores,
implying nonpolar amino acids are driven to
be sequestered from water. (b) Model com-
pound studies show 12 kcal/mol for trans-
ferring a hydrophobic side chain from wa-
ter into oil-like media (234), and there are
many of them. (c) Proteins are readily de-
natured in nonpolar solvents. (d ) Sequences
that are jumbled and retain only their cor-
rect hydrophobic and polar patterning fold to
their expected native states (39, 98, 112, 118),
in the absence of efforts to design packing,
charges, or hydrogen bonding. Hydrophobic
and polar patterning also appears to be a key
to encoding of amyloid-like bril structures
(236).
What stabilizes secondary structures? Be-
fore any protein structure was known, Linus
Pauling and colleagues (180, 181) inferred
from hydrogen-bonding models that proteins
might have -helices. However, secondary
structures are seldom stable on their own in
solution. Although different amino acids have
different energetic propensities to be in sec-
ondary structures (6, 41, 55, 100), there are
also many chameleon sequences in natural
proteins, which are peptide segments that can
assume either helical or conformations de-
pending on their tertiary context (158, 162).
www.annualreviews.org The Protein Folding Problem 291
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2

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(
[

]
2
2
2
)
15
a b c
0 5 10
0
15
5
10
(radius)
3
Native fold
Partially folded
structures
Figure 1
(a) Binary code. Experiments show that a primarily binary hydrophobic-polar code is sufcient to fold
helix-bundle proteins (112). Reprinted from Reference 112 with permission from AAAS.
(b) Compactness stabilizes secondary structure, in proteins, from lattice models. (c) Experiments
supporting panel b, showing that compactness correlates with secondary structure content in nonnative
states of many different proteins (218). Reprinted from Reference 218 with permission.
Three-helix bundl e
[Alcohol]
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1.0
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Q
FP
H
O R
N
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2
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H
R
O
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NH
2
n
a
b
Peptide
Peptoid
c
Methanol
Propanol
Ethanol
Designed molecule
Experimentally determined
structure
Figure 2
(a) A novel protein fold, called Top7, designed by Kuhlman et al. (129).
Designed molecule (blue) and the experimental structure determined
subsequently (red ). From Reference 129; reprinted with permission from
AAAS. (b) Three-helix bundle foldamers have been made using
nonbiological backbones (peptoids, i.e., N-substituted glycines).
(c) Their denaturation by alcohols indicates they have hydrophobic cores
characteristic of a folded molecule (134).
Studies of lattice models (25, 29, 51) and
tube models (11, 12, 159) have shown that
secondary structures in proteins are substan-
tially stabilized by the chain compactness,
an indirect consequence of the hydrophobic
force to collapse (Figure 1). Like airport se-
curity lines, helical and sheet congurations
are the only regular ways to pack a linear chain
(of people or monomers) into a tight space.
Designing New Proteins
and Nonbiological Foldamers
Although our knowledge of the forces of fold-
ing remains incomplete, it has not hindered
the emergence of successful practical pro-
tein design. Novel proteins are now designed
as variants of existing proteins (43, 94, 99,
145, 173, 243), or from broadened alphabets
of nonnatural amino acids (226), or de novo
(129) (Figure 2). Moreover, folding codes are
used to design new polymeric materials called
foldamers (76, 86, 120). Folded helix bundles
have now been designed using nonbiological
backbones (134). Foldamers are nding appli-
cations in biomedicine as antimicrobials (179,
185), lung surfactant replacements (235),
292 Dill et al.
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ANRV343-BB37-14 ARI 9 April 2008 11:34
cytomegalovirus inhibitors (62), and siRNA
delivery agents (217). Hence, questions of
deep principle are no longer bottlenecks to
designing foldable polymers for practical ap-
plications and new materials.
COMPUTATIONAL PROTEIN
STRUCTURE PREDICTION IS
INCREASINGLY SUCCESSFUL
A major goal of computational biology has
been to predict a proteins three-dimensional
native structure fromits amino acid sequence.
This could help to (a) accelerate drug dis-
covery by replacing slow, expensive struc-
tural biology experiments with faster, cheaper
computer simulations, and (b) annotate pro-
tein function from genome sequences (9).
With the rapid growth of experimentally de-
termined structures available in the Protein
Databank (PDB), protein structure predic-
tion has become as much a problem of infer-
ence and machine learning as it is of protein
physics.
Among the earliest uses of protein
databases to infer protein structures were
secondary structure prediction algorithms
(33, 34, 190). In the mid-1980s, several groups
began using the methods of computational
physicsatomic force elds plus Monte Carlo
samplingto compute the structures of the
Met-enkephalin, a ve-residue peptide (95,
141). The early 1990s saw signicant strides
in using databases and homology detection
algorithms to assemble structures from ho-
mologous sequences (192) and to recognize
folds by threading unknown sequences onto
three-dimensional structures from a database
(111). A key advance was the exploitation of
evolutionary relationships among sequences
through the development of robust sequence
alignment methods (32, 64, 224).
CASP: A Community-Wide
Experiment
In 1994, John Moult invented CASP (Critical
Assessment of Techniques for Protein Struc-
ture Prediction) (165), a biennial, community-
wide blind test to predict the unknown struc-
tures of proteins. Organizers identify pro-
teins likely to be solved or whose structures
have not yet been released, and predictors
have roughly 35 weeks to predict their native
structures. CASP has grown from 35 predic-
tor groups and 24 target sequences in CASP1
in 1994 to over 200 groups and 100+ targets
in CASP7 in 2006.
Over the seven CASPs, two trends are
clear (164, 219). First, although much re-
mains to be done, there has been substantial
improvement in protein structure prediction.
Web servers and software packages often
predict the native structure of small, single-
domain proteins to within about 26

A of
their experimental structures (8, 17, 242). In
addition, fast-homology methods are com-
puting approximate folds for whole genomes
(182, 214). Figure 3 shows a quantitative
assessment of performance at the rst ve
CASP meetings. The most signicant gains
have occurred in the alignments of targets
to homologs, the detection of evolutionarily
distant homologs, and the generation of
reasonable models for difcult targets that do
CASP5
Target difficulty
Easy Difficult
P
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(
%
)
100
0
CASP4
CASP3
CASP2
CASP1
Figure 3
Progress in protein structure prediction in CASP15 (219). The y-axis
contains the GDT TS score, the percentage of model residues that can be
superimposed on the true native structure, averaged over four resolutions
from 1 to 8

A (100% is perfect). The x-axis is the ranked target difculty,
measured by sequence and structural similarities to proteins in the PDB at
the time of the respective CASP. This shows that protein structure
prediction on easy targets is quite good and is improving for targets of
intermediate difculty. Reprinted from Reference 219 with permission.
www.annualreviews.org The Protein Folding Problem 293
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ANRV343-BB37-14 ARI 9 April 2008 11:34
not have templates (new folds). Since CASP5,
predictions have also beneted from the use
of metaservers, which solicit and establish
consensus among predictions from multiple
algorithms. Second, while most methods
rely on both physics and bioinformatics,
the most successful methods currently draw
heavily from knowledge contained in native
structural databases. Bioinformatics methods
have beneted from the growth in size of the
PDB (9, 219).
The following challenges remain (8, 164,
219): (a) to rene homology models beyond
those of the best template structures; (b) to
reduce errors to routinely better than 3

A,
particularly for proteins that are large, have
signicant content, are new folds, or have
low homology; (c) to handle large multido-
main or domain-swapped proteins; (d ) to ad-
dress membrane proteins; and (e) to predict
protein-protein interactions. Structural ge-
nomics is likely to help here (87, 222). In
any case, the current successes in computer-
based predictions of native protein struc-
tures are far beyond what was expected thirty
years ago, when structure prediction seemed
impossible.
ARE THERE MECHANISMS
OF PROTEIN FOLDING?
In 1968, Cyrus Levinthal rst noted the puz-
zle that even though they have vast confor-
mational spaces, proteins can search and con-
verge quickly to native states, sometimes in
microseconds. How do proteins nd their na-
tive states so quickly? It was postulated that
if we understood the physical mechanism of
protein folding, it could lead to fast computer
algorithms to predict native structures from
their amino acid sequences. In its description
of the 125 most important unsolved problems
in science, Science magazine framed the prob-
lem this way: Can we predict how proteins
will fold? Out of a near innitude of possible
ways to fold, a protein picks one in just tens of
microseconds. The same task takes 30 years
of computer time (1).
The following questions of principle have
driven the eld: How can all the denatured
molecules in a beaker nd the same na-
tive structure, starting from different con-
formations? What conformations are not
searched? Is folding hierarchical (10, 119)?
Which comes rst: secondary or tertiary
structure (80, 239)? Does the protein col-
lapse to compact structures before struc-
ture formation, or before the rate-limiting
step (RLS), or are they concurrent (7, 89,
101, 195, 205, 213)? Are there folding nuclei
(58, 152)?
Several models have emerged. In the
diffusion-collision model, microdomain
structures form rst and then diffuse and
collide to form larger structures (115, 116).
The nucleation-condensation mechanism
(70) proposes that a diffuse transition state en-
semble (TSE) with some secondary structure
nucleates tertiary contacts. Some proteins,
such as helical bundles, appear to follow a
hierarchical diffusion-collision model (155,
169) in which secondary structure forms and
assembles in a hierarchical order. In hierar-
chic condensation (139), the chain searches
for compact, contiguous structured units,
which are then assembled into the folded
state. Or, proteins may fold via the stepwise
assembly of structural subunits called foldons
(22, 126), or they may search for topomers,
which are largely unfolded states that have
native-like topologies (45, 150). These
models are not mutually exclusive.
There Have Been Big
Advances in Experimental
and Theoretical Methods
The search for folding mechanisms has driven
major advances in experimental protein sci-
ence. These include fast laser temperature-
jump methods (22); mutational methods
that give quantities called -values (71, 84,
106) [now also used for ion-channel kinet-
ics and other rate processes (42)] or -
values (204), which can identify those residues
most important for folding speed; hydrogen
294 Dill et al.
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ANRV343-BB37-14 ARI 9 April 2008 11:34
exchange methods that give monomer-level
information about folding events (125, 149);
andthe extensive explorationof proteinmodel
systems, including cytochrome c, CI2, bar-
nase, apomyoglobin, the src, -spectrin, and
fyn SH3 domains, proteins Land G, WWdo-
mains, trpzip, and trp cage (154). In addition,
peptide model experimental test systems pro-
vide insights into the fast early-folding events
(14, 109, 124). Furthermore, single-molecule
methods are beginning to explore the con-
formational heterogeneity of folding (23, 133,
166, 194).
There have been corresponding advances
in theory and computation. Computer-based
molecular minimization methods were rst
applied to protein structures in the 1960s
(18, 79, 171), followed by molecular dynam-
ics (140, 155), improved force elds (40) (re-
viewed in Reference 114), weighted sampling
and multi-temperature methods (130, 210),
highly parallelized codes for supercomputers
(2, 57), and distributed grid computing meth-
ods such as Folding@home (198, 241). Mod-
els having less atomic detail also emerged to
address questions more global and less de-
tailed in nature about protein conformational
spaces: (a) The Go model (82), which was in-
tended to see if a computer could nd the
native structure if native guiding constraints
were imposed, is now widely used to study
folding kinetics of proteins having known na-
tive structures (37, 38, 113, 197, 225). (b) More
physical models, typically based on polymer-
like lattices, are used to study the static and
dynamic properties of conformational spaces
(19, 50). (c) Master-equation approaches can
explore dynamics in heterogeneous systems
(26, 36, 77, 138, 161, 176, 231, 232). Below,
we describe some of what has been learned
from these studies.
The PSB Plot: Folding Speed
Correlates with the Localness of
Contacts in the Native Structure
One of the few universal features of protein
folding kinetics was rst observed by Plaxco,
0
14
8 22
R = 0.75
RCO (%)
l
n

k
f
Figure 4
Folding rate versus relative contact order (a
measure of localness of contacts in the native
structure) for the 48 two-state proteins given in
Reference 91, showing that proteins with the most
local contacts fold faster than proteins with more
nonlocal contacts.
Simons, and Baker (PSB), namely, that the
folding speed of a protein is correlated with
a topological property of its native struc-
ture (88, 184). As shown in Figure 4, Plaxco
et al. found that the folding rates of two-state
proteinsnowknown to vary more than 8 or-
ders of magnitudecorrelate with the aver-
age degree to which native contacts are local
within the chain sequence: Fast-folders usu-
ally have mostly local structure, such as he-
lices andtight turns. Slow-folders usually have
more nonlocal structure, such as -sheets
(184), although there are exceptions (237).
Folding rates have been subsequently found
to correlate well with other native topologi-
cal parameters such as the proteins effective
chain length (chain length minus the num-
ber of amino acids in helices) (107), secondary
structure length (104), sequence-distant con-
tacts per residue (90), the fraction of contacts
that are sequence distant (163), the total con-
tact distance (245), and intrinsic propensities,
for example, of -helices (131). And, there
are now also methods that predict the folding
rate from the sequence (91, 186). It follows
that a protein typically forms smaller loops
and turns faster than it forms larger loops
and turns, consistent with the so-called zip-
pingandassembly (ZA) mechanism, described
below, which postulates that search speed is
www.annualreviews.org The Protein Folding Problem 295
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ANRV343-BB37-14 ARI 9 April 2008 11:34
governed by the effective loop sizes [the ef-
fective contact order (ECO) (53, 73)] that the
chain must search at any step.
Proteins Fold on Funnel-Shaped
Energy Landscapes
Why has folding been regarded as so chal-
lenging? The issue is the astronomical num-
ber of conformations a protein must search
to nd its native state. Models arose in the
1980s to study the nature of the conforma-
tional space (19, 47), i.e., the shape of the
energy landscape, which is the mathemati-
cal function F(, , X) that describes the
intramolecular-plus-solvation free energy of
a given protein as a function of the micro-
scopic degrees of freedom. A central goal has
beentoquantify the statistical mechanical par-
tition function, a key component of which is
the density of states (DOS), i.e., the number of
conformations at each energy level. In simple
cases, the logarithm of the DOS is the con-
formational entropy. Such entropies have not
been determinable through all-atom model-
ing, because that would require astronomi-
cal amounts of computational sampling (al-
though replica-exchange methods can nowdo
this for very small peptides). Hence under-
standing a proteins DOS and its entropies
has required simplied models, such as mean-
eldpolymer andlattice treatments (51), spin-
glass theories (19, 47), or exact enumerations
in minimalist models (132).
A key conclusion is that proteins have
funnel-shaped energy landscapes, i.e., many
high-energy states and few low-energy states
(19, 49, 50, 52, 138). What do we learn from
the funnel idea? Funnels have both qualita-
tive and quantitative uses. First, cartooniza-
tions of funnels (Figure 5) provide a use-
ful shorthand language for communicating
the statistical mechanical properties and fold-
ing kinetics of proteins. Figure 5 illustrates
fast folding (simple funnel), kinetic trapping
(moats or wells), and slow random searching
(golf course). These pictures show a key dis-
tinction between protein folding and simple
classical chemical reactions. A simple chemi-
cal reaction proceeds from its reactant A to its
product B, through a pathway, i.e., a sequence
of individual structures. A protein cannot do
this because its reactant, the denatured state,
is not a single microscopic structure. Fold-
ing is a transition from disorder to order, not
from one structure to another. Simple one-
dimensional reaction path diagrams do not
capture this tremendous reduction in confor-
mational degeneracy.
A funnel describes a proteins conforma-
tional heterogeneity. Conformational het-
erogeneity has been found in the few experi-
ments that have been designed to look for it
a b c d
N N N N
Figure 5
One type of energy landscape cartoon: the free energy F(, , ) of the bond degrees of freedom. These
pictures give a sort of simplied schematic diagram, useful for illustrating a proteins partition function
and density of states. (a) A smooth energy landscape for a fast folder, (b) a rugged energy landscape with
kinetic traps, (c) a golf course energy landscape in which folding is dominated by diffusional
conformational search, and (d ) a moat landscape, where folding must pass through an obligatory
intermediate.
296 Dill et al.
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ANRV343-BB37-14 ARI 9 April 2008 11:34
(16, 143, 157, 206, 209, 244). For example, us-
ing time-resolved FRET with four different
intramolecular distances, Sridevi et al. (206)
found inBarstar that (a) that the chainentropy
increases as structures become less stable,
(b) that there are multiple folding routes, and
(c) that different routes dominate under differ-
ent folding conditions. Moreover, changing
the denaturant canchange the dominant path-
way, implying heterogeneous kinetics (143).
Figure 6 shows the funnel landscape that
has been determined by extensive mutational
analysis of the seven ankyrin sequence repeats
of the Notch ankyrin repeat domain (16, 157,
209).
A funnel describes a proteins chain
entropy. The funnel idea rst arose to ex-
plain denaturation, the balance between the
chain entropy and the forces of folding (48).
Proteins denature at high temperatures be-
cause there are many states of high energy
and fewer states of low energy, that is, the
landscape is funneled. For cold unfolding, the
shape of the funnel changes with temperature
because of free-energy changes of the aqueous
solvent. When you can accurately compute a
-6
0
6

G

k
c
a
l
/
m
o
l
Figure 6
The experimentally determined energy landscape of the seven ankyrin
repeats of the Notch receptor (16, 157, 209). The energy landscape is
constructed by measuring the stabilities of folded fragments for a series of
overlapping modular repeats. Each horizontal tier presents the partially
folded fragments with the same number of repeats. Reprinted from
Reference 157 with permission.
proteins DOS, you can predict the proteins
free energy of folding and its denaturationand
cooperativity properties. Figure 7 shows an
example in which the DOS (set onto its side
to illustrate the funnel) was found by exten-
sive lattice enumeration for F13W

, a three-
helix bundle, with predictions compared to
experiments (146).
Native
E
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)
ln (conformation count)
50
0 80
2 0
0
1
0.5
50 0 100
0
1
0.5
4 6
Transition
states
GuHCl (M)
Temperature (C)
F
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Figure 7
(Left) The density of states (DOS) cartoonized as an energy landscape for the three-helix bundle protein
F13W

: DOS (x-axis) versus the energy (y-axis). (Right) Denaturation predictions versus experiments
(146). The peak free energy (here, where the DOS is minimum), typically taken to be the transition state,
is energetically very close to native.
www.annualreviews.org The Protein Folding Problem 297
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Funnels provide a microscopic framework
for folding kinetics. Folding kinetics is tra-
ditionally described by simple mass action
models, such as D I N (on-path inter-
mediate I between the denatured state D and
native state N) or X D N (off-path in-
termediate X), where the symbol I or X rep-
resents macrostates that are invoked for the
purpose of curve-tting experimental kinet-
ics data. In contrast, funnel models or master-
equation models aim to explain the kinetics
in terms of underlying physical forces. They
aim to predict the microstate composition of
those macrostates, for example. The states in
master-equation models differ from those in
mass-action models insofar as the former are
more numerous, more microscopic, are de-
ned by structural or energetic criteria, and
are arranged kinetically to reect the underly-
ing funnel-like organization of the dynamical
ows.
For example, Figure 8b shows a master-
equation model for the folding of SH3, illus-
trating the apparently paradoxical result that
folding can be serial and parallel at the same
time. The protein has multiple routes avail-
able. However, one of the dominant series
pathways is UB BDBDEBCDE
N. BD is the TSE because it is the dynam-
ically least populated state. B precedes BD
diagrammatically inseries inthis pathway. Yet,
the probability bucket labeled B does not rst
ll up and then empty into BD; rather the lev-
els in both buckets, B and BD, rise and fall to-
gether and hence are dynamically in parallel.
Such series and parallel steps are also seen in
computer simulations of CI2 (189), for exam-
ple. Sometimes a chain contact Aforms before
b
ABCE
1.9
ACDE
BDE
0.2
ABDE
1.3
B
2.0
ABD
2.9
ABCD
2.4
BD
2.4
A
2.5
ACE
2.9 1.3
AC
4.0
0.0
C
1.5
BCD
1.9
ABCDE
2.3
BCDE
0.3
BC
3.5
0
a
R TS P
R
Time
10
-4
10
-2
1
0.1 10 1000
0
B
BD
BDE
BCDE
ABCDE
P
i
(
t
)
10
-6
10
-4
10
-2
1
0.1 10 1000
Time
P
i
(
t
)
TS
P
A
D
E
B
C
10 20 30 40 50
10
20
30
40
50
strand
Diverging turn DT
3-10 helix
Loop
Figure 8
(a) A simple single-pathway system. R, reactant; TS, transition state; P, product. (b) Pathway diagram of
SH3 folding from a master-equation model. The native protein has ve contact clusters: A = RT loop;
B =
2

3
; C =
3

4
; D = RT loop-
4
; and E =
1

5
. Combined letters, such as BD, mean that
multiple contact clusters have formed. Funneling occurs toward the right, because the symbols on the left
indicate large ensembles, whereas the symbols on the right are smaller ensembles. The numbers indicate
free energies relative to the denatured state. The arrows between the states are colored to indicate
transition times between states. The slowest steps are in red; the fastest steps in green. BD is the
transition state ensemble because it is the highest free energy along the dominant route. While B and BD
would seem to be obligatorily in series, the time evolutions of these states show that they actually rise and
fall in parallel (232).
298 Dill et al.
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ANRV343-BB37-14 ARI 9 April 2008 11:34
another contact B in nearly all the simulation
trajectories (series-like). But another contact
C may form before B in some trajectories and
after B in others (parallel-like). Some folding
is sequential, as inFynSH3 (123), cytochrome
(66), T4 lysozyme (24), and Im7 (74), and
some folding is parallel, as in cytochrome C
(83) and HEW lyzosyme (151).
Or, consider the traditional idea that a re-
actions RLS coincides with the point of the
highest free energy, G

, along the reaction

coordinate. As a matter of principle, the RLS
need not necessarily coincide with the free-
energy barrier. There is evidence that they
may not coincide for some protein folding
processes (13, 15, 27). Whats the distinction?
To nd the RLS, you need a dynamical model.
Youwouldndthe eigenvector corresponding
to the slowest eigenvalue (in two-state kinet-
ics). Microscopic master-equation modeling
typically nds that this eigenvector only iden-
ties the process U N, with no ner pin-
pointing of any special structures along the
way. In contrast, G

is a thermodynamic
quantitythe maximumfree energy alongthe
fastest route, which usually does correspond
to some specic ensemble of structures. Be-
low, we describe why these matters of princi-
ple are important.
How do we convert folding experiments
into insights about molecular behavior?
To interpret data, we must use models. -
value experiments aim to identify RLSs in
folding. But how we understand the molec-
ular events causing a given -value depends
on whether we interpret it by funnels or path-
ways. A -value measures how a folding rate
changes when a protein is mutated (42, 67,
71, 105, 144, 153, 154, 176, 178, 193) (see
Reference 231andreferences 124 therein).
equals the change in the logarithmof the fold-
ing rate causedby the mutation, dividedby the
change in the logarithm of the folding equi-
librium constant. If we then seek a structural
interpretation of , we need a model. Us-
ing the Bronsted-Hammond pathway model
of chemical reactions, is often assumed to
indicate the position of the TSE along the
folding reaction coordinate: = 0 means
the mutation site is denatured in the TSE;
= 1 means the mutation site is native in
the TSE. In this pathway view, can never
lie outside the range from 0 to 1; in the fun-
nel view, is not physically restricted to this
range. For example, < 0 or > 1 has been
predicted for mutations that stabilize a helix
but that destabilize the bundles tertiary struc-
ture (231). Unfortunately, experiments are not
yet denitive. While some -values are in-
deed observed to be negative or greater than
1 (44, 85, 176, 193), those values might be ex-
perimental artifacts (193). Other challenges in
interpreting have also been noted (65, 188).
To resolve the ambiguities in interpreting ,
we need to deepen our understanding beyond
the single-reaction-coordinate idea.
How do we convert computer simulations
into insights about molecular behavior?
Similarly, insights about folding events are
often sought from computer modeling. It is
much easier to calculate structural or ener-
getic quantities than kinetic quantities. For
example, some modeling efforts compute -
values by assuming some particular struc-
ture for the TSE (78, 233) or some partic-
ular reaction coordinate, such as the RMSD
to native structure, radius of gyration, num-
ber of hydrogen bonds, or number of na-
tive contacts (196). Alternatively, a quantity
called pfold (56), which denes a separatrix
(a sort of continental divide between folded
and unfolded states), is sometimes computed.
Although pfold predicts well the RLSs for
simple landscapes (147), it can give less in-
sight into protein landscapes having multi-
ple barriers or other complexity (30). To go
beyond classical assumptions, there has been
an extensive and growing effort to use master-
equationapproaches (13, 26, 31, 36, 60, 61, 77,
110, 161, 172, 176, 201, 202, 208, 211, 212,
231, 232) to explore underlying assumptions
about reaction coordinates, pathways, transi-
tion states, and RLSs.
www.annualreviews.org The Protein Folding Problem 299
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ANRV343-BB37-14 ARI 9 April 2008 11:34
Funnel models can explain some non-
canonical behaviors in ultrafast folding.
More than a dozen proteins fold in microsec-
onds (128). Some foldinhundreds of nanosec-
onds (127, 237). Is there a state of protein
folding that is so fast that there is no free-
energy barrier at all (156)? This has some-
times been called downhill folding (93, 122,
128, 187). There is currently an intensive
search for downhill foldersand much con-
troversy about whether or not such folding
has yet been observed, mainly in BBL, a
40-residue helical protein. That controversy
hinges on questions of experimental analysis
(68, 69, 75, 103, 168, 170, 191): establishing
proper baselines and ionization states to nd
denaturation temperatures and to determine
whether the equilibrium is two-state, for ex-
ample, which would imply a barrier between
D and N.
Remarkably, all known ultrafast-folders
have anti-Arrhenius thermal kinetics. That
is, heating those proteins at high tempera-
tures slows down folding, the opposite of what
is expected from traditional activation barri-
ers. Here too, any molecular interpretation
requires a model, and the common expecta-
tion is based on the classical Arrhenius/Eyring
pathway model. Is the Arrhenius model suf-
cient for funnels involving many fast pro-
cesses? Ultrafast folding kinetics has recently
been explored in various models (59, 77, 122,
148, 167). One funnel model (77) explains
that the reason why increasing the tempera-
ture leads to slower folding is because of ther-
mal unfolding of the denatured chain, leading
to a larger conformational space that must be
searched for the chain to nd route to native
downhill. It predicts that the ultimate speed
limit to protein folding, at temperatures that
will disappear all other barriers, is the confor-
mational search through the denatured basin.
Near the speed limit of protein folding, the
heterogeneity and searching that are intrinsic
to funnels can be an important component of
the folding physics. That model also explains
that helical proteins fold faster than -sheets,
on average, because helices have more paral-
lel microscopic folding routes (because a he-
lix can nucleate at many different points along
the chain).
The Zipping and Assembly
Hypothesis for the Folding Routes
Protein folding is a stochastic process: One
protein molecule in a beaker follows a
different microscopic trajectory than another
molecule because of thermal uctuations.
Hence, protein folding is often studied us-
ing Monte Carlo or molecular dynamics sam-
pling. However, computations seeking the
native state using purely physical models
are prohibitively expensive, because this is a
challenging needle-in-a-haystack global opti-
mization problem (96, 132, 216). Since the
beginnings of experimental folding kinetics,
there has been the view that the Levinthal
paradoxof how a protein searches its con-
formational space so quicklymight be ex-
plained by a folding mechanism, i.e., by some
higher-level description (beyond the state-
ment that it is stochastic) that claries how
the protein decides which structures to form
and avoids searching vast stretches of the con-
formational space in the rst place.
Zipping and assembly (ZA) is a hypothesis
for a general folding mechanism. On fast
timescales, small fragments of the chain can
search their conformations more completely
than larger fragments can (53, 73). There are
certain problems of global optimization
including the ZA mechanism of protein
folding and the Cocke-Kasami-Younger
method for parsing sentences (54, 102)in
which the globally optimal solution (native
structure, in this case) can almost always
be found (although not guaranteed) by a
divide-and-conquer strategy, a fast process
of cobbling together smaller locally optimal
decisions. Accordingly, in the earliest time
steps after folding is initiated (picoseconds to
nanoseconds), each of the different peptide
fragments of the chain searches for small
300 Dill et al.
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local metastable structures, such as helical
turns, -turns, or small loops. Each peptide
segment searches its own conformations, at
the same time that other segments are search-
ing. Not stable on their own, a few of those
local structures are sufciently metastable
to survive to the next longer timescale,
where they grow (or zip) into increasingly
larger and more stable structures. On still
longer timescales, pairs or groups of these
substructures can assemble into structures
that are still larger and more native-like, and
metastability gives way to stability (97, 102,
103, 199, 215, 223, 228230, 232).
The ZA mechanism shares much in
common with other mechanisms, such as
diffusion-collision, hierarchical, and foldon
models. The last two mechanisms, however,
are descriptors of experiments. They do not
prescribe how to compute a proteins fold-
ing route from its amino acid sequence. In
contrast, the ZA mechanism is such a micro-
scopic recipe, starting from the amino acid
sequence and specifying a time series of en-
sembles of conformations the chain searches
at each stage of folding. ZAis a funnel process:
There are many parallel microscopic routes at
the beginning, and fewer and more sequential
routes at the end. The ZA mechanism pro-
vides a plausible answer to Levinthals para-
dox of what vast stretches of conformational
space the protein never bothers to search.
For any compact native polymer structure,
there are always routes to the native state that
take only small-conformational-entropy-loss
steps. ZAotherwise explores very little of con-
formational space. These few routes consti-
tute the dominant folding processes in the
ZA mechanism. One test of this mechanismis
the prediction of the change of folding routes
(229), measured by the change in -value dis-
tributions (143), upon circular permutation
of the chain. Proteins can be circularly per-
muted if the chain termini are adjacent to each
other inthe wild-type native structure. Insuch
cases, the ends are covalently linked and the
chain is broken elsewhere. This alters the na-
tive topology (contact map) dramatically, and
sometimes the folding routes, but does not ap-
pear to substantially change the native struc-
ture (20, 142, 143, 160, 221).
PHYSICS-BASED MODELING
OF FOLDING AND
STRUCTURE PREDICTION
Computer simulations of purely physics-
based models are becoming useful for struc-
ture prediction and for studying folding
routes. Here the metric of success is not purely
performance in native structure prediction; it
is to gain a deeper understanding of the forces
and dynamics that govern protein properties.
When purely physical methods are success-
ful, it will allow us to go beyond bioinfor-
matics to (a) predict conformational changes,
such as induced t, important for computa-
tional drug discovery; (b) understand protein
mechanisms of action, motions, folding pro-
cesses, enzymatic catalysis, and other situa-
tions that require more than just the static
native structure; (c) understand how proteins
respond to solvents, pH, salts, denaturants,
and other factors; and (d ) design synthetic
proteins having noncanonical amino acids
or foldameric polymers with nonbiological
backbones.
A key issue has been whether semiem-
pirical atomic physical force elds are good
enough to fold up a protein in a computer.
Physics-based methods are currently limited
by large computational requirements ow-
ing to the formidable conformational search
problemand, to a lesser extent, by weaknesses
in force elds. Nevertheless, there have been
notable successes in the past decade enabled
by the development of large supercomputer
resources and distributed computing systems.
The rst milestone was a supercomputer sim-
ulation by Duan and Kollman in 1998 of
the folding of the 36-residue villin headpiece
in explicit solvent, for nearly a microsec-
ond of computed time, reaching a collapsed
state 4.5

A from the NMR structure (57).
www.annualreviews.org The Protein Folding Problem 301
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Another milestone was the development by
Pande and colleagues of Folding@home, a
distributed grid computing system running
on the screensavers of volunteer comput-
ers worldwide. Pande and colleagues (241)
have studied the folding kinetics of villin.
High-resolution structures of villin have re-
cently been reached by Pande and colleagues
(110) and Duan and colleagues (136, 137). In
addition, three groups have folded the 20-
residue Trp-cage peptide to 1

A: Simmer-
ling et al. (200), the IBM Blue Gene group
of Pitera and Swope (183), and Duan and
colleagues (35). Recently, Lei & Duan (135)
folded the albumin-binding domain, a 47-
residue, three-helix bundle, to 2.0

A. Physics-
based approaches are also folding small he-
lices and -hairpin peptides of up to 20
residues that have stable secondary structures
(63, 81, 108, 240, 246; M.S. Shell, R. Ritterson
MTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE
815
617
2839
4354
4156
2856
156
120
3037 4552
a
b
Figure 9
The folding routes found in the ZAM conformational search process for
protein G, from the work described in Reference 177. The chain is rst
parsed into many short, overlapping fragments. After sampling by replica
exchange molecular dynamics, stable hydrophobic contacts are identied
and restrained. Fragments are then either (a) grown or zipped though
iterations of adding new residues, sampling, and contact detection, or
(b) assembled together pairwise using rigid body alignment followed by
further sampling until a completed structure is reached.
& K.A. Dill, unpublished data). Physical po-
tential models have also been sampled using
non-Boltzmann stochastic and deterministic
optimization strategies (121, 174, 207, 220).
Here are some of the key conclusions.
First, a powerful way to sample conforma-
tions and obtain proper Boltzmann aver-
ages is replica exchange molecular dynamics
(REMD) (210). Second, although force elds
are good, they need improvements in back-
bone torsional energies to address the bal-
ance between helical and extended conforma-
tions (81, 108, 240), and in implicit solvation,
which dramatically reduces the expense rel-
ative to explicit water simulations but which
frequently overstabilizes ion-pairing interac-
tions, in turn destabilizing native structures
(63, 246).
Can modern force elds with Boltzmann
sampling predict larger native structures? Re-
cent work indicates that, when combined with
a conformational search technique based on
the ZA folding mechanism, purely physics-
based methods can arrive at structures close
to the native state for chains up to 100
monomers (177; M.S. Shell, S.B. Ozkan, V.A.
Voelz, G.H.A. Wu & K.A. Dill, unpublished
data). The approach, called ZAM (zipping
and assembly method), uses replica exchange
and the AMBER96 force eld and works by
(a) breaking the full protein chain into small
fragments (initially 8-mers), which are sim-
ulated separately using REMD; (b) then
growing or zipping the fragments having
metastable structures by adding a few new
residues or assembling two such fragments
together, with further REMD and itera-
tions; and (c) locking in place any stable
residue-residue contacts with a harmonic
spring, enforcing emerging putative physi-
cal folding routes, without the need to sam-
ple huge numbers of degrees of freedom at a
time.
ZAM was tested through the folding
of eight of nine small proteins from the
PDB to within 2.5

A, using a 70-processor
cluster over 6 months (177), giving good
302 Dill et al.
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ANRV343-BB37-14 ARI 9 April 2008 11:34
agreement with the -values known for
four of them. Figure 9 shows the ZAM
folding process for one of these proteins, and
Figure 10 shows the predicted versus experi-
mental structures for all nine. In a more strin-
gent test, ZAM was applied in CASP7 to the
folding of six small proteins from 76 to 112
residues (M.S. Shell, S.B. Ozkan, V.A. Voelz,
G.H.A. Wu & K.A. Dill, unpublished data).
Of the four proteins attempted in CASP7 that
were not domain-swapped, ZAM predicted
roughly correct tertiary structures, segments
of more than 40 residues with an average
RMSDof 5.9 angstroms, andsecondary struc-
tures with 73% accuracy. From these stud-
ies it has been concluded that ZA routes can
identify limited-sampling routes to the native
state from unfolded states, directed by all-
atomforce elds, and that the AMBER96 plus
a generalized Born implicit solvent model is a
reasonable scoring function. Fragments that
adopt incorrect secondary structures early in
the simulations are frequently corrected in
later-stage folding because the emerging ter-
tiary structure of the protein often will not
tolerate them.
SUMMARY
The protein folding problem has seen enor-
mous advances over the last fty years.
Newexperimental techniques have arisen, in-
cluding hydrogen exchange, -value meth-
ods that probe mutational effects on fold-
ing rates, single-molecule methods that can
explore heterogeneity of folding and en-
ergy landscapes, and fast temperature-jump
methods. New theoretical and computa-
tional approaches have emerged, includ-
ing methods of bioinformatics, multiple-
sequence alignments, structure-prediction
Web servers, physics-based force elds of
good accuracy, physical models of energy
landscapes, fast methods of conformational
sampling and searching, master-equation
methods to explore the physical mecha-
nisms of folding, parallel and distributed
grid-based computing, and the CASP
community-wide event for protein structure
prediction.
Protein folding no longer appears to be
the insurmountable grand challenge that it
once appeared to be. Current knowledge of
folding codes is sufcient to guide the suc-
cessful designs of newproteins and foldameric
materials. For the once seemingly intractable
Levinthal puzzle, there is now a viable hy-
pothesis: A protein can fold quickly and
solve its big global optimization puzzle
by piecewise solutions of smaller compo-
nent puzzles. Other matters of principle
are now yielding to theory and physics-
based modeling. And current computer algo-
rithms are now predicting native structures of
small proteins remarkably accurately, promis-
ing growing value in drug discovery and
proteomics.
Protein A Albumin-binding domain protein 3 D
Protein G -spectrin SH3 src-SH3
YJQ8 WW domain (res 731) FBP28 WW domain (res 631) 35-mer unit of ubiquitin
Figure 10
Ribbondiagrams of the predictedproteinstructures using the ZAMsearchal-
gorithm( purple) versus experimental PDBstructures (orange). The backbone
C-RMSDs with respect to PDB structures are protein A (1.9

A), albumin
domain binding protein (2.4

A), alpha-3D [2.85

A (excluding loop residues)
or 4.6

A], FBP26 WW domain (2.2

A), YJQ8 WW domain (2.0

A), 135
residue fragment of Ubiquitin (2.0

A), protein G (1.6

A), and -spectrin SH3
(2.2

A). ZAM fails to nd the src-SH3 structure: Shown is a conformation
that is 6

A from the experimental structure. The problem in this case appears
to be overstabilization of nonnative ion pairs in the GB/SA implicit solvation
model.
www.annualreviews.org The Protein Folding Problem 303
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ANRV343-BB37-14 ARI 9 April 2008 11:34
SUMMARY POINTS
1. The protein folding code is mainly embodied in side chain solvation interactions.
Novel protein folds and nonbiological foldamers are now being successfully designed
and are moving toward practical applications.
2. Thanks to CASP, the growing PDB, and fast-homology and sequence alignment
methods, computer methods now can often predict correct native structures of small
proteins.
3. The protein folding problem has both drivenand beneted frombig advances in
experimental and theoretical/computational methods.
4. Proteins fold on funnel-shaped energy landscapes, which describe the conformational
heterogeneity among the nonnative states. This heterogeneity is key to the entropy
that opposes folding and thus to folding equilibria. This heterogeneity is also impor-
tant for understanding folding kinetics at the level of the individual chain processes.
5. A protein can fold quickly to its native structure by ZA, making independent local
decisions rst and then combining those substructures. In this way, a protein can avoid
searching most of its conformational space. ZA appears to be a useful search method
for computational modeling.
DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of
this review.
ACKNOWLEDGMENTS
For very helpful comments and insights, both on this review and through ongoing discussions
over the years, we are deeply grateful to D. Wayne Bolen, Hue Sun Chan, John Chodera, Yong
Duan, Walter Englander, Frank Noe, Jos e Onuchic, Vijay Pande, Jed Pitera, Kevin Plaxco,
Adrian Roitberg, George Rose, Tobin Sosnick, Bill Swope, Dave Thirumalai, Vince Voelz,
Peter G Wolynes, and Huan-Xiang Zhou. We owe particular thanks and appreciation to Buzz
Baldwin, to whom this volume of the Annual Review of Biophysics is dedicated, not only for his
interest and engagement with us on matters of protein folding over the many years, but also
for his pioneering and founding leadership of the whole eld. We appreciate the support from
NIH grant GM 34993, the Air Force, and the Sandler Foundation.
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AR343-FM ARI 10 April 2008 7:2
Annual Review of
Biophysics
Volume 37, 2008
Contents
Frontispiece
Robert L. Baldwin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p xiv
The Search for Folding Intermediates and the Mechanism
of Protein Folding
Robert L. Baldwin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
How Translocons Select Transmembrane Helices
Stephen H. White and Gunnar von Heijne p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 23
Unique Rotary ATP Synthase and Its Biological Diversity
Christoph von Ballmoos, Gregory M. Cook, and Peter Dimroth p p p p p p p p p p p p p p p p p p p p p p p p 43
Mediation, Modulation, and Consequences
of Membrane-Cytoskeleton Interactions
Gary J. Doherty and Harvey T. McMahon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 65
Metal Binding Afnity and Selectivity in Metalloproteins:
Insights from Computational Studies
Todor Dudev and Carmay Lim p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 97
Riboswitches: Emerging Themes in RNA Structure and Function
Rebecca K. Montange and Robert T. Batey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 117
Calorimetry and Thermodynamics in Drug Design
Jonathan B. Chaires p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 135
Protein Design by Directed Evolution
Christian Jckel, Peter Kast, and Donald Hilvert p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 153
PIP
2
Is A Necessary Cofactor for Ion Channel Function:
How and Why?
Byung-Chang Suh and Bertil Hille p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 175
RNA Folding: Conformational Statistics, Folding Kinetics,
and Ion Electrostatics
Shi-Jie Chen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 197
Intrinsically Disordered Proteins in Human Diseases: Introducing
the D
2
Concept
Vladimir N. Uversky, Christopher J. Oldeld, and A. Keith Dunker p p p p p p p p p p p p p p p p 215
Crowding Effects on Diffusion in Solutions and Cells
James A. Dix and A.S. Verkman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 247
vii
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AR343-FM ARI 10 April 2008 7:2
Nanobiotechnology and Cell Biology: Micro- and Nanofabricated
Surfaces to Investigate Receptor-Mediated Signaling
Alexis J. Torres, Min Wu, David Holowka, and Barbara Baird p p p p p p p p p p p p p p p p p p p p p p 265
The Protein Folding Problem
Ken A. Dill, S. Banu Ozkan, M. Scott Shell, and Thomas R. Weikl p p p p p p p p p p p p p p p p p p 289
Translocation and Unwinding Mechanisms of RNA
and DNA Helicases
Anna Marie Pyle p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 317
Structure of Eukaryotic RNA Polymerases
P. Cramer, K.-J. Armache, S. Baumli, S. Benkert, F. Brueckner, C. Buchen,
G.E. Damsma, S. Dengl, S.R. Geiger, A.J. Jasiak, A. Jawhari, S. Jennebach,
T. Kamenski, H. Kettenberger, C.-D. Kuhn, E. Lehmann, K. Leike, J.F. Sydow,
and A. Vannini p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 337
Structure-Based View of Epidermal Growth Factor Receptor
Regulation
Kathryn M. Ferguson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 353
Macromolecular Crowding and Connement: Biochemical,
Biophysical, and Potential Physiological Consequences
Huan-Xiang Zhou, Germn Rivas, and Allen P. Minton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 375
Biophysics of Catch Bonds
Wendy E. Thomas, Viola Vogel, and Evgeni Sokurenko p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 399
Single-Molecule Approach to Molecular Biology in Living Bacterial
Cells
X. Sunney Xie, Paul J. Choi, Gene-Wei Li, Nam Ki Lee, and Giuseppe Lia p p p p p p p p p 417
Structural Principles from Large RNAs
Stephen R. Holbrook p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 445
Bimolecular Fluorescence Complementation (BiFC) Analysis
as a Probe of Protein Interactions in Living Cells
Tom K. Kerppola p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 465
Multiple Routes and Structural Heterogeneity in Protein Folding
Jayant B. Udgaonkar p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 489
Index
Cumulative Index of Contributing Authors, Volumes 3337 p p p p p p p p p p p p p p p p p p p p p p p p 511
Errata
An online log of corrections to Annual Review of Biophysics articles may be found at
http://biophys.annualreviews.org/errata.shtml
viii Contents
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ANNUAL REVIEWS
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Editor: Stephen E. Fienberg, Carnegie Mellon University
Associate Editors: Nancy Reid, University of Toronto
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The Annual Review of Statistics and Its Application aims to inform statisticians and quantitative methodologists, as
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TABLE OF CONTENTS:
What Is Statistics? Stephen E. Fienberg
A Systematic Statistical Approach to Evaluating Evidence
from Observational Studies, David Madigan, Paul E. Stang,
Jesse A. Berlin, Martijn Schuemie, J. Marc Overhage,
Marc A. Suchard, Bill Dumouchel, Abraham G. Hartzema,
Patrick B. Ryan
The Role of Statistics in the Discovery of a Higgs Boson,
David A. van Dyk
Brain Imaging Analysis, F. DuBois Bowman
Statistics and Climate, Peter Guttorp
Climate Simulators and Climate Projections,
Jonathan Rougier, Michael Goldstein
Probabilistic Forecasting, Tilmann Gneiting,
Matthias Katzfuss
Bayesian Computational Tools, Christian P. Robert
Bayesian Computation Via Markov Chain Monte Carlo,
Radu V. Craiu, Jefrey S. Rosenthal
Build, Compute, Critique, Repeat: Data Analysis with Latent
Variable Models, David M. Blei
Structured Regularizers for High-Dimensional Problems:
Statistical and Computational Issues, Martin J. Wainwright
High-Dimensional Statistics with a View Toward Applications
in Biology, Peter Bhlmann, Markus Kalisch, Lukas Meier
Next-Generation Statistical Genetics: Modeling, Penalization,
and Optimization in High-Dimensional Data, Kenneth Lange,
Jeanette C. Papp, Janet S. Sinsheimer, Eric M. Sobel
Breaking Bad: Two Decades of Life-Course Data Analysis
in Criminology, Developmental Psychology, and Beyond,
Elena A. Erosheva, Ross L. Matsueda, Donatello Telesca
Event History Analysis, Niels Keiding
Statistical Evaluation of Forensic DNA Profle Evidence,
Christopher D. Steele, David J. Balding
Using League Table Rankings in Public Policy Formation:
Statistical Issues, Harvey Goldstein
Statistical Ecology, Ruth King
Estimating the Number of Species in Microbial Diversity
Studies, John Bunge, Amy Willis, Fiona Walsh
Dynamic Treatment Regimes, Bibhas Chakraborty,
Susan A. Murphy
Statistics and Related Topics in Single-Molecule Biophysics,
Hong Qian, S.C. Kou
Statistics and Quantitative Risk Management for Banking
and Insurance, Paul Embrechts, Marius Hofert
Access this and all other Annual Reviews journals via your institution at www.annualreviews.org.
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