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Journal of Molecular Biology

Published as: J Mol Biol. 2010 April 16; 397(5): 1329–1338.
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Protein Folding Requires Crowd Control in a Simulated Cell

Benjamin R. Jefferys⁎, Lawrence A. Kelley, and Michael J.E. Sternberg

Division of Molecular Biosciences, Biochemistry Building, Imperial College London, South
Kensington, London SW7 2AZ, UK.

Abstract
Macromolecular crowding has a profound effect upon biochemical processes in the cell. We have
computationally studied the effect of crowding upon protein folding for 12 small domains in a
simulated cell using a coarse-grained protein model, which is based upon Langevin dynamics,
designed to unify the often disjoint goals of protein folding simulation and structure prediction. The
model can make predictions of native conformation with accuracy comparable with that of the best
current template-free models. It is fast enough to enable a more extensive analysis of crowding than
previously attempted, studying several proteins at many crowding levels and further random
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repetitions designed to more closely approximate the ensemble of conformations. We found that
when crowding approaches 40% excluded volume, the maximum level found in the cell, proteins
fold to fewer native-like states. Notably, when crowding is increased beyond this level, there is a
sudden failure of protein folding: proteins fix upon a structure more quickly and become trapped in
extended conformations. These results suggest that the ability of small protein domains to fold
without the help of chaperones may be an important factor in limiting the degree of macromolecular
crowding in the cell. Here, we discuss the possible implications regarding the relationship between
protein expression level, protein size, chaperone activity and aggregation.

Abbreviation
TM, template modelling

Keywords
macromolecular crowding; protein structure prediction; protein misfolding; protein aggregation;
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protein expression

Introduction
It is widely acknowledged that there are two fundamental questions in modelling protein
folding:
What is the biologically active conformation of a protein sequence?

© 2010 Elsevier Ltd.

This document may be redistributed and reused, subject to certain conditions.
⁎
Corresponding author. Fax: +44 (0) 20 7594 5789. [email protected].
This document was posted here by permission of the publisher. At the time of deposit, it included all changes made during peer review,
copyediting, and publishing. The U.S. National Library of Medicine is responsible for all links within the document and for incorporating
any publisher-supplied amendments or retractions issued subsequently. The published journal article, guaranteed to be such by Elsevier,
is available for free, on ScienceDirect.
Jefferys et al. Page 2

How does a protein find that state in the cell?

These two problems have generally been approached using radically different methods. The
most successful methods for predicting the biologically active state are through finding a
homologue of the sequence to another sequence with known structure or through assembly of
fragments of structure when such homology cannot be detected. These are valuable tools for
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suggesting the structure of a protein sequence where no experimental structure is available,

and they consistently perform well at CASP (Critical Assessment of techniques for protein
Structure Prediction) experiments.

However, such methods provide little, if any, insight into the principles underlying the search
for that state: the speed and reliability of folding, the stability and flexibility of the final
conformation, the effect of conditions in the cell, misfolding and aggregation and error-
correcting processes. Molecular dynamics is used for investigating these elements of protein
folding. Although progress is being made toward predicting protein structure through
molecular dynamics, such methods are not yet among the most successful predictive tools and
the large computational resources required make their application to large-scale analysis
impractical.

Simplifications of molecular dynamics that reduce the number of particles modelled, simplify
the force field and use time-saving heuristics in the search method enable larger-scale analyses
of protein folding. Ultimately, the goal is to study the mechanism of protein folding and obtain
the biologically active conformation with a unified model for protein structure, which predicts
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the native state through accurate modelling of the folding process. We present here a protein
model developed for this purpose and demonstrate its effectiveness in both protein structure
prediction and the study of the protein folding process. The structural model reduces the
backbone to a series of particles representing Cα atoms and particles representing the centroid
of side-chain atoms. The folding model is a simplified iterative solution of Newtonian equations
of motion based upon Langevin dynamics, with linear elastic springs modelling the majority
of effects in the force field. Solution of this equation aims to simulate protein folding over time
through a putative pathway. The Langevin equation includes a term for random bombardment
by implicit solvent; therefore, the generated folding pathway is dependent upon the seed for a
random number generator and multiple pathways must be produced to assess the ensemble of
conformations. Such a model can be used to investigate many aspects of protein folding,
including the effect of conditions in the cell, such as macromolecular crowding.

The cell is a crowded and chaotic environment: it is estimated that between 10% and 40% of
the cell's volume is occupied by macromolecules. However, complex and intricate biochemical
processes must be performed reliably in this environment. A minimum concentration of
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molecules in the cell is necessary for interacting partners to associate. There is also a maximum
concentration beyond which normal cellular function would be prevented due to restriction of
molecular motion. Evidence suggests that crowding enhances protein stability, protein
association and chaperonin action but that it also increases protein aggregation and lowers
diffusion rates. The macromolecular concentration observed in cells may have been selected
to balance these factors, according to requirements in different cellular localities. In this study,
we investigated the role of protein folding in this balance.

Previous work suggests that macromolecular crowding has a complex effect upon protein
folding. Computational models and experimental work show that, at a crowding level similar
to that of the cell as a whole, crowding can make proteins fold more rapidly and stabilise the
native state at secondary and tertiary levels. This effect is interpreted as being due to the
reduction of conformation space by exclusion of extended conformations.

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Jefferys et al. Page 3

Computational studies of the effect of crowding upon protein folding commonly use a Gō-like
term in the energy function for the protein, which favours interactions known to occur in the
native conformation. Clearly, such terms disqualify these methods as tools for predicting the
final conformation. As a consequence of using a Gō-like potential, terms may be omitted for
non-native but favourable interactions that might be encountered on the way to the native state
—interactions that might be important in finding the native state or in creating a non-native
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local energy minimum. If the effect of crowding were to trap a protein in such a minimum,
then this effect would not be observed with a Gō-like potential.

Simulations making use of molecular dynamics do not have this disadvantage. However, due
to computational limitations, they often study only the change in stability of proteins in their
native state when subjected to crowding, and where proteins have been refolded from a partially
denatured state, only very small proteins were studied. A study using molecular dynamics has
shown that crowding may destabilise the native state of the protein by forcing the solvent into
a more ordered state, reducing the entropic advantage of the native over other
conformations. Native stability and refolding rates are important aspects of the protein folding
problem. However, in the cell, proteins are synthesized slowly into a crowded environment.
While computational analysis has found that co-translational folding appears to have a small
effect on protein folding rate and has only weakly imprinted motifs in structures that might be
expected if it did, experimental observations are more mixed. Slow synthesis may allow
proteins to find a compact state more easily than from a denatured state, an effect that may be
even more important in a crowded environment. Additionally, computational and experimental
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constraints have limited previous studies to one or two structures and few crowding levels.
This raises the possibility that effects observed are specific to the chosen conditions.

To address these issues, we have studied the effect of crowding using a protein model that does
not include knowledge of long-range interactions, can predict protein tertiary structure with
accuracy comparable with that of the best current template-free methods, includes a simulation
of the folding pathway during slow synthesis of a protein by a large, heavy ribosome and is
fast enough to allow study of many proteins at several crowding levels. The aim of this work
was to find the upper limit of crowding in which proteins can successfully fold and compare
this with the level of crowding observed in the cell.

Results
Model verification through structure prediction
We tested our model by making predictions of the tertiary structure for a set of 30 small protein
domains (listed in Supplementary Material) that were chosen for the quality of experimental
structure, size and fold class. The structure prediction protocol is described in Materials and
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Methods. Modelling is based upon the predicted secondary structure using psipred. Five
structure predictions are made for each protein, and the best TM (template modelling) score
and largest fraction of residues alignable to within 5 Å to the native are given in Fig. 1. The
TM score is a measure of structural similarity that is more sensitive to the similarity of protein
fragments than the more commonly used global RMSD (root-mean-square deviation). The
mean TM score between randomly chosen Protein Data Bank structures is 0.17, and a TM
score exceeding 0.3 indicates a roughly native-like topology. It is unusual to exceed a TM score
of 0.4 in template-free modelling. The best of the five predictions for 24 of the proteins has a
TM score higher than 0.3, and for 4 of those proteins, the score is higher than 0.4. Predictions
and the best structures generated for 3 of the proteins are illustrated in Fig. 2.

These results mean this method is comparable with the best template-free structure prediction
methods. Each prediction is made from simulations using at most 40 CPU hours, a tiny fraction
of the time that is used by many other successful template-free predictive methods. It is this

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Jefferys et al. Page 4

short processing time that has enabled this work studying protein folding in detail under
hundreds of experimental conditions.

Our model iteratively generates putative folding pathways using basic physical principles.
While we have not analysed how these pathways compare with experimental data that might
suggest the natural folding process for a protein, the good results from predicting tertiary
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structure using conformations sampled from these pathways suggest that they are reasonable.
Although the amount of good-quality data regarding the mechanism of folding is growing, it
is still small compared with the huge and reliable database of protein structures, and therefore
those data are used as a metric to assess the performance of our model.

Effect of crowding upon protein folding

The effect of crowding upon protein folding was investigated for a subset of the proteins used
to test the model through structure prediction. Of the 30 proteins, 12 (listed in Supplementary
Material) for which the model performs well were chosen, producing structures with a TM
score higher than 0.3 for at least 10% of the total simulation during 100 syntheses of the protein.
These proteins were then studied under a range of crowding conditions. We assume that the
primary crowding agent at the time of protein folding is another previously synthesized protein
of size similar to that which is being folded. Other crowding macromolecules are likely to be
present in the cell; however, the effect of heterogeneous crowding as compared with
homogeneous crowding is beyond the scope of this work and left for future investigation. It is
observed from experimental work that the specific physicochemical properties of the crowding
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macromolecules are not especially important, rather it is the volume of solvent excluded by
the molecules that has the greatest influence upon protein folding. Therefore, crowding was
simulated by spheres with radius and mass designed to simulate the volume excluded by a
small domain. Modelling a protein-like crowding macromolecule at this coarse level of detail
makes this study computationally feasible: a more detailed model would slow simulations by
orders of magnitude. Since the primary concern at this stage is folding rather than structure
prediction, we used secondary structure assigned from the known native using Stride. Each of
the 12 proteins was synthesized 100 times into a cell with a number of crowding
macromolecules according to the desired crowding level. After synthesis, simulation continued
for 1 million iterations and conformations were sampled at 1000 iteration intervals, this being
sufficient to allow local rearrangement of secondary structure elements. In summary, 1000
conformation samples were taken from each of 100 folding simulations, producing 100,000
protein conformations for each protein at each crowding level. Since 12 proteins were selected
for this analysis, 1.2 million conformations in total were produced to assess the effect of a given
level of crowding. The simulation is illustrated in Fig. 3, and a series of images taken at regular
iteration intervals from a simulation synthesizing a protein into a cell with the maximum
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experimentally observed crowding level is shown in Fig. 4. To determine how the changing
environment affects different aspects of folding, we made measurements of conformation size,
conformational freedom and time spent in a native-like state as volume excluded by crowding
macromolecules increases. Results are summarised in Fig. 5.

Effect of crowding upon conformation size—Crowding may result in more compact

proteins through exclusion of extended conformations or in less compact proteins through
trapping in extended conformations. In order to investigate this, we measured conformation
size using radius of gyration relative to the radius of gyration of the native state. The median
(black line) and middle 50th (blue region) percentiles of the distribution of these radii are given
in the top graph in Fig. 5. Conformations are generally compact up to 45% excluded volume.
At 50% excluded volume, many conformations are less compact. Visual examination of the
most extended conformations shows that they are trapped in narrow corridors of solvent
between the crowding particles, as illustrated in (f) in Fig. 5.

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Jefferys et al. Page 5

Effect of crowding upon conformational freedom—Crowding may change the

conformational freedom of proteins—they may be stabilised or destabilised by collisions with
macromolecules. We assess the time step of each simulation at which a protein finds a
conformation that does not change significantly for the rest of the simulation. Conformations
are defined as being similar if the mean difference in pairwise distances between corresponding
amino acids is less than 4 Å. The reported time step for a given simulation is that at which a
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conformation that is similar to all conformations after it until the end of the simulation is found.
The median (black line) and middle 50th (blue region) percentiles of this number are shown
in the middle graph in Fig. 5. Freedom remains fairly constant up to 30% excluded volume,
with most of the simulated proteins free to explore conformation space until around 800,000
iterations (out of 1 million). Above 40% excluded volume, conformational freedom decreases
sharply: most simulated proteins fix upon a structure before 200,000 iterations. Combining
this with the observations on conformation size, at 50% excluded volume, it would seem that
proteins are becoming trapped in an extended state. At slightly below this level, proteins are
generally trapped in a compact state.

Effect of crowding upon native-like time—Crowding may increase or decrease the

similarity of the protein to the native state during the simulation. We assess this effect to be
measuring native-like time, which we define as the percentage of sampled conformations that
are higher than 0.3 in TM score to the native state. The percentage across all proteins is shown
in the bottom graph of Fig. 5. Native-like time remains fairly constant up to 25% excluded
volume, with over 20% of conformations being above 0.3 in TM score. There is an initial slight
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drop up to 30% crowding, which cannot be attributed to more extended conformations or

becoming trapped in a conformation early. Above this level, native-like time reduces rapidly,
until at 50% excluded volume, just over 10% of conformations have a TM score above 0.3.
The large drop in native-like time at 35% coincides with a slight reduction in conformational
freedom, although conformation size remains fairly constant. Some proteins become stabilised
in compact but non-native conformations by the presence of crowding particles, illustrated in
(e) in Fig. 5. This effect becomes more acute at 45% excluded volume, when conformational
freedom drops sharply. Finally, when crowding is so great the proteins cannot find a compact
conformation, proteins very quickly become trapped in extended states that are clearly less
likely to be similar to the native [illustrated in (f) in Fig. 5].

In summary, there is little effect on the measured features up to 20% excluded volume. At
around 25%, native-like time begins to reduce, although not due to being fixed early or trapped
in extended conformations. Above 40% crowding, proteins are becoming stuck in structures
earlier in the simulation, and around 15 of the structures are in native-like topologies, compared
with 14 with no crowding. At 50% crowding, proteins are stabilised in extended conformations
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early in the simulation in narrow gaps between macromolecules.

Discussion
We have presented a protein structure model designed to predict tertiary structure without the
use of a template or detailed knowledge of the native state. The model demonstrates accuracy
comparable with that of the most successful methods, such as fragment folding, with the added
benefit that it is fast and models a putative folding pathway. Using this model, we have tested
the effect of macromolecular crowding upon the folding of 12 small protein domains
synthesized into a simulated cell.

Crowding macromolecules in the simulated cell were simulated using same-sized spheres up
to an excluded volume of 50%. As crowding is increased to experimentally observed levels
(20% to 40%), a slow decrease in native-like time, due to the stabilisation of non-native but
compact states, is observed. However, when the crowding level surpasses the maximum

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Jefferys et al. Page 6

observed experimentally, conformation size and conformational freedom change sharply and
native-like time drops substantially. This represents a level of crowding beyond that observed
experimentally, yet still far less than the maximum achievable by close packing spheres
(74%), leaving more than sufficient space for a protein to fold—especially given that the
modelled crowding macromolecules can move to accommodate the growing protein.
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The results presented here have shown the effect of folding upon the protein folding pathway
at a coarse level. The general finding that proteins fix upon a protein structure very much earlier
in an overcrowded cell, relative to uncrowded conditions, is not intended to be a prediction of
the level of conformational frustration but is indicative that this is a possible cause of the
reduction in successful protein folding at extreme crowding levels. The folding pathways
produced by the iterative process in our model have not been verified in detail; therefore, a
study of the effect of crowding upon such detail would be premature at this stage. However,
future work based upon such verification may reveal more about the specific cause of the effect
we have observed. In particular, the relative influence of two-state versus cooperative folding
processes under crowded conditions, as compared with proteins in pure water, may be
important.

The importance of crowding to cellular processes is clear. High levels of crowding are known
to increase protein aggregation, which can lead to cell death, and substantial evidence that
prevention of aggregation is a key factor in protein evolution exists. It is clear that reliable
protein folding is critical to the survival of the cell. Hence, factors that detrimentally affect this
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process are expected to be under strong selective pressure. This work suggests that crowding
above 40% excluded volume severely hinders folding. The agreement between this maximum
value of crowding tolerated in our simulation and that found experimentally suggests that the
ability to fold proteins reliably in the cell may be an important evolutionary constraint upon
the level of macromolecule concentration.

Macromolecular crowding is influenced by two key factors: protein size and expression level.
The well-established inverse relationship between these two factors is generally interpreted as
being due to evolutionary minimization of transcriptional and translational costs. However,
the results of this work suggest that this relationship may also be in part due to the balancing
of size and expression to maintain a crowding level that permits folding. This suggestion is
supported by the strong anti-correlation between expression level and propensity to
aggregate and by evidence that larger proteins are more prone to aggregation.

Crowding may have been controlled during evolution to allow the folding of small domains
without recourse to error-correcting mechanisms, such as chaperones and directed proteolysis.
Chaperones associate preferentially with larger domains, generally above 200–300 amino
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acids, suggesting that error-correcting mechanisms such as chaperones may have evolved in
part to enable crowding to increase beyond the level tolerated by larger domains.

This work demonstrates a theoretical model that unifies the often disjoint goals of protein
structure prediction and modelling folding dynamics, using it to study the effect of crowding
to a level of detail that would be difficult to achieve experimentally. Further development of
such a model can enable study of protein flexibility, complex formation, sequence design for
synthetic biology and disease-causing misfolding and aggregation.

Materials and Methods

Protein structure and force field
The model, known as poing, reduces a protein structure to Cα points plus side-chain
centroids. It models structures through iterative prediction of a folding pathway that enforces

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Jefferys et al. Page 7

a number of heuristic constraints representing effects important in protein folding. poing uses
the Langevin equation for the motion of a particle in the system:

(1)
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where a⃗ is the acceleration of the particle, F⃗ is the force field at the coordinates of the particle
(the sum of all forces acting on the particle), γ is a drag factor due to motion through the implicit
solvent, v⃗ is the current velocity of the particle, R⃗ is a random force vector designed to model
the effect of kicks from the implicit solvent and m is the mass of the particle. This equation is
solved iteratively. The force field consists of a set of pairwise force functions that act upon the
particles:

(2)

F⃗cov represents stiff springs linking backbone and side-chain particles that are directly
covalently bonded. F⃗bb represents springs between backbone particles with sequence
separations of 2 or 3, with a range of equilibrium lengths based on secondary structure assigned
to the relevant amino acids, either from secondary structure prediction (when used as a
predictive tool) or from knowledge of the native (when used to simulate folding pathways).
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F⃗sc represents springs linking side-chain particles to neighbouring backbone particles,

controlling the orientation of side chains relative to the backbone. F⃗vdw is a repulsive force
derived from the probability that atoms in an all-atom model of those particles at a given
distance apart would clash sterically, based upon analysis of side-chain and backbone
conformations in the Protein Data Bank. F⃗hb is an attractive force between backbone particles
designed to bring virtual backbone hydrogen bonding O and H atoms into closer proximity, if
they already come within a distance threshold.

Hybrid implicit–explicit solvent with hydrophobic effect

The standard Langevin has an implicit solvent model, with drag (−γv⃗) and kick (R⃗) terms in
the main equation. We have enhanced this by ensuring that drag and kicks only act upon parts
of a particle exposed to solvent, with solvent-accessible surfaces modelled by spheres around
each particle of a radius dependent upon the side-chain type. This ensures that the internal parts
of a protein are not subject to solvent effects, a key advantage of modelling an explicit solvent.
The solvent-accessible radii used have been optimised to maximise the difference in accessible
area between known native and a set of non-native states for a small test set of proteins, to
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destabilise non-native states.

The solvent kick model is modified from the normal Langevin to enable this process. At each
time step, a kick is initiated upon a particle with some probability per Å2 accessible surface
area. All kicks are of the same velocity. If the kick does not come from a direction that is
blocked by other particles, it is added to the acceleration for that particle. The probability of
kicks is increased for hydrophobic side-chain particles. This results in preferential burial of
hydrophobic residues away from solvent and therefore the hydrophobic collapse of a protein
molecule. This is the only effect of the hydrophobicity of a side chain in the model.

Structure prediction
Given a sequence of amino acids, the initial step is to predict the secondary structure using
psipred, a neural network-based secondary structure prediction tool that takes as input position-
specific scoring matrices derived from homologous sequences found with PSI-BLAST. The

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Jefferys et al. Page 8

accuracy of this method generally reduces where no homologue is found. A simple three-state
output (helix, extended/strand and coil) is used to assign secondary structure to the amino acids
in the poing model. The protein is then modelled in poing, for (6000l + 400,000) iterations,
where l is the number of amino acids in the protein. Forty structures are sampled at equal
intervals from iteration 6000l to the end of the simulation. As a computational shortcut to a
compact structure, the protein is slowly synthesized by adding an amino acid to the C-terminus
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of the protein every set of 1000 iterations and tethering this growing end to the edge of a large
heavy sphere representing a ribosome. This process is repeated 150 times with different random
seeds for the generation of kicks from solvent in the Langevin equation, producing many
folding trajectories. This produces the final pool of structures from which predictions are made.

Determination of structure predictions from generated conformations is performed in three

stages. First, a mean of the contact maps of the backbone particles of the pool of structures is
generated. The contact maps are calculated based upon backbone traces smoothed with a
window of nine amino acids. The contact maps disregard contacts between amino acids within
the same secondary structure unit. Different distance cutoffs are used depending on the
secondary structure: 8 Å between amino acids that are both assigned as strand and 11 Å in all
other cases. This reflects the fact that the backbones of hydrogen-bonded strands are closer
than other secondary structure elements. All 6000 structures are sorted according to their
similarity to this mean contact map—the contact map similarity score penalises a lack of contact
where there should be one and rewards the presence of a contact where there should be one.
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The top 20 structures selected by similarity to a consensus contact map are scored using
ProQ, a neural net-based structure scoring program trained to predict the MaxSub score of a
protein as compared with its (unknown) native state. If any of the top 5 structures picked by
ProQ are very similar to one another, the one with the lowest score is eliminated and the next
structure down is brought in to the top 5. Two proteins are judged to be very similar if more
than 90% of the residues can be aligned to less than 7 Å RMSD. This process is repeated until
the top 5 represents a range of the best structures produced by poing, or there are no more
structures.

Model for protein synthesis into a crowded cell

This is illustrated in Fig. 3. The model consists of a simulated protein (described above) and
crowding macromolecule spheres inside a containing sphere and a heavy ribosome with a
synthesis exit point located on its surface, with the exit point tethered to the centre of the
containing sphere. The ribosome, crowding macromolecules and containing sphere all move
iteratively under forces applied through the Langevin equation, including the solvent model.
Details of these elements are given below.
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In poing, the ribosome is modelled with a heavy sphere of radius 100 Å. All simulated particles
are excluded from this sphere. The mass is set to make the ribosome an essentially immovable
object. The protein emerges from an exit tunnel on the surface of a ribosome, to which it is
tethered: this is modelled by a particle attached to the surface of the ribosome. This particle
has the same mass as the ribosome in order to approximate the moment of inertia of the
ribosome as a whole. The backbone particle at the growing end of a protein is attached to this
particle. This set of strong springs and massive particles presents a major constraint to the
protein's freedom, which is designed to approximate the effect of the ribosome on protein
folding.

Protein synthesis is modelled by periodically adding backbone and side-chain particles to the
C-terminus chain at the position of the exit tunnel. The new backbone point is tethered to the
exit point, and the old backbone point to which it is attached is no longer tethered (however,
its location is still restricted by its backbone link to the new backbone particle). We used a

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Jefferys et al. Page 9

synthesis rate of one amino acid per 1000 time steps. This is an artificially fast synthesis rate,
relative to the rate of protein collapse in our model; however, short tests have found that slower
synthesis rates do not alter our observations. This relatively fast rate was chosen as a
computational optimisation.

Macromolecular crowding is designed to simulate the crowding of a protein by other proteins

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of a similar size. Previous work has suggested that the specific physicochemical properties of
the crowding macromolecules are not especially important and that the principal effect is
volume exclusion. It would be computationally prohibitive and unnecessary to crowd a protein
with other complete protein models if there is no requirement to simulate specific interactions.
Therefore, in poing, a crowding macromolecule is reduced to a large, heavy sphere. The
macromolecules crowding a protein are assumed to be of size similar to the protein being
crowded. The set of small domains used in this study is between 43 and 90 amino acids long.
All particles in the protein model have the same mass, and each amino acid has two particles
associated with it. This is simplified to a single crowding particle that models a 75-amino-acid
protein, of diameter 30 Å (the approximate size of a 75-amino-acid protein) and with mass 150
times that of a single particle. The entire system of particles (excluding the ribosome) is
contained within a sphere of diameter 100 Å.

The ribosome exit point is tied to the centre of the crowding containment sphere with a spring,
ensuring that the protein is synthesized into the centre of the crowding area. This ensures that
any observed effects upon folding from crowding are due to the crowding rather than boundary-
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specific effects that might be observed if the protein is synthesized at the boundary.

Appendix A Supplementary data

Refer to Web version on PubMed Central for supplementary material.

Appendix A Supplementary Data

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
This work was supported by the Wellcome Trust and the Biotechnology and Biological Sciences Research Council.
We thank Maxime Huvet and Mark Wass for valuable comments and suggestions.

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Fig. 1.
Structure prediction results. TM scores for the proteins are shown on the left. The shape and
colour indicate broad protein class: red circles are all-helical, blue squares are all-strand and
green diamonds are mixed. Twenty-four targets were predicted with structures better than 0.3
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in TM score, and four of those were predicted with structures better than 0.4 in TM score. For
comparison, we plotted the largest proportion of the protein that can be aligned to the native
at less than 5 Å RMSD on the right. The point shape and colour are as those for the graph on
the left.
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Fig. 2.
Best of five predictions and the best structure (by TM score) generated by the model during
structure prediction for an all-helical protein, a mixed protein and an all-sheet protein. Proteins
are shown coloured from red at the C-terminus to blue at the N-terminus, with arrows
representing strands and ribbons representing helices. The best TM score structure in the right-
hand column shows the best conformation, as compared with the known native structure, out
of several thousands of structures produced by 150 separate folding trajectories and therefore
does not represent a prediction but rather the theoretical best prediction that could be made by
poing. Images were generated using PyMOL.

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Fig. 3.
Illustration of model for synthesizing protein into a crowded container. The white curly lines
represent springs that hold together the ribosome, ribosome exit point and container and keep
particles inside the container. The ribosome is the large red circle (only partially visible), the
ribosome synthesis exit point is the small blue circle on the ribosome's surface, the crowding
macromolecules are in translucent gray and the protein is shown with colours going from red
at the C-terminus to purple at the N-terminus.

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Fig. 4.
A series of images showing the synthesis of a protein into a cell with 41% volume excluded
by macromolecules. Each image is approximately 10,000 time steps apart. The protein is shown
in cartoon form, with ribbons showing the assigned secondary structure. Colour scheme is as
that for Fig. 3, with translucent crowding macromolecules. Note that the protein is becoming
squeezed between the crowding macromolecules as it is synthesized. Images were generated
by poing, the protein model used for this work.
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Fig. 5.
Effects of crowding upon conformation size, conformational freedom and native-like time.
N = 1.2 million for each crowding level for all graphs, although structures produced during
simulation of a single folding trajectory are not strictly independent, and 1200 folding
trajectories were simulated. The illustrations at the bottom show how crowding changes the
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space remaining for the protein to fold. Colour scheme is as that for Fig. 3. All elements have
proportions designed to show the relative excluded volume resulting from crowding as relative
excluded area in two dimensions—for example, at 50% crowding, 50% of the area within the
container not occupied by the ribosome is occupied by macromolecules. (Row A) These
illustrate the space that remains for a successfully folded protein. Space is limited at 50% (c),
but there is still space for more proteins in the three-dimensional case. (Row B) These illustrate
the largest effect of crowding upon folding. At 14% (d), there is no measurable effect on the
folded protein. At 36% (e), proteins are starting to get trapped in non-native but compact
conformations. At 50% (f), proteins are becoming trapped in extended conformations.

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