Cysteine-Cysteine Contact Preference Leads to Target-Focusing in
Mihaela E. Sardiu,*zMargaret S. Cheung,yand Yi-Kuo Yuz
*Stowers Institute for Medical Research, Kansas City, Missouri;yDepartment of Physics, University of Houston, Houston, Texas;
andzNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland
interactions. We include in the analysis only tertiary contacts, because they are less constrained—compared to secondary
contacts—by proteins’ backbone rigidity. Using proteins from the protein data bank, our analysis reveals an unusually high fre-
quency of cysteine pairings relative to that expected from random. To elucidate the possible effects of cysteine interactions in
folding, we perform molecular simulations on three cysteine-rich proteins. In particular, we investigate the difference in folding
dynamics between a G? o-like model (where attraction only occurs between amino acids forming a native contact) and a variant
model (where attraction between any two cysteines is introduced to mimic the formation/dissociation of native/nonnative di-
sulfide bonds). We find that when attraction among cysteines is nonspecific and comparable to a solvent-averaged interaction,
they produce a target-focusing effect that expedites folding of cysteine-rich proteins as a result of a reduction of conformational
search space. In addition, the target-focusing effect also helps reduce glassiness by lowering activation energy barriers and
kinetic frustration in the system. The concept of target-focusing also provides a qualitative understanding of a correlation be-
tween the rates of protein folding and parameters such as contact order and total contact distance.
We perform a statistical analysis of amino-acid contacts to investigate possible preferences of amino-acid
Important tasks in a cell are mostly carried out by proteins.
Given a cellular environment, the linear arrangement of
amino acids in a protein determines its native structure and
there is a characteristic time for this protein to reach its native
state to conduct biological functions (1,2). However, despite
decades of investigations in the research community, it still
remains a challenge to use only the knowledge of the primary
amino-acid sequence to either predict the relationship be-
tween structure and function or justify a characteristic fold-
ing time. In this regard, using bioinformatics approaches to
extract information from protein structure database can be
useful in investigation of functional roles of particular con-
tact pairs in a protein. Using this strategy, several groups
(3–5) have identified—using computational/experimental
interactions. Here we follow these paths to perform a tertiary
amino-acid contact analysis and find an unusually high
contact frequency among cysteines. We further extend this
information into molecular dynamics (MD) simulations, at
least qualitatively, to investigate how protein dynamics in
living systems may possibly benefit from cysteine-cysteine
The importance of disulfide bond formation in protein
folding has been discussed (6,7). It was argued that disulfide
bonds may enhance thermal stability of many disulfide-rich
proteins. For instance, Mallick et al. (5) recently showed that
the intracellular proteins of hyperthermophilic archea are
disulfide-rich. Further, one may ask whether there could be
other interesting roles for a cysteine pair to play. In this
article, we investigate whether the cysteine-cysteine interac-
tion can promote folding.
In terms of protein folding, disulfide bonds can be helpful
if they form at a correct folding nucleus (4). On the other
hand, nonnative disulfide bonds, if formed, can also hinder
the folding. Nevertheless, in a radical environment such as in
living systems, the bonds can form and break frequently at a
biological timescale (8) if the bonding energy between a pair
of cysteines is of order kBT. This somewhat fast exchange
rate makes it possible to offset a potentially detrimental con-
sequence of misfolded proteins as a result of forming non-
native disulfide pairs. In this article, we investigate the
possibility for such frequent formations and dissociation of
disulfide bonds to assist protein folding at least in generic
Given the difficulties encountered in the pursuit of ac-
curate quantification of the interactions among amino acids,
many studies in this area use either statistics-based energetics
or structure-specific energetics. A classic example of statistics-
based potential is the Miyazawa-Jernigan interaction matrix
(3,9). Inevitably, many specific features such as orientational
dependence of the interactions and side-chain contact/packing
are averaged out. The G? o model (10) is a representative of
structure-specific models. Basically, given a protein p and
its native structure S(p), the force between two amino acids
in p isattractive(repulsive)iftheirthree-dimensionalsepara-
tion in S(p) is within (outside) the so-called contact distance.
An extra criterion needed for the two amino acids to be
attractive is when no other amino acids stand between them.
Aiming to provide a minimally frustrated folding energy
landscape (11,12), the G? o potential has been commonly used
Submitted September 20, 2006, and accepted for publication April 3, 2007.
Address reprint requests to M. E. Sardiu, E-mail: email@example.com.
Editor: Angel E. Garcia.
? 2007 by the Biophysical Society
938Biophysical JournalVolume 93 August 2007938–951
in protein folding simulations (13,14). Structure-specific
potentials, however, lack the generality of the fundamental
physical forces. Apparently, there is a trade-off between
strengths and limitations in both types of approaches.
Since our goal is to examine the generic effect of disulfide
bond formation/dissociation on folding, we would like to
employ a model that can fold protein within reasonable com-
putational time. Designed for minimal frustration, the G? o
model is known to quickly fold many proteins under MD
simulations and is thus chosen as our starting model (wild-
type model). A variant model that includes the nonspecific
cysteine interactions can be readily constructed. We simply
replace specific interactions between any cysteine pairs in a
protein sequence with nonspecific attraction while leaving
the rest of the pairwise interactions G? o-like. Intuitively, if
the cysteine-cysteine attraction is much larger than solvent-
averaged interactions between any two amino acids, nonna-
tive contacts formed among cysteines will introduce energetic
traps and the folding kinetics will be hindered. However, we
find that if this nonspecific interaction is comparable to a
solvent-averaged interaction, it helps proteins fold even faster
than a standard G? o model.
This interesting finding prompts us to seek the possible
with .100 amino acids) to efficiently find their equilibrium
conformations. A useful concept, termed ‘‘target-focusing,’’
is therefore introduced to elucidate, at least qualitatively, a
plausible mechanism. The targets refer to monomer (e.g.,
amino acid) pairs whose effective mutual attractions are
stronger than others. When the effective attraction is not too
strong, the interacting targets on the polymer will loosely
focusing helps reduce the size of search space that a hetero-
polymer needs to explore before reaching its equilibrium
In addition to search space reduction, target-focusing also
enables a related feature: reduction of glassiness in folding.
This phenomenon, resulting from lowering kinetic frustra-
tion and activation energy barriers, is analyzed in Results and
Discussion. In the same section, we also further describe how
the target-focusing concept may help us to understand the
observed correlation between protein folding rate and other
parameters such as contact order (15) and total contact dis-
MODELS AND METHODS
Pairwise tertiary contact analysis
From the Protein DataBank (PDB), we downloaded 4143 proteins (12,455
chains in total) with known three-dimensional structures. Because a protein
may contain several chains (subunits), the number of chains is much larger
than the number of proteins. To avoid overrepresentation of almost identical
chains, we retain only one chain among highly similar chains. Using a score
threshold of 200 bits, this procedure is done by ‘‘purge,’’ a preprocessing
program of Gibbs motif sampling (17). After purging, the remaining 4142
proteins (5398 chains) are used for the contact analysis.
Compared to the contacts formed within a secondary structure, tertiary
contacts among amino acids are less constrained by peptide backbone
rigidity. Tertiary contact analysis is thus expected to provide information
less relevant to secondary assembly within proteins but perhaps more rel-
evant to proteins’ tertiary assembly. In our analysis, a contact is defined
plainly. Excluding the case when they are in the same secondary structure
unit, two residues (amino acids) i and j (i, j ¼ 1, 2, ..., 20), are considered in
contact if the distance between their Cais smaller than a cutoff (7 A˚) and if
these two residues are separated by more than two amino acids in the pri-
mary sequence. This contact analysis is also useful in other applications such
as multiple sequence alignment.
To quantify the tertiary contact preference between secondary structures
of the same type, we first estimate the joint probability of contact involving
amino acids i and j by
Qi;j¼ Ci;j= +
where Ci,jis the number of contacts found between amino acids i and j while
+i9$j9Ci9;j9sums the total number of contacts. The likelihood for an amino
acid i to participate in a tertiary contact is estimated by the secondary-
structure-specific background frequencies
where Cicounts amino acid i in one type of secondary structure. When con-
sidering contacts resulting from different types of secondary structures, Ciin
Eq. 2 sums the counts of amino acid i in both types of secondary structures.
For a pair of amino acids i and j, the ratio of Qi,j(the observed contact
frequency) to pipj(the expected contact frequency by chance),
quantifies the preference of residue contacts. The relative error d associated
Ri,jcan be estimated by 1=Ci;j
a-helices and among b-sheets, the 10 most preferred tertiary contact pairs
are given in Table 1. The tertiary contacts resulting from different secondary
structures are much less from our analysis of PDB data. Due to insufficient
sample size, we refrain ourselves from showing those numbers in Table 1.
Nonetheless, the major feature, such as the cysteine-pair ranks among top
probability ratios, remains the same.
It is natural to ask how the probability ratios change when we change the
cutoff distanceused. Figs. 1–3 provide suchinformation with cutoff distance
ranging from 5 A˚to 8 A˚. Note that in both Fig. 1 and Fig. 2, the change of
cutoff distance has little effect on the dominant pairs, indicating the gen-
erality of conclusions drawn.
The overwhelming preference for cysteine-cysteine contact indicates that
if a protein contains cysteines, the cysteines tend to be close in the folded
: For contacts among
TABLE 1 Top probability ratios
16.55 6 2.02
3.51 6 0.38
4.86 6 0.19
3.35 6 0.24
4.05 6 0.22
3.40 6 0.37
4.20 6 0.19
3.08 6 0.48
3.70 6 0.57
3.05 6 0.41
36.95 6 3.05
4.91 6 0.29
C-W V-IF-W V-F
8.38 6 1.27
4.91 6 0.45
7.54 6 0.30
4.83 6 0.57
6.02 6 0.65
4.82 6 0.49
4.99 6 0.27
4.5 6 0.20
The top 10 probability ratios in contacts formed between various secondary
Violation of the FDT in a Protein System939
Biophysical Journal 93(3) 938–951
structure of the protein. It is this observation that motivates our study of the
role of cysteine contacts in the folding of cysteine-rich proteins.
It is worth noting that in our analysis the cysteine-cysteine pairing ratio is
larger than observed in other analyses (3,9), where they actually documented
otheramino-acid pairs tohave larger pairingratiosthan the cysteine-cysteine
pair. We attribute this difference to the fact that we use only tertiary contacts
while the other analyses include secondary contacts. As we mentioned
earlier, the tertiary contact is less constrained by the backbone rigidity than
the secondary contacts, thus they may be able to better reflect, albeit statis-
tically, the intrinsic interaction strengths among various amino acids.
We first choose cysteine-rich proteins whose PDB files include the keyword
SSBOND. We screen proteins based on the following criteria: 1), proteins
with structures determined by x-ray crystallography but not solely deter-
mined by NMR; and 2), each protein must contain at least two pairs of
cysteine-cysteine contacts in its native structure. Table 2 lists some details of
three proteins selected: hen egg-white lysozyme (1AT5), Ustilago maydis
killer toxin kp6 a-subunit (1KP6), and bovine pancreatic ribonuclease A
(7RSA). The structures of these three proteins are displayed in Fig. 4.
We use a simple G? o model (10) where each amino acid is represented by
its Caatom (13). The local structural Hamiltonian includes the regular bond-
stretching, bond-rotation, bond-angle, and dihedral rotation terms describing
the backbone deformation energy. For the pairwise interaction between two
residues i and j separated by distance r ¼ jr ~i? r ~jj; the potential is given by
Here r0is the contact distance between the G? o-pair residues i and j in native
structure, and s0is a parameter with dimensions of length. The G? o-type
pairwise interaction is aimed to minimize energetic frustration, and thus is
often expected to fold proteins the fastest. We call the model with this G? o-
like potential the wild-type (wt).
As suggested by our tertiary contact analysis, we introduce unbiased
interactions among all the cysteine residues in place of the G? o-like potential
to produce a variant model. Precisely, cysteine mand cysteinen separated by
distance r will have potential energy
Vm;n¼ e 5
regardless of whether m and n form a G? o pair or not. The energy parameter e
is allowed to vary from 2e0to 20e0to parameterize the strength of disulfide
bond formation. Small e mimics an environment that is more reducing for
disulfide bonds. The distance parameter r0(m, n) is defined as follows. When
cysteines m and n form a G? o pair in the native structure, the native distance
between these two cysteines has two equivalent names: RN(m) and RN(n),
with RN(m) ¼ RN(n), of course. In this case, the quantity r0(m, n) is defined
to be RN(m), which is also equal to RN(n). We then assume that cysteine m,
influenced by its nearby amino acids, would contribute a preferred bonding
length RN(m)/2 while bonding to any another cysteine. This is a reasonable,
albeit ad hoc,extrapolation fromthe originalG? omodel. Consequently, when
cysteine m and cysteine n do not form a G? o pair, r0(m, n) is chosen to be
(RN(m) 1 RN(n))/2.
For thermodynamic simulations,we employa standardmolecular simulation
method using AMBER6 program as an integrator (18). Descriptions of para-
meters and time steps can be found elsewhere (14). Thermodynamic prop-
erties, such as folding temperatures (Tf), are calculated by the weighted
A˚increment. Thus, panel A summarizes the results for using 5 A˚as the cutoff distance, while panel G summarizes the results for using 8 A˚as the cutoff distance.
940Sardiu et al.
Biophysical Journal 93(3) 938–951
histogram analysis method (19). To study the kinetic effect of nonspecific
cysteine-cysteine attraction in each protein, we employ Langevin dynamics
(20) to simulate folding of both the wt and the variant.
For folding kinetics studies, initial structures are quenched at a high
temperature (2.8 Tf). To avoid overrepresentation of similar initial config-
urations, we accept a new initial configuration only if the root mean-square
distance (RMSD) between the new one and every existing one is larger than
a phenomenological cutoff ;1:17
acids in the protein considered. The idea here is to approximate the con-
formation of a denatured protein by that of a Gaussian chain. Since the
gyration radius is proportional to the square root of the length of the chain,
the natural length scale to discriminate two denatured states is proportional
: The numerical factor 1.17 A˚associated with the RMSD cutoff is
chosen, after manually looking into many configurations differed by various
RMSDs, to ensure that any two initial configurations are sufficiently distinct.
We generated 100 initial configurations for each protein studied.
To minimize the errors due to biased sampling in initial configurations,
for each protein studied we dictate both the wt and the variant to use the
same set of initial configurations and the same temperature Tsfor Langevin
dynamics simulations. Tsis 0.9Tf, 0.9Tf, and 0.8Tffor 1AT5, 1KP6, and
7RSA, respectively. At Ts, the optimal folding temperature, folding rate
reaches maximum for each wt model. The folding time in a simulation run is
defined by a first passage time: when the potential energy first becomes
lower than a threshold Ecutand all the native pairs of cysteines are formed.
Loosely speaking, if the potential energy is lower than Ecut, it means that the
current configuration and the lowest energy configuration shares .90%
similarity in terms of amino-acid contacts.
˚A; where nAis the number of amino
Contact formation analysis
It was suggested that the cysteine-cysteine contacts, native or not, may play
an important role in the folding of cysteine-rich proteins. For example, there
exists phenomenological theory (21) that attempts to explain folding of
cysteine-rich proteins considering only interactions among cysteines. For
each protein, we analyze contact formation in all cysteine pairs to investigate
the importance of individual cysteine pairs at various stages of the folding.
For each starting configuration s in the MD simulations, one may follow
its time evolution and define the t-dependent contact percentage, averaged
over a window size W, between two cysteines i and j as
ij? jr ~iðt1nÞ ? r ~jðt1nÞjÞ;
Heaviside step function taking value 1 if x $ 0 and value 0 otherwise, and
r ~iðtÞ is the position vector of cysteine i at time t in a MD simulation. This
running average reveals which contact pairs are formed at various stages of
Taking the window size W to be the folding time for each of the folded
trajectories, we may further calculate
ijis the native distance between cysteine i and cysteine j, u(x) is the
a:c:ði;j;t ¼ 1Þæ[ð1=NfoldedÞ +
a:c:ði;j;t ¼ 1Þ;
which is the contact percentage averaged over the folded ensemble and
abbreviated by Æpa.c.æ.
Moreover, we investigate how nonspecific cysteine interactions influence
the contact between cysteines and other noncysteine residues. To investigate
this effect, we monitor individual MD trajectories with special focus on
cysteines and the noncysteine residues with the largest number of native
contacts. For the ithamino acid wialong the primary sequence, we define its
kinetic radius r(wi) to be the largest residue-residue separation among all G? o
pairs containing wi. All the amino acids, whose Caatoms are within distance
r(wi) of wiin the native structures, are divided into two sets: those that form
is given by the heat map. In the alphabetical order of the panels, from A to G, we display the probability ratios with different cutoff distances ranging from 5 A˚
to 8 A˚with a 0.5 A˚increment. Thus, panel A summarizes the results for using 5 A˚as the cutoff distance, while panel G summarizes the results for using 8 A˚as
the cutoff distance.
The probability ratios (explained in pairwise tertiary contact analysis) for tertiary contacts among b-sheet secondary structures. The number key
Violation of the FDT in a Protein System941
Biophysical Journal 93(3) 938–951
native G? o contacts with wiand those that do not. The number of residues in
the first set defines the expected contact numbers (ECN) of native kind,
while the number in the second set defines ECN of nonnative kind. The
deviations from ECN (DFECN) indicate whether the local region associated
with a certain residue is crowded by native (nonnative) contacts or not.
RESULTS AND DISCUSSION
For the ease of referencing, we summarize all the abbrevi-
ations employed in this article in Table 3 before discussing
Folding rates and folding kinetics
For small to intermediate attraction strength 0.25 , e , 2
(see Eq. 5), we find that the folding rates of the variants
are larger than those of the wt. This behavior dramatically
changes (data not shown), as expected, once the amplitude
of nonspecific attraction becomes very large (e . 10). The
effect, due to the nonnative cysteine-cysteine attraction, on
the folding of a protein is studied in detail using an attraction
strength that is approximately two-times the solvent-averaged
energy in the G? o model (wt) used. Fig. 5 shows the percent-
ages of not-yet-folded (NYF) trajectories versus time steps
for the three proteins studied.
We plot the percentage of NYF trajectories at Ts(defined
in Models and Methods) versus time. As shown in Fig. 5, A
and B, for proteins 1AT5 and 1KP6, the folding kinetics is
largely characterized by single-transition-state behavior (the
percentage of NYF trajectories is exponential in time Pnot yet
folded(t) ; exp(?t/t)). However, the folding kinetics also
exhibits a power-law tail Pnotyetfoldedðt ? 1Þ;t?a(insets,
Fig. 5, A and B) at large time, indicating the possibility of
glassy kinetics. For the wt case of protein 7RSA, the per-
centage of NYF trajectories is almost purely power-law.
When the nonspecific cysteine attraction is used, we see an
increase in the number of data points characterizable by a
single transition state. However, the majority of the points
still fall in the realm that is characterizable by power law (see
inset, Fig. 5 C). The large time kinetics, being closer to a
power law than a single exponential, does indicate the pos-
sibility of glassy kinetics. However, we must emphasize that
what we meant by glassiness here is in a broad sense. For
example, a system with a large number of intermediate traps
of energies not much higher than that of the ground state will
TABLE 2 Three cysteine-rich proteins selected
identifier No. A.A. No. Cys. Native cysteine-cysteine contact pairs
(6,127) (30,115) (64,80) (76,94)
(5,12) (16,74) (18,65) (35,51)
(26,84) (40,95) (58,110) (65,72)
contacts between helices and sheets. The number key is given by the heat map. In the alphabetical order of the panels, from A to G, we display the probability
ratios with different cutoff distances ranging from 5 A˚to 8 A˚with a 0.5 A˚increment. Thus, panel A summarizes the results for using 5 A˚as the cutoff distance,
while panel G summarizes the results for using 8 A˚as the cutoff distance.
The probability ratios (explained in Pairwise Tertiary Contact Analysis) for tertiary contacts formed between different secondary structures, i.e.,
942 Sardiu et al.
Biophysical Journal 93(3) 938–951
be termed glassy in our definition. Therefore, systems that
are kinetically frustrated by many potential traps will fall in
this broad definition of glass. Kinetic frustration analysis will
be made in the next subsection, followed by more discus-
sions regarding other alternative explanations for the non-
exponential kinetics as well as glassiness analysis.
Compared to its wt, the variant either reaches 100% fold-
ing within shorter simulation steps (1AT5) or enjoys a higher
overall folded percentage (1KP6 and 7RSA) within the same
maximum simulation steps. This result, documented in Table
4, demonstrates a special role played by cysteine contacts in
protein folding kinetics. The MD simulation of 7RSA fold-
ing shows that a simple, minimally frustrated model is not
enough to be rid of glassiness (in a broad sense) in the fold-
Contact formation analysis and kinetic frustration
The analysis of folding rates substantiates the importance of
cysteine-cysteine contacts in protein folding. It is possible
that both the native and nonnative cysteine contacts con-
tribute positively to folding. We therefore analyzed contact
formation of all cysteine pairs (four native and 24 nonnative)
for each of the three proteins to investigate the importance of
individual cysteine pairs at various stages of folding.
Fig. 6 A shows how nonspecific cysteine-cysteine inter-
actions may facilitate the folding of protein 1AT5. Two
folding trajectories, one for the wt and one for the variant,
with identical initial configuration are used. DFECN is com-
puted for noncysteine residue 53 that has the largest number
of native G? o contacts and for residue 94 that is a cysteine.
Fig. 6, A(a) and A(b), shows DFECN of native contacts and
nonnative contacts for the wt, respectively; Fig. 6, A(c) and
A(d), show DFECN of native contacts and nonnative con-
tacts, respectively, for the variant. The variant folds faster
of the nonnative kind for the variant.
Also shown in Fig. 6 B is another example, where foldings
of the variant reached the native state but the wt did not, at
least up to the maximum simulation time (i.e., 30 3 106time
steps). The legends of Fig. 6 B(a) to 6 B(d) are the same as
those of Fig. 6 A(a) to 6 A(d). For the wt, DFECN of both
native and nonnative contacts for residue 53 is large and
negative (Fig. 6 B(a,b)) while DFECN of nonnative contacts
for residue 94 is frequently positive (Fig. 6 B(b)). It indicates
that for the wt, native contact pairing to 53 is deficient and
contact pairing to 94 is overwhelmed by nonnative ones.
Such conformations form kinetic traps that impede folding.
However, when nonspecific attraction between residue 94
and other cysteines is introduced, it helps to circumvent such
kinetic traps. First, the number of native contact pairing to
residue 53 increases (Fig. 6 B(c)). Second, the overwhelming
number of nonnative contact pairing to residue 94 decreases.
Consequently, the variant reaches the native state in a much
shorter time. Similar analyses for the other two proteins,
namely 1KP6 and 7RSA, can be found in Figs. 7 and 8 and
In Table 5, we document Æpa.c.æ, the contact percentage
averaged over the folded ensemble, for all cysteine pairs and
seek qualitative connection to experimentally observed data.
For protein 1AT5, the nonnative Cys64-Cys76pair has
highest contact percentage (;70.60% in wt and 77.40% in
its variant). The other higher contact percentages come from
the native cysteine pairs Cys64-Cys80and Cys76-Cys94.
Interestingly, almost 30 years ago Anderson and Wetlaufer
(22) suggested that two disulfide bonds involving Cys64-
Cys80and Cys76-Cys94formed earlier than the pairs Cys6-
Cys127and Cys30-Cys115in the folding of hen lysozyme.
Further, Shioi et al. (23) suggested that the preferential
formation of Cys64-Cys76might facilitate the formation of
Cys64-Cys80and Cys76-Cys94. Upon introducing the non-
specific attraction among cysteines, we see a significant in-
crease in contact percentage for all three pairs: Cys64-Cys76,
Cys64-Cys80, Cys76-Cys94. The essential features of our re-
sults agree reasonably well with the experimental observa-
tions, indicating the important role of cysteine contacts in
For protein 1KP6, the very high contact percentage for
nonnative pair Cys16-Cys18might be an artifact due to their
closeness in the primary structure. The nonspecific attraction
among cysteines again increases the contact percentage of
native pairs but decreases that of Cys16-Cys51, a nonnative
Bank, of the three proteins studies. Displayed from left to right are: the hen
egg-white lysozyme (1AT5), U. maydis killer toxin kp6 a-subunit (1KP6),
andbovinepancreatic ribonucleaseA(7RSA).Whilethe bulkof the proteins
are in ribbon (b-strand) and cylinder (a-helix) representations, cysteine
residues are shown using bond representation.
The native structures, downloaded from the Protein Data-
TABLE 3 Abbreviation summary
Expected contact number
Deviation from ECN
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Biophysical Journal 93(3) 938–951
For protein 7RSA, the nonspecific attraction among
cysteines again increases the contact percentages of native
cysteine pairs. The native pair Cys65-Cys72has the highest
contact percentage. Among other native pairs, the contact
percentages of pairs Cys26-Cys84and Cys40-Cys95in the
variant increase significantly when compared to their wt
counterparts. From the structural point of view, the Cys40-
Cys95pair increases the protein core stability. For other pairs
with high contact percentages, interesting comparisons to
experiments may also be made. Shin et al. (24) showed that
the pair Cys65-Cys72occurs in the early stage of folding.
Further, Klink et al. (25) suggested that Cys26-Cys84is very
important to conformational stability. These experimental
evidences lend support to the generic features of our results.
Nonexponential kinetics, glassiness,
and barrier height
As we have show in the insets of Fig. 5, at long time all the
protein models seem to display nonexponential kinetics, at
least not describable by a single exponential. Although the
late time kinetics data displayed seem to be easily charac-
terized by a power law, indicating possible glassiness, we
should first examine other alternative models that are known
to exhibit nonexponential kinetics before firmly dwelling on
the idea of glassiness.
It has been observed that small proteins may exhibit
fast but noncooperative folding that display nonexponential
kinetics. Basically, in this type of process, it is believed that
proteins will take trajectories strictly downhill in the free
energy landscape, even though the downhill folding ensem-
ble may consist of folding paths of different converging
speed toward the native state (36). The question is: could this
be the case in our minimally frustrated protein model? If
folding is entirely downhill in a free-energy sense and free of
glassy traps before reaching the native state, then the en-
semble of intermediate structures becomes progressively
more nativelike, indicating a reduction of entropy. Conse-
quently, the energy gradient must completely overcome the
entropy loss (37,38) to maintain the downhill folding in the
free energy landscape. That said, for glassiness-free downhill
folding, the energy itself, compared to free energy, must be
an even steeper downhill toward native state for each tra-
jectory. It turns out that testing this possibility is quite
straightforward. We have randomly picked a few folding
trajectories whose folding time fall in the range that is de-
scribable by power law. We found no evidence of glassiness-
free downhill folding. This is shown in Fig. 9. As we can see
from the two examples, typical energy variation over 1500
time steps is much smaller than the typical energy difference
between nearby spikes and troughs in the figure. This indi-
cates that the roughness in energy versus time cannot be
attributed to stochastic noise and the scenario of glassiness-
free downhill folding seems unlikely in the protein models
proteinsconsidered.Note that the percentageof NYFis alwaysplottedin log
scale while the time step is plotted in linear scale in the figure but in log scale
in the insets. The exponents’ a-values are obtained by fitting the power law
in the insets. Both the a-values and the inverse characteristic timescales
t?1are given in Table 4.
Percentage of NYF trajectories versus time for the three
944Sardiu et al.
Biophysical Journal 93(3) 938–951
The other possibility would be to use multiexponential
instead of a single exponential in describing the folding
kinetics. However, we also need to remember that any power
points (100for 1AT5 for bothwt and variant,53for 7RSA wt,
and 63 for 7RSA variant), we limit ourselves to triple
exponential (which already contains six free parameters as
Fig. 10, A and B, replot, respectively, the data for protein
models associated with 7RSA and 1AT5. Theoretically
speaking, a triple-exponential fitting should take the form
PnotyetfoldedðtÞ ¼ +
1 1 ? +
with Ai$ 0, ti. 0 " i and +2
free parameters, we still cannot get any decent fit even for the
wt models. For better fitting, we therefore modify Eq. 7 to
to allow one more free parameter t0. This modified triple
exponential is only shown for wt protein models since it still
does not fit the variant to any reasonable extent. However,
power-law tails are fitted for both wt and variant models.
Relevant fitting parameters are given in the figure caption.
Although the triple-exponential fit for 7RSA wt model
shown seems reasonably good, we have noticed that the third
exponential (with t3¼ 5 3 1049and (1 – A1– A2) ? 0.4314)
essentially is a constant over the range plotted. That is, if we
allow those NYF trajectories to continue, extrapolating the
triple-exponential fit will rule out the possibility for any of
them to fold. Any appreciable folding event can only occur at
another 1048time steps. This essentially means that there will
be a large portion of denatured configurations that will never,
in any realistic number of time steps, fold into the native
state, contradicting the fundamental reason of introducing
multiexponential fit instead of adopting the glassiness
picture. Fig. 10 B shows the fitting results for protein model
1AT5. In this case, it is apparent that the triple-exponential fit
does not fit as well as the power law. After examining two
alternatives, we now proceed to examine the possibility of
i¼1Ai#1: However, with five
PnotyetfoldedðtÞ ¼ +
Aiexpð?ðt ? t0Þ=tiÞ
1 ? +
expð?ðt ? t0Þ=t3Þ
primary sequence is rigid and in fact acts like quenched
disorder within the relevant temperature range for protein
folding. The folding of a protein thus bears similarity to
ground state formation in glass systems (26–30). The type of
glassiness associated with protein folding, also termed struc-
glass is formed (31). Despite the seemingly difference be-
tween structural glasses and spin glasses, many experimental/
protein problems. In particular, a hierarchical structure in
energy (similar to ultrametric structure) has been observed
(26) in myoglobin of 153 amino acids. One important char-
acteristic of a glassy system is the existence of many nearly
degenerate ground states, which have been shown to exhibit
ultrametric topology (32) and whose relaxation dynamics
have been modeled and studied in detail (33).
A G? o-like potential, in some way, is designed to minimize
the glassiness of the protein model by minimizing the en-
ergetic frustration. The insets in Figs. 5 and 10, however,
suggest that the tail of the percentage of NYF trajectories is
still characteristic of a power law. If we assume that the en-
ergetic frustration of structural glasses is largely similar to
that of the regular spin glasses, as suggested by several
studies (26–28), then the interesting study in Ogielski and
Stein (33) will suggest that the percentage of NYF trajec-
tories at large time t ? 1 behaves as
with T being temperature, D being the activation energy
barrier, and d the number of neighboring states that are
separated by an energy barrier D from one another. In
comparison to the power-law behavior of Pnot yet folded(t) ;
t?aat large time t, we find a } 1/D. For each protein, the MD
simulations of both the wt and the variant are performed at
the same temperature, the optimal folding temperature Tsof
the wt. The ratio
reveals the change in the activation barrier.
The a-values in Table 4 suggest that nonspecific attraction
among cysteines increases the protein-folding rate by low-
ering the activation energy barriers. Further, an interesting
observation now becomes obvious. Despite the power-law
TABLE 4Summary of kinetics and glassiness analysis
MS (3 108) wt/variant
t?1(3 10?6) wt/variant
a wt/variantTotal folded % wt/variant
0.23 6 0.02/0.32 6 0.03
0.038 6 0.002/0.055 6 0.003
NA/0.23 6 0.02
0.90 6 0.08/1.32 6 0.09
0.42 6 0.04/0.56 6 0.05
0.20 6 0.02/0.23 6 0.02
The maximum number of simulation steps (MS), folding rate 1/t, power-law exponent a, and overall folded percentage of the three selected cysteine-rich
proteins. The 1/t entry for 7RSA in wt is not available because of the lack of sufficient data points to make a reliable estimate.
Violation of the FDT in a Protein System 945
Biophysical Journal 93(3) 938–951
kinetics, the glassy picture actually suggests a larger
probability of folding at long time than suggested by triple-
exponential fitting. It is possible that in the context of G? o
model and the variant model, the level of glassiness may in-
crease as the size of the protein increases.
THE TARGET-FOCUSING CONCEPT
Nonspecific attraction among all cysteines creates apparent
energetic frustration in an otherwise G? o-like protein model.
How can the frustrated proteins (variant) actually fold more
effectively than the less frustrated proteins (wt) even at the
optimal folding temperature Tsof the wt? It is commonly
postulated that a foldable protein should have TF=TG? 1;
i.e., the glass transition temperature TGis much lower than
the protein-folding temperature TF, making glassiness less
important at the relevant temperature range. Our simulations,
however, indicate the existence of nonnegligible glassiness
even when using the least frustrated protein model simulated
(DFECN) versus integration time steps for protein 1AT5.
DFECN is computed for noncysteine residue 53 that has
the largest number of native G? o contacts and for residue 94
that is a cysteine. Panels A(a) and A(b) show DFECNs of
native contacts and nonnative contacts, respectively, from
a folding trajectory of a wild-type protein; and panels A(c)
and A(d) show DFECNs of native contacts and nonnative
contacts, respectively, from another folding trajectory of
the variant. The same initial structure is given for both the
wild-type and the variant in folding simulations, and the
variant folds faster than the wild-type. In addition, another
set of folding simulations (B(a)–B(d)) is given to show that
nonspecific cysteine-cysteine interactions facilitate fold-
ing. Particularly in this case, the wild-type trajectory did
not reach the native state within the maximum folding time
(i.e., 30 3 106time steps). However, the variant did. The
legends of B(a)–B(d) are the same as those of panels A(a)–
A(d). DFECN of native contacts associated with 53 is large
and negative in panel B(a) while DFECN of nonnative
contacts associated with 94 became frequently positive in
panel B(b). It indicates that for a wild-type protein, contact
pairing to 53 is far from nativelike, and contact pairing to
94 is overwhelmed by nonnativeones. Such conformations
form kinetic traps that impede folding (B(a) and B(b)).
However, when the nonspecific attraction among cysteines
is introduced (i.e., variant B(c) and B(d)), it helps in
circumventing such kinetic traps and allows the variant
model to reach the native state in a much shorter time.
DFECN is averaged over a window size of W ¼ 1.5 3 105.
Deviation from expected contact numbers
946 Sardiu et al.
Biophysical Journal 93(3) 938–951
Nonspecific attraction among cysteines, once introduced,
seems to be able to alleviate glassiness in folding. We found
that this nonspecific attraction does induce a qualitative
change in folding behavior of the three cysteine-rich proteins
studied, namely, 1AT5, 1KP6, and 7RSA. Not only do they
fold faster, all three proteins have at least one nonnative
cysteine pair that shows a higher percentage in contact for-
mation than one of native cysteine pairs. These results
(DFECN) versus integration time steps of protein 1KP6. In
general, the variant model folds faster than the wild-type.
DFECNs of residue 61 (which has the most content of
native G? o contacts) and residue 5 (a cysteine) are plotted.
Using the same initial configurations, we run MD simu-
lations for the wt model and for the variant model. Panel A
shows the DFECN of native kind of the wt; panel B shows
the DFECN of nonnative kind of the wt; panel C shows the
DFECN of native kind of the variant; and panel D shows
the DFECN of nonnative kind of the variant. In essence,
slow folders usually suffer more frequent kinetic frustra-
tion compared to the fast folders.
Deviation from expected contact number
(DFECN)versus integrationtime stepsof protein 7RSA.In
general, the variant model folds significantlyfaster than the
wild-type. DFECNs of residue 6 (which has the most
content of native G? o contacts) and residue 72 (a cysteine)
are plotted. Using the same initial configurations, we run
MD simulations for the wt model and for the variant
model. Panel A shows the DFECN of native kind of the wt;
panel B shows the DFECN of nonnative kind of the wt;
panel C shows the DFECN of native kind of the variant;
and panel D shows the DFECN of nonnative kind of the
variant. DFECN is averaged over a window size of W ¼
1.5 3 105.
Deviation from expected contact number
Violation of the FDT in a Protein System947
Biophysical Journal 93(3) 938–951
suggest a concept, we termed ‘‘target-focusing’’, as far as
folding of a large protein is concerned.
What we meant by target-focusing is actually rather
simple. Basically, the nonspecific attraction among cysteines
tends to bring cysteines closer and thus reduce the available
phase space of the peptide segment in between cysteines.
When all the cysteine pairs formed are those in the native
structure, the remaining trial space for noncysteine mono-
mers is greatly reduced. When incorrect cysteine pairs are
formed, the same reduction of phase space also turns out to
be useful in reducing the basin of trapping. Therefore, we
believe the native cysteine pairs (primary targets) are focused
through the nonspecific attraction among cysteines. This
effect is pertinent to the folding mechanism of large, cysteine-
rich proteins where the system bears glassiness as mentioned
However, one may also ask whether the same effect,
within the protein models we studied, can be easily produced
by choosing a different amino-acid pair to have a nonspecific
attractive potential (see Eq. 5). To answer that, it is natural to
seek an alternative amino-acid pair to introduce the nonspe-
cific interaction in one of our studied protein models. We
therefore apply the tertiary contact analysis to a single pro-
tein 1AT5. As expected, one should anticipate a much larger
statistical fluctuation since the sample size is now very small.
We find that cysteine-cysteine pair, mainly due to a larger
cysteine count, no longer has significantly larger probability
ratio than others. There are 21 other pairs with larger
probability ratios than the cysteine pair. We then randomly
pick a methionine-tryptophan (MW) pair, with probability
ratio only slightly larger than that of the cysteine pair. In
1AT5, there are eight cysteines, two methionines, and six
tryptophans. Starting from G? o-like pairwise potential, we
construct a new variant model for protein 1AT5 by replacing
the G? o-like pairwise potential for each MW pair with non-
specific attractive potential. We study how differently the
new variant behaves from our previous studies.
Interestingly, the MW mutant folds much slower than the
wt. It exhibits a glassy behavior, as shown in Fig. 11.Folding
of protein 1AT5 did not benefit from the addition of non-
specific MW attraction. This result indicates that cysteines in
fact do play the target roles in cysteine-rich proteins and it
seems nontrivial to find other alternatives. We should also
point out that in our study the addition of nonnative cysteine
interaction is based on the current database rather than on
randomly chosen pairs (34). The nonspecific cysteine at-
traction may have an effect in terms of native state stability in
the context of the G? o model. However, to study such an
TABLE 5 Contact formation analysis for all cysteine pairs
Cysteine pairWild-type Æpa.c.æ
Cysteine pairWild-type Æpa.c.æ
Cysteine pairWild-type Æpa.c.æ
The contact percentage of each pair is first calculated for each folded trajectory and then averaged over all folded trajectories to yield Æpa.c.æ. In addition to the
native cysteine pairs, we also highlight, in boldface type, all the Æpa.c.æ values that are .15%.
948Sardiu et al.
Biophysical Journal 93(3) 938–951
effect is beyond the scope of the current article. Generically
speaking, the native state stability may be studied in terms of
denaturing processes. In terms of folding process, enhanced
native state stability may, in principle, increase the chance of
pulling the protein conformation to be near its native state.
We cannot, and probably should not, rule out this possibil-
ity. However, if we were to believe that faster folding is
solely due to enhanced native state stability, we immediately
learn from studying the MW pair a nontrivial lesson: despite
the apparent lowering of contact energy in native state, not
all the nonspecific attraction can increase the native state
It is likely that the phenomenon of target-focusing can also
be present in many other proteins. However, identification of
the targets is most likely more difficult than that in the
cysteine-rich proteins. Nevertheless, Table 1 suggests other
amino-acid pairs—such as F-C and F-W—as generic target
candidates. To test those new target candidates, however,
one needs to select protein models based on the abundance of
fitting. The plots are shown in log-log scale. For visual clarity, we have
divided the time steps associated with the variant models by a factor of two,
resulting in a parallel shift to the left for all the variant models. (A) We plot
the percentage of NYF trajectories versus simulation time steps for protein
model 7RSA wt and 7RSA variant. At large time range, both the wt and
variant are well fitted by power law. The wt is also fitted by triple
exponentials with coefficients (see Eq. 8) given by t0¼ 3.92 3 106, A1¼
0.3515, A2¼ 0.21711,t1¼ 1.2277 3 107, t2¼ 9.5925 3 107, andt3¼ 5 3
1049. Although triple exponential seems a reasonable fit in the data range
displayed, the largeness of t3seems to contradict the purpose of triple-
exponential fitting (see text for detail). (B) We plot the percentage of NYF
trajectories versus simulation time steps. The best triple-exponential fitting,
withparameterst0¼ 1.7713 106,A1¼ 2.193 10?5,A2¼ 0.933,t1¼ 2.013
106, t2¼ 4.12 3 106, and t3¼ 5 3 1042, apparently does not fit the large
time part. However, the large time regions for both the wt and the variant are
well fitted by a power law.
Comparison of triple-exponential fitting and power-law
the energy versus time of a slow folding trajectoryfrom protein model 7RSA
wt; the folding time of this trajectory is within the range describable by
power law. Panel B plots the energy versus time of a slow folding trajectory
from protein model 1AT5 wt; the folding time of this trajectory again is
within the range describable by power law. These typical energy versus time
plots do not show any clear descending trend in energy and thus do not lend
support to the glassiness-free down-hill folding scenario. In particular, the
typical energy differences, 2.9 and 3.4 units for 7RSA wt and 1AT5 wt,
respectively, over a time interval of 1500 time steps for both trajectories are
approximately one order-of-magnitude smaller than their respective peak-to-
Energy versus time step for wt protein models. Panel A plots
Violation of the FDT in a Protein System949
Biophysical Journal 93(3) 938–951
those target pairs, just as we studied the cysteine target pair
using cysteine-rich proteins. Thus, to study the effect of F-C
pair, one may need to choose proteins containing more F-C
contacts in its native structure.
Some additional support for the generality of the target-
focusing effect in protein folding is obtained from the studies
(16,35) on the statistically significant correlation between a
protein’s folding rate and its contact order (CO) (15) or its
total contact distance (TCD) (16). For any given protein,
both CO and TCD are proportional to
where nAdenotes the total number of amino acids of the
protein, ncdenotes the total number of native contacts, dk
denotes the separation on the primary sequence between the
two residues that form contact k, and lcutdenotes the cutoff
separation on primary sequence. Qualitatively speaking, F is
larger when the protein chain has a more complex/tangled
topology (e.g., when native contacts are mainly formed by
residues that are far apart on the primary sequences). In an
average sense, a larger F therefore indicates a larger con-
formational barrier for the two amino acids of any target to
form contact. When this is the case, the folding slows down
because the power of target-focusing is weakened. The ob-
servation made in Plaxco et al. (35)—some mutations that do
not significantly alter CO still affect folding rates—can also
be understood using the target-focusing idea. Even if the
mutation does not affect the CO defined in Plaxco et al. (15),
the folding rate can still have a nonnegligible change if the
mutation does affect the targets.
From a bioinformatics study of tertiary contact, we have
identified, along with other groups, that cysteine-cysteine
contacts have a frequency much higher than expected by ran-
dom pairing. Using molecular simulations, we investigate
the effects of nonspecific cysteine attraction on the course of
folding. Using three cysteine-rich protein models that are
larger than a typical fast folding protein (e.g., containing
,100 amino acids), we have found that an addition of non-
specific interactions can help promote folding and reduce
glassiness of a protein. We come forward with the ‘‘target-
focusing’’ concept, in which an addition of nonspecific inter-
actions from evolutionarily selected contact pairs will help a
large protein fold more efficiently. This is because interac-
tions among ‘‘targets’’ are able to collectively reduce the
search space of other nontarget monomers. Consequently,
the effective time spent by a protein to search in conforma-
tional space to reach its native state is reduced.
Finally, as a cautionary note, one must acknowledge that
the concept of target-focusing cannot enhance the prediction
of how proteins fold given their primary structures unless
(primary) targets can be identified via correct characteriza-
tion of molecular interactions. Nevertheless, the notion of
target-focusing may still be useful in analyzing protein
evolution or even in protein design.
We thank Dr. John Wootton for valuable discussions, and Dr. David
Landsman and Dr. Steve Bryant for helpful comments. M.E.S. thanks
Dr. Timothy Doerr for constant help during the course of this study.
M.S.C. thanks the University of Houston for a new faculty start-up fund
and A. P. Sloan Foundation for a postdoctoral fellowship while visiting the
University of Maryland. We also thank the administrative group of the
National Institutes of Health Biowulf clusters, where all the computational
tasks were carried out. This work was partially supported by the Intramural
Research Program of the National Library of Medicine at National Institutes
of Health/Department of Health and Human Services.
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