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RESEARCH ARTICLEOpen Access

Modeling coding-sequence evolution within

the context of residue solvent accessibility

Michael P Scherrer, Austin G Meyer and Claus O Wilke*

Abstract

Background: Protein structure mediates site-specific patterns of sequence divergence. In particular, residues in the

core of a protein (solvent-inaccessible residues) tend to be more evolutionarily conserved than residues on the

surface (solvent-accessible residues).

Results: Here, we present a model of sequence evolution that explicitly accounts for the relative solvent accessibility

of each residue in a protein. Our model is a variant of the Goldman-Yang 1994 (GY94) model in which all model

parameters can be functions of the relative solvent accessibility (RSA) of a residue. We apply this model to a data set

comprised of nearly 600 yeast genes, and find that an evolutionary-rate ratio ω that varies linearly with RSA provides a

better model fit than an RSA-independent ω or an ω that is estimated separately in individual RSA bins. We further

show that the branch length t and the transition–transverion ratio κ also vary with RSA. The RSA-dependent GY94

model performs better than an RSA-dependent Muse-Gaut 1994 (MG94) model in which the synonymous and

non-synonymous rates individually are linear functions of RSA. Finally, protein core size affects the slope of the linear

relationship between ω and RSA, and gene expression level affects both the intercept and the slope.

Conclusions: Structure-aware models of sequence evolution provide a significantly better fit than traditional models

that neglect structure. The linear relationship between ω and RSA implies that genes are better characterized by their

ω slope and intercept than by just their mean ω.

Background

Substitution patterns in protein-coding genes are shaped

by the 3-dimensional structure of the expressed pro-

teins. To account for this influence of structure on

sequence evolution, evolutionary biologists increasingly

aim to combine sequence analysis with structural infor-

mation or to develop models of sequence evolution that

incorporate structural features of the expressed protein.

Some authors calculate amino-acid substitution matri-

ces as a function of protein structure [1,2] or correlate

sequence variability in alignments with structural features

[3,4]. Others subdivide proteins into broad categories by

solvent exposure (buried/exposed) or secondary structure

(α-helix, β-sheet, etc.) and then use standard maximum-

likelihood models of sequence evolution to infer evolu-

tionary rates as a function of structural features [5-9].

Some authors employ more complex methods that allow

*Correspondence: wilke@austin.utexas.edu

Center for Computational Biology and Bioinformatics, Institute for Cellular and

Molecular Biology, and Section of Integrative Biology The University of Texas at

Austin, Austin, TX 78712, USA

for non-independence among sites, and use energy func-

tions to model how substitutions at one site influence

substitutions at others [10-13]. Finally, a few groups have

attempted a variety of other approaches to link sequence

variability with protein structure [14-17].

These various analyses differ in their specific results

as well as in the approaches taken. However, one pat-

tern consistently emerges: Residues in the core of pro-

teins are more conserved than residues on the surface.

This finding agrees with our understanding of protein

biochemistry. Substitutions in the core of a protein are

more likely to disrupt fold stability than substitutions on

the surface, and the loss of the structural integrity of a

protein is frequently the underlying cause of loss of func-

tion [18,19]. Further, the observed relationship between

residue buriedness and evolutionary conservation seems

surprisingly simple. When evolutionary rate is plotted as

a function of relative solvent accessibility (RSA, a num-

ber between 0 and 1 measuring how exposed a residue

is to the solvent surrounding the protein), one finds a

near-perfect linear relationship [9,20].

© 2012 Scherrer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative

Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and

reproduction in any medium, provided the original work is properly cited.

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Inspired by the observed linear relationship between

evolutionary conservation and RSA, we here take the

standard Goldman-Yang model of coding-sequence evo-

lution (GY94, [21]) and introduce to it a dependency

of the model parameters on RSA. We find that the

RSA-dependent GY94 model provides a substantially bet-

ter fit to yeast sequence data than the standard, RSA-

independentmodel.Wefurtherfindthatforseveralmodel

parameters, a simple, linear dependency on RSA provides

the best fit. In particular, the ratio of non-synonymous

to synonymous evolutionary rates ω is a linear, increas-

ing function of RSA. Thus, we can characterize protein

evolutionary rates by the slope and intercept of the ω–

RSA relationship rather than by just a single ω value. We

show that slope and intercept of the ω–RSA relationship

vary among proteins with different structures or different

expression levels.

Results

An RSA-dependent Markov model of coding-sequence

evolution

Previous works assessing the relationship between evolu-

tionary rate and RSA subdivided sites into groups with

comparable RSA and then calculated evolutionary rates

separately for each group [9,20]. This approach yields a

setofindependentevolutionary-rateestimatesthatcanbe

plotted against representative RSA values for each group.

While this approach has provided valuable new insight,

it is not satisfactory from a methodological perspec-

tive. First, some model parameters (such as parameters

describing the nucleotide-level mutation process, e.g. the

transition–transversion bias) could be conserved among

groups. Yet they are estimated individually for each group.

Second, a consistent framework for hypothesis testing is

lacking. For example, in order to test whether evolution-

ary rates vary linearly with RSA, one would have to do a

regression analysis on the previously estimated rates. In

this regression analysis, sample size corresponds to the

number of RSA groups rather than to the number of sites

intheoriginaldataset.Consequently,theP valueresulting

from the regression would likely be incorrect.

To resolve these shortcomings, we developed a vari-

ant of the GY94 model [21] in which model parameters

are functions of RSA. We write the infinitesimal gener-

ator Q = (Qij) of the Markov process describing the

substitution process as (for i ?= j)

⎧

κ(r)πj, if synonymous transition

ω(r)πj, if nonsynonymous transversion

κ(r)ω(r)πj, if nonsynonymous transition

Qij=

⎪⎪⎪⎪⎪⎪⎨

⎪⎪⎪⎪⎪⎪⎩

0, if more than one nucleotide change

πj, if synonymous transversion

,

(1)

where κ is the ratio of transitions to transversions, ω is

the ratio of the nonsynonymous to synonymous substitu-

tion rates, and r stands for the RSA of a site. The indices

i and j run over all 61 sense codons, and πjis the fre-

quencyofcodonj.(Wedonotestimatesite-specificcodon

frequencies). The finite-time transition matrix is given by

P = exp[t(r)Q] ,

where t corresponds to evolutionary time, in arbitrary

units. The parameter t measures the branch length in

the phylogenetic tree; it is broadly related to the rate of

synonymoussubstitutions.Onfirstglance,itmightbesur-

prising that we allow t to vary with RSA. However, as

we will see below, models with site-dependent t fit the

data better than models with a single t across all sites.

The reason for the improved fit is that RSA influences

both amino-acid level processes and nucleotide-level pro-

cesses.

We implemented this model in the phylogenetic mod-

eling language HyPhy [22]. One problem we faced was

that HyPhy does not allow a continuous co-variable (such

as r) in the model matrix. To overcome this technical

problem, we binned RSA values into n bins and repre-

sented all RSA values within bin k by the bin mid-point,

which we denote by rk. In this way, we approximate

a single matrix Q(r) that changes continuously with

r by a set of n discrete matrices Qk= Q(rk), with

k = 1,...,n. HyPhy allows us to simultaneously fit mul-

tiple discrete matrices, and it also allows us to share

parameters among these matrices. In the limit of large

n, our discretized model converges to the model that is

continuous in r.

Our model contains three fitted parameters: ω(r), κ(r),

and t(r). For each parameter, we considered three types

of RSA dependency. First, a parameter can be constant,

i.e., not actually depend on RSA. In this case, we have

ω(r) = ω0, κ(r) = κ0, or t(r) = t0. Second, a parame-

ter can be a linear function of RSA. In this case, we have

ω(r) = ω0+ ω1r, κ(r) = κ0+ κ1r, or t(r) = t0+ t1r.

(But note that we actually use only n discrete RSA val-

ues rk, because of the binning procedure). Finally, we can

allow for separate ω, κ, and t values in each bin. (We refer

to this case as per-bin parameter estimation). In this case,

we fit n distinct ω values, one for each bin (which we refer

to as ωrk), and likewise for κ and t. Figure 1 illustrates

the various modeling choices for ω, κ, and t, in various

combinations.

(2)

A linear RSA ependency for all estimated parameters

provides the best model fit

We fitted our model to a data set of yeast sequences

with available structural information. We identified 587

Saccharomyces cerevisiae genes with known ortholog

in Saccharomyces paradoxus and with a representative

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A

B

C

Figure 1 Examples of RSA-dependent sequence-evolution

models considered. All models have three parameters, evolutionary-

rate ratio ω, branch length t, and transition–transversion ratio κ. All

three parameters can be estimated as an individual value within each

RSA bin (per-bin), as a linear function of RSA (linear), or as a constant

across all RSA values (constant). The examples here are illustrated for

n = 10 RSA bins. (A) All parameters are estimated per-bin. (B) ω is

estimated as linear function, t is estimated per-bin, and κ is estimated

as a constant. (C) All paramters are estimated as linear functions.

structure in the Protein Data Bank (PDB). We calculated

RSAforeachsiteasdescribed[7].Unlessnotedotherwise,

we used n = 20 evenly-spaced RSA bins.

Since we considered three different functional forms of

RSA dependence (constant, linear, and per-bin) for each

of the three parameters ω, κ, and t, we had 27 possi-

ble models. We fit all these models to our data set and

ranked them by their Akaike Information Criterion (AIC

[23,24]). Results for all models are shown in Table 1. The

top-scoring model was one in which ω and t depended

linearly on RSA while κ was estimated per bin. The dif-

ferences in AIC were quite substantial among models, and

the top-scoring model was clearly better than the next-

best model (in which all parameters were estimated as

linear functions).

In general, we found that all parameters varied signifi-

cantly with RSA. The top eight models did not contain a

single model in which even one parameter was constant

over RSA. This result shows that it is not sufficient to just

make ω a function of RSA, the transition–transversion

bias κ and the branch-length t also depend on RSA.

Among the models with constant parameters, models

with constant t ranked the highest. Models with constant

ω rankedconsistentlythelowest.Thisresulthighlightsthe

strongdependencyofamino-acidsubstitutionpatternson

RSA.

Whenever the transition–transversion bias κ was

allowed to vary with RSA, either linearly or per-bin, we

found that it generally had a negative slope (decreased

with increasing RSA). The branch length t tended to have

a positive slope (increased with increasing RSA), unless κ

was made constant, in which case t assumed a negative

slope (Table 1).

Figure 2 shows ω as a function of RSA as estimated

for the overall best model (with linear ω and t and per-

bin κ) and, for comparison, for the overall best model

with per-bin ω (with linear t and per-bin κ). We see

that the estimates from both models are highly con-

sistent with each other, and that the per-bin estimates

strongly support a linear relationship between ω and

RSA.

To assess the effect of the binning procedure on model

estimation, we re-fitted the fully linear model (with linear

ω, κ, and t) using different numbers of bins, from n = 4 to

n = 20. Parameter estimates were nearly independent of

n and varied smoothly in n (Table 2). We obtained similar

results when we used a model with linear ω and t and per-

bin κ (data not shown).

Surprisingly, the log-likelihood did not vary smoothly in

n(Table2).Forexample,weobservedtheoverallbestlike-

lihood score for n = 11, while n = 10 had a comparatively

poor likelihood score. We believe that the discontinuity

in likelihood scores was caused by aliasing issues. A site’s

RSA can be high or low relative to the range of RSA val-

ues within a bin. After a small change in the number

of bins (for example from n = 10 to n = 11), some

sites that previously had a relatively low RSA for their

bin will now have a relatively high RSA or vice versa. If

those sites are particularly variable or particularly con-

served, the change in their location relative to the bin

center can substantially affect the quality of the model

fit. For this reason, we do not think that it is reason-

able to select the number of bins based on the likelihood

score of the model. Instead, we opted for using a rela-

tively large bin number (n = 20), which more accurately

approximates a smooth dependency of model parameters

on RSA.

GY94 model provides a better model-fit than MG94 model

The GY94 model describes evolutionary rates using the

two parameters t and ω. An alternative model, the Muse–

Gaut model (MG94 [25]), uses instead the parameters α

and β. The parameter α in MG94 corresponds to t in

GY94 and the parameter β in MG94 corresponds to tω

in GY94. If we fit a model without site variability (all

parameters are constant across sites), the MG94 model

and the GY94 model are identical. However, when we

allow for site variability, the two models become dif-

ferent. The GY94 model is usually set up with a con-

stant t and a variable ω [26,27]. This set-up implicitly

assumes that the synonymous rate is constant across

sites whereas the nonsynonymous rate is variable. The

MG94 model, on the other hand, has been used to explic-

itly model both nonsynonymous and synonymous site

variability [28].

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Table 1 Fitted models, in order of ascending AIC

ω

t

κ

lnL df

AIC

t slope

κ slope

linear linear per-bin

−839713.86

−839736.74

−839701.37

−839722.37

−839723.27

−839707.75

−839710.08

−839694.42

−839757.23

−839740.64

−839742.62

−839727.25

−839825.99

−839809.70

−839817.06

−839800.41

−839867.98

−839856.43

−840468.84

−840459.99

−840479.14

−840524.57

−840697.41

−840688.35

−840738.77

−840740.37

−840726.86

24 1679476+

−

−

−

−

−

−

−

−

−

−

−

−

0

linear linearlinear6 1679485+

per-binlinear per-bin42 1679487+

per-bin linear linear24 1679493+

linearper-binlinear24 1679495+

linear per-binper-bin42 1679499+

per-bin per-binlinear 42 1679504+

per-bin per-bin per-bin60 1679509+

linear constant linear5 16795240

per-binconstantlinear 231679527+

linear constantper-bin 23 16795310

per-bin constantper-bin4116795370

linear linearconstant5 1679662

−

−

−

−

0

per-bin linearconstant2316796650

linearper-bin constant 2316796800

per-bin per-binconstant41 16796830

linearconstantconstant4 16797440

per-bin constantconstant 22167975700

constantlinearper-bin23 1680984+

−

−

−

−

0

constant per-binper-bin 411681002+

constantper-bin linear23 1681004+

constantlinearlinear5 1681059+

constantlinear constant4 1681403+

constantper-bin constant221681421+0

constantconstantlinear 416814860

−

0

constantconstantconstant

316814870

constantconstantper-bin22168149800

Here, we have allowed both ω and t to vary with RSA,

so we have considered both nonsynonymous and synony-

mous rate variation. However, in using the GY94 model,

we have assumed that the two quantities that vary linearly

with RSA are the synonymous rate and the ratio of the

nonsynonymous to synonymous rates. A priori, it is just

as reasonable to assume that the synonymous rate α and

the nonsynonymous rate β are linear functions of RSA. In

this case,the ratio ω = β/α would of course not be linear

in RSA.

To assess whether the nonsynonymous rate β or the

ratio ω = β/α is linear in RSA, we fitted a model in

which α and β were linear functions of RSA. (κ was

estimated per-bin). The resulting relationship of ω vs.

RSA was similar but not identical to the one observed

for linear ω (Figure 3). The log-likelihood score for this

model fit was −839720.75, compared to a log-likelihood

score of −839713.86 for the model with linear ω. The

two models are not nested, so we cannot compare them

using a likelihood ratio test. However, they are compara-

ble via AIC, and the model with linear ω was clearly better

(?AIC = 14).

Effect of relative solvent accessibility on synonymous and

nonsynonymous substitution rates

The previous subsections have shown that substitu-

tion rates at both synonymous and nonsynonymous

sites are affected by RSA, and that the ratio ω

dN/dS changes linearly with RSA. If ω is linear in

RSA and both dN and dS vary with RSA, then

we expect dN and dS individually to not be linear

in RSA.

The quantities dN and dS are not parameters that

are estimated in the model fit. Instead, they are derived

quantities that we can calculate once the model has

been fit to the data. One complication in calculating

=

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Relative Solvent Accessibility

dN/dS

0.000.250.500.75 1.00

0.00

0.03

0.06

0.09

0.12

0.15

0.18

Per−bin

Linear

Figure 2 Evolutionary-rate ratio increases linearly with RSA. The

solid line shows ω = dN/dS versus RSA as estimated by the best

model (linear ω, linear t, per-bin κ). The dots show the same for the

best model with per-bin ω (which has linear t and per-bin κ). Both

models are consistent with each other and strongly support a linear

relationship between ω and RSA.

dN and dS arises, however: There are multiple defini-

tions of these parameters. For example, dS is defined

as the number of synonymous differences divided by

the number of synonymous sites in the sequence. We

obtain the number of synonymous differences by sum-

ming over appropriate elements in the matrix Q [29]. The

number of synonymous sites can be obtained in two dif-

ferent ways. First, we can simply count the number of

sites atwhich a mutation would lead to a synonymous

change, using fractional counts for sites at which muta-

tions can cause either a synonymous or a nonsynonymous

change. This method of counting gives us the physical-

sites definition of dS [30]. Second, we can weigh each

site with the probability that a synonymous mutation will

occur at this site under the fitted model. This method

of counting sites gives us the mutational-opportunity

definition of dS [30]. The same two definitions exist

for dN.

The mutational-opportunity and the physical-sites def-

initions gave nearly identical results for dN (Figure 4A).

In both cases, dN showed a strong increasing trend with

RSA, with a slight deviation from linearity for higher RSA

values.Bycontrast,thetwodefinitionsgavesomewhatdif-

ferent results for dS. Under the mutational-opportunity

definition, dS was decreasing with RSA, whereas under

the physical-site definition it showed no obvious trend

(Figure 4B).

The effect of core size and expression level on evolutionary

rate

In yeast, the primary determinant of evolutionary rate

is gene expression level [31,32]. A second determinant

is protein structure, measured either by contact density

[7] or by core size [9]. Thus, we investigated how the

Table 2 Effect of the number of bins on parameter estimates

n

ω0

ω1

t0

t1

κ0

κ1

lnL

4 0.12050.01060.71102.4706 -2.54875.3465-839824.56

50.1208 0.01160.69672.4734 -2.59485.3547 -817178.82

6 0.1162 0.01350.70122.4828 -2.53615.3136-839781.41

7 0.1149 0.01430.7034 2.4849 -2.51025.2976-839764.54

8 0.11380.0148 0.7269 2.4805-2.5336 5.2996 -839760.69

9 0.1123 0.01540.7062 2.4900-2.48315.2759-835407.29

100.1129 0.0156 0.7020 2.4898-2.50035.2811 -839745.29

11 0.1132 0.01590.6742 2.4879-2.44975.2669 -797981.33

120.1119 0.0161 0.6706 2.5007-2.44515.2571 -837291.42

13 0.1110 0.01620.7114 2.4902 -2.48465.2703-836692.33

140.11080.0164 0.69562.5005 -2.46325.2532 -837806.63

150.1115 0.01640.6959 2.4941 -2.47595.2653-839684.07

160.11020.0167 0.7174 2.4897 -2.4858 5.2666 -839740.91

17 0.10980.01690.7146 2.4886-2.46095.2562 -835852.76

180.1097 0.01700.70742.4942-2.4652 5.2548-839148.15

19 0.11000.0169 0.70382.4937 -2.4785 5.2627-839318.45

200.1097 0.0171 0.7038 2.4943-2.4732 5.2592-839736.74

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Relative Solvent Accessibility

dN/dS

0.00 0.250.500.751.00

0.00

0.03

0.08

0.13

GY94

MG94

Figure 3 Comparison of the GY94 and the MG94 models. The

solid line shows ω = dN/dS versus RSA, as estimated by the GY94

model. The dashed line shows the same for the MG94 model. Under

the MG94 model, ω shows moderate curvature. The GY94 model

provides a better fit to the data (?AIC = 14).

slope and the intercept of the linear function ω = ω0+

ω1r changed with protein core size (measured by average

RSA) and with gene expression level (measured by mRNA

abundance).

Franzosa and Xia showed that the slope of ω changed

with core size while the intercept remained nearly

unchanged. We repeated their analysis by identifying

the proteins with the 33% largest and smallest cores

and fitting a joint evolutionary model to these pro-

teins. We fitted one line each for κ and t but fitted two

separate lines for ω, one for the large-core proteins

(ωlc= ωlc

teins (ωsc

=

We foundthat small-core

smaller slope than large-core proteins (ωsc

ωlc

icant (likelihood ratiotest,

contrast, the intercepts were not significantly differ-

ent (likelihood ratio test, P = 0.136), and we found

(ωsc

The two slopes we found were more similar to each

other than the ones found by Franzosa and Xia [9].

The main difference between our data set and theirs was

that we used more stringent criteria to match sequences

tostructures.Toverifythatwecouldreproducetheresults

of Ref. [9], we relaxed our criteria for alignment length

to 70%, thereby increasing our dataset to 870 sequence-

structure pairs. For this larger data set, we found a similar

slopeforlarge-coreproteinsasfoundbefore(ωlc

but the slope for small-core proteins was reduced (ωsc

0.058). These slopes were consistent with the findings of

Ref. [9].

We carried out a similar analysis on high-expression

and low-expression genes, fitting a separate line to each

group of proteins (ωhe

genes, ωle

1

We found a substantial difference in slope between these

two groups of genes (ωhe

1

The difference was significant (likelihood ratio test, P =

1.75 × 10−62). We also found a difference in intercept

(ωhe

0

significant as well (likelihood ratio test, P = 6.05 ×

10−12). Similar results were found when we used codon

adaptation index as a proxy for gene expression level

(data not shown).

0+ ωlc

1r) and one for the small-core pro-

ωsc

0

proteins

+ ωsc

1r), as shown in Figure 5.

displayed

1= 0.082 vs.

a

1= 0.127). This difference in slopes was signif-

P = 6.41 × 10−9).By

0= ωlc

0= 0.018).

1= 0.124),

1=

1= ωhe

0+ ωle

0+ ωhe

1r for low-expression genes).

1r for high-expression

=

ωle

= 0.047 vs. ωle

1

= 0.164).

= 0.011 vs. ωle

0= 0.023) and this difference was

Relative Solvent Accessibility

dS

0.000.25 0.500.75 1.00

0.00

0.06

0.12

0.18

0.24

rtunity

Physical Sites

A

B

Relative Solvent Accessibility

dN

0.00 0.25 0.50 0.75 1.00

0.000

0.006

0.012

0.018

0.024

Figure 4 Evolutionary rates dN and dS. (A) The nonsynonymous rate, dN, correlates strongly with RSA under both the mutational-opportunity

definition and the physical-sites definition. (B) The synonymous rate, dS, shows a moderate negative correlation with RSA under the

mutational-opportunity definition and no slope under the physical-sites definition. The fitted model had linear ω, linear t, and per-bin κ.

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A

B

Relative Solvent Accessibility

dN/dS

0.00 0.250.500.751.00

0.00

0.05

0.10

0.15

0.20

0.25

Large Core

Small Core

All Genes

Relative Solvent Accessibility

dN/dS

0.000.250.500.75 1.00

0.00

0.05

0.10

0.15

0.20

0.25

High Expression

Low Expression

All Genes

Figure 5 Dependency of ω = dN/dS on protein core size and expression level. (A) Core size affects evolutionary rate on the surface of the

protein but not in the core. (B) Expression level affects evolutionary rate both on the surface and in the core. However, it has a bigger effect on the

surface of the protein. In both figures, the solid lines were estimated jointly from the data using a linear dependency of ω on RSA. Points for

individual bins are shown for illustration purposes only. They were estimated using a per-bin model for ω. The dashed black line represents the

genome-wide trend, as shown in Figure 2, and is provided as a reference.

Finally, we carried out a joint analysis of core size and

expression level by extracting four groups of proteins

from our data set: proteins with (1) high expression level

and large core, (2) high expression level and small core,

(3) low expression level and large core, and (4) low

expression level and small core. Figure 6 shows the result-

ing model fit. Clearly, expression level plays a larger role

in determining evolutionary rate than core size. However,

the model with core-size-dependent slope showed a bet-

ter fit than a model in which the slope depended only on

Relative Solvent Accessibility

dN/dS

0.000.25 0.500.751.00

0.00

0.05

0.10

0.15

0.20

Small

Small

Large

Large

High

Low

Figure 6 Joint analysis of the effects of both core size (small or

large) and expression level (low or high) on the relationship

between ω = dN/dS and RSA. Only the fitted lines are shown.

Surprisingly, for low-expression genes, small-core proteins evolve

faster than larger-core proteins. This relationship is reversed in a larger

dataset obtained with less-stringent criteria (see text).

expression level (likelihood ratio test, P = 5.33 × 10−4).

Surprisingly, the effect of core size on slope was reversed

for high- and low-expression genes. For high-expression

genes, proteins with larger core size showed a larger slope

in ω than did proteins with smaller core size, consistent

with prior results. By contrast, low-expression proteins

with larger core size showed a smaller slope than did pro-

teins with smaller core size. However, this unexpected

pattern disappeared when we repeated the above analy-

sis on our expanded data set with 870 sequence-structure

pairs. There, the large-core-size proteins had the larger

slope in all cases, consistent with prior results (data not

shown).

Discussion

We have developed a method that models the evolution-

ary rate of a coding sequence within the context of the

protein’s 3-dimensional structure. Our method is a sim-

ple extension of the standard GY94 model, modified such

that all parameters are functions of relative solvent acces-

sibility (RSA). We have found that the evolutionary-rate

ratio ω = dN/dS, the branch length t, and the transition–

transversion bias κ all depend on RSA. The overall best

fitting model had a linear relationship of ω and t with

RSA, while κ showed small deviations from strict linear-

ity. In the second-best model, all parameters had a linear

relationship with RSA.

Our method presents a unified statistical framework for

comparing RSA-dependent model parameters among dif-

ferent groups of proteins. Using this framework, we have

shown that protein core size affects only the slope of ω as

a function of RSA, but not the intercept. The most buried

residues have—on average—the same ω value regardless

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of protein core size. By contrast, expression level affects ω

even for the most buried residues.

We have found that the variation in ω with RSA is

substantial; for the most exposed residues, ω was on aver-

age 5-10 times larger than it was for the most buried

residues. This observation highlights the importance of

incorporating protein structure into models of coding-

sequence evolution. Traditional models of rate varia-

tion [27,29,33] cannot distinguish between rate variation

caused by protein structure and rate variation caused

by other factors (e.g., positive or negative selection on

sites of functional importance). As an obvious exten-

sion to the work presented here, we can combine the

present model with more traditional models of rate vari-

ation by allowing for additional rate variation among

sites with similar RSA. This work will be presented

elsewhere [34].

Our findings here are broadly consistent with the find-

ingsofFranzosaandXia[9].Wehaveconfirmed thelinear

relationshipbetweendN/dS andRSAinanindependently

derived data set; we have also confirmed that proteins

with larger core size show a faster increase of dN/dS

with increasing RSA than proteins with smaller core size.

Our work goes beyond Franzosa and Xia’s findings by

demonstrating that the evolutionary rate of fully buried

residues is independent of protein core size, that expres-

sion level affects evolutionary rate at all RSA values, and

that the GY94 model provides a better model fit than the

MG94modelwhenRSA-dependentevolutionaryratesare

considered. Our work also suggests that nucleotide-level

processes vary systematically with protein structure.

In our joint analysis of core size and expression level,

we made the unexpected observation that the effect of

core size on the slope of ω is reversed for genes with low

expression level. However, this observation disappeared

in a larger data set obtained under slightly less strin-

gent criteria for matching sequences to PDB structures.

We can offer no good explanation for this observation.

It could be a statistical fluke. The number of genes in

each of the four groups (low expression and small core

size, low expression and large core size, high expression

and small core size, high expression and large core size)

is relatively small in this analysis, so a few unusual pro-

teins could skew the analysis. What exactly is the cause

of this unexpected observation may have to be clarified in

future analyses, either using expanded data sets—as more

structures become available—or using data from different

organisms.

Our approach is conceptually related to other recent

works attempting to combine protein structure with

sequence evolution [10-13]. These works imposed struc-

tural constraints on sequence evolution via sophisticated

energy functions describing how protein fold stability

changes as amino acids are replaced. In comparison, our

approach is much more simplistic. However, we believe

that this simplicity has substantial benefits. First, our

approach is simple and fast. All the models we have

used here can be fit within 10–15 minutes on an off-

the-shelf laptop. Second, our approach yields results that

can be interpreted easily. Instead of a single ω value per

gene, we obtain two values, an intercept and a slope.

The intercept tells us to what extent selection constrains

the most buried residues; the slope tells us by how

much selection relaxes as we move towards more exposed

residues. Third, our approach can be implemented with

relative ease in existing modeling frameworks such

as HyPhy [22].

Following Franzosa and Xia [9], we used a model that

fit a single rate ratio ω, regardless of which amino acids

were substituted into which other ones. A recent study

has shown that such models can always be improved

upon with amino-acid dependent transition rates, even if

aminoacidsaregroupedintoexchangeabilitycategoriesat

random [35]. This finding is not entirely surprising, con-

sidering that amino-acid substitution matrices have con-

sistentlybeenfoundtodependsubstantiallyontheamino-

acid identity (e.g. Refs. [36-38]). Therefore, it would be

desirable to develop codon-level substitution models that

accurately capture this rate variation, without adding too

many additional parameters. Approaches that have been

suggested include automatically grouping amino acids

into exchangeability categories [39,40] and decompos-

ing amino-acid substitution rates into components corre-

sponding to biophysical properties of amino acids (LCAP

model, Ref. [41]). Yet substitution rates also depend on

protein structure [1,2,6,42], and thus one would want to

incorporatestructureintothesemodelsaswell.Onestudy

developed a variant of the LCAP model where parame-

ters were fit separately to buried and exposed sites and

found to be significantly different [17]. Since we have seen

here that substitution rates seem to depend continuously

(and linearly) on RSA, it might be worth it to investigate

a variant of the LCAP model in which rate parameters

are linear functions of RSA. Such a model would have

the same number of parameters as the model in Ref. [17]

but would quite possibly provide a better fit to the data.

Alternatively, one could attempt to incorporate an RSA-

dependence into models that automatically group amino

acids [39,40].

We found that in our model, both t and κ varied with

RSA. We believe that this finding reflects the effect of

selection on nucleotide-level processes. First, equilibrium

amino-acid frequencies vary with RSA [20,43], and this

variation will have some effect on equilibrium codon fre-

quencies. Second, protein structure also seems to exert

a direct selection pressure on synonymous codon choice

[44-50], most likely through an interaction between the

translation process and protein folding. A more realistic

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model could represent this relationship between protein

structure and the nucleotide-level substitution process

more accurately, for example via a structure-dependent

variant of the FMutSel model [51] or by extending models

such as the LCAP model [17,41] to contain structure-

dependent terms for nucleotide-level processes.

The challenge in developing any such models will be to

make them realistic yet sufficiently simple so they can be

fit to moderately sized data sets. An alternative, simpler

strategy could be to calculate equilibrium codon frequen-

cies in an RSA-dependent manner. We considered calcu-

lating codon frequencies per bin and found that doing so

generally improved AIC scores but did not eliminate the

need for RSA-dependent t or κ, nor did it alter any of our

other results in a substantive way (not shown).

Our method requires a solved crystal structure to cal-

culate RSA values. Although the Protein Data Bank (PDB)

has been growing rapidly over the past decade, the num-

ber of available structures is still small compared to the

number of available sequences. For example, many of the

yeast sequences we used in our analysis did not have a

corresponding structure. For those sequences, we relied

onhomologousproteinstructuressolvedinrelatedorgan-

ism. Homology mapping performs relatively well in pre-

dicting relative solvent accessibility [49] but clearly it is

not perfect. Further, certain proteins or regions of pro-

teins, such as membrane proteins or intrinsically disor-

dered regions, can usually not be crystalized. Thus, our

method cannot be applied to such proteins or regions of

proteins.

Our method assumes that RSA remains constant

throughout evolution. Yet every amino-acid replacement

will cause some distortion in the protein structure [52],

and RSA values at homologous sites will slowly diverge

with increasing sequence divergence [49]. In the future, if

either the number of available PDB structures increases

drastically or if atom-level computational modeling of

protein structures becomes sufficiently reliable, we will

able to study how changes in structure correlate with

evolutionary rate.

Conclusions

Our work has shown that the evolutionary rate ratio ω,

the branch length t, and the transition–transversion bias

κ all vary significantly with relative solvent accessibility

(RSA). All three parameters show an approximately linear

RSA dependency. In general, both the slope and inter-

cept of the ω–RSA dependency differ according to the

specifics of individual genes, such as protein structure and

geneexpressionlevel.Ourworkdemonstratesthatprotein

structure can be an important ingredient in comparative

sequenceanalysis.Ourworkfurthersuggeststhatatighter

integration of structural and sequence data will improve

the performance of comparative analysis methods.

Methods

Homology mapping and categorization of genes

In order to construct a large data set of sequences

with corresponding structures, we obtained open read-

ing frames (ORFs) of the yeast Saccharomyces cerevisiae

from the Saccharomyces Genome Database [53] and

aligned them with orthologous Saccharomyces paradoxus

sequences using MUSCLE [54]. Each ORF was translated

and searched against the Protein Data Bank (PDB) [55]

using the PSI-BLAST algorithm [56] and then paired with

the structural chain with the lowest alignment E-value. To

ensure that enough of the yeast protein was represented

in the chain and that the PDB structure was a reasonable

homology model, we only considered pairs with > 80%

alignment length and > 40% sequence identity for analy-

sis. Our final data set had 587 sequence–structure pairs.

A data set with relaxed criteria used > 70% alignment

length and > 40% sequence identity. This data set had 870

sequence–structure pairs.

The percent solvent-accessible surface area (ASA) for

each aligned residue was calculated using DSSP [57]. We

obtained relative solvent accessibility (RSA) by normal-

izing ASA values with the surface areas of an extended

Gly-X-Gly peptide [58].

Calculation of evolutionary rates

The codons from the yeast alignments were binned by

the RSA value of their respective residues, as described

[9]. Protein core size was estimated by the average RSA

value over all residues in a protein. We considered a struc-

ture to have a large core if its average RSA ranked within

the bottom third of all average RSA values and to have

a small core if ranked within the top third of all average

RSA values [9]. Yeast expression data measured in mRNA

abundance per cell was obtained from [59]. Codon adap-

tation index (CAI), a measure of the strength of codon

usage bias, was used as an alternative for expression level,

since the latter may be biased by laboratory growth con-

ditions of the yeast cells [60]. Both expression level and

CAIwererankedanddividedintothirdswiththetopthird

representing high-expression genes and the bottom third

low-expression genes.

We implemented the model described by Eq. (1) in the

HyPhy batch language [22]. We estimated codon frequen-

cies (πj) using F3×4 model.

We calculated synonymous (dS) and nonsynonymous

(dN) substitution rates according to the mutational-

opportunity andthe physical-sites

described [29,30].

definitions,as

Statistical analysis

WeusedtheAkaikeinformationcriterion(AIC)[23,24]to

rank models by their quality of fit. For pairwise compari-

son of nested models, we also carried out likelihood-ratio

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tests. All statistical analyses were performed using the

statistics software R [61].

Abbreviations

AIC: Akaike information criterion; CAI: Codon adaptation index; GY94:

Goldman-Yang 1994; HyPhy: Hypothesis testing using Phylogenies (software);

MG94: Muse-Gaut 1994; ORF: Open reading frame; PDB: Protein data bank;

RSA: Relative solvent accessibility.

Authors’ contributions

MPS collected data, developed HyPhy batch files, ran analyses, prepared

figures, and wrote the manuscript. AGM developed HyPhy batch files. COW

conceived of the study, participated in its design and coordination, and wrote

the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by NIH grant R01 GM088344, NSF grant EF-0742373,

and NSF Cooperative Agreement No. DBI-0939454.

Received: 11 April 2012 Accepted: 3 September 2012

Published: 12 September 2012

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doi:10.1186/1471-2148-12-179

Cite this article as: Scherrer et al.: Modeling coding-sequence evolution

within the context of residue solvent accessibility. BMCEvolutionaryBiology

2012 12:179.

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