doi:10.1006/jmbi.2001.5226 available online at http://www.idealibrary.com on J. Mol. Biol.

(2002) 315, 479±484

Why Are Proteins So Robust To Site Mutations?

Darin M. Taverna1 and Richard A. Goldstein2*
1
Biophysics Research Division
and
2 with little change in structure, stability, or function. These results are
Department of Chemistry
University of Michigan, Ann
Arbor, MI 48109-1055, USA
*Corresponding author
There have been repeated observations that proteins are surprisingly
robust to site mutations, enduring signi®cant numbers of substitutions
almost paradoxical in light of what is known about random heteropoly-
mers and the sensitivity of their properties to seemingly trivial mutations.
To address this discrepancy, the preservation of biological protein prop-
erties in the presence of mutation has been interpreted as indicating the
independence of selective pressure on such properties. Such results also
lead to the prediction that de novo protein design should be relatively
easy, in contrast to what is observed. Here, we use a computational
model with lattice proteins to demonstrate how this robustness can result
from population dynamics during the evolutionary process. As a result,
sequence plasticity may be a characteristic of evolutionarily derived pro-
teins and not necessarily a property of designed proteins. This suggests
that this robustness must be re-interpreted in evolutionary terms, and
has consequences for our understanding of both in vivo and in vitro
protein evolution.
# 2002 Academic Press
Keywords: site substitution; mutagenesis; molecular evaluation; protein
stability; protein folding

Introduction site substitutions are destabilizing, many result in

There has been much interest in probing the
relationship between a protein's sequence and its
resulting structural, thermodynamic, and func-
tional properties. It is hoped that insights resulting
from these pursuits will lead to the ability to pre-
dict protein properties based on sequence infor-
mation as well as how these properties could be
altered by changes in the sequence. Such insights
are also crucial in developing the ability to design
proteins with prescribed or altered structures, stab-
ilities, and functionalities.
One of the major methods of investigating the
relationship of protein sequence to the correspond-
ing properties is to alter naturally occuring pro-
teins through site mutagenesis. Often the
substitution is chosen so as to modify a speci®c
interaction, although more exhaustive and random
substitutions have been studied. One of the sur-
prising results of such studies is the robustness of
protein properties to mutations. Although most
Present address: D. Taverna, Protein Pathways, Inc.,
1145 Gayley Ave., Suite 304, Los Angeles, CA 90024,
USA.
E-mail address of the corresponding author:
richard@umich.edu
essentially unchanged stabilities, and a signi®cant
fraction of mutations actually result in increased
stability over the wild type (e.g. see Reddy et al.1).
The conclusion drawn from these studies is that
there is an inherent robustness to the mapping of
sequence to structure, and that sequence space con-
sists of large regions of possible sequences corre-
sponding to proteins with essentially equivalent
properties. The general level of sequence plasticity
has also led researchers to conclude that the robust
properties must not be under active selection
during evolution.2 This plasticity provides opti-
mism for de novo protein design, in that it indicates
that there are large numbers of amino acid
sequences consistent with a given stable structure;
the ability of a protein to fold despite changed
interactions means that the interactions do not
have to be formulated precisely in advance. Protein
design may correspond to ®nding a needle in a
haystack, but at least it is a quite sizable needle.
In contrast, de novo engineering of well-packed
proteins has proven ``surprisingly dif®cult''.3 Why
does the sequence plasticity observed in site-
directed mutagenesis not translate into ease in pro-
tein engineering? Perhaps we are interpreting the
results of these mutagenesis experiments in the
wrong context. Proteins are the result of a long

0022-2836/02/030479±6 $35.00/0 # 2002 Academic Press

480
evolutionary process, involving the dynamics of a
population cluster, or pseudospecies, in sequence
space. During the past few years we (among
others) have looked at how population-based evol-
utionary dynamics can alter the structures, func-
tions, and thermodynamics of proteins and other
biological macromolecules.4 ± 10
Here, we use a simple computational model to
demonstrate how population dynamics can explain
why proteins are so robust to changes in sequence.
We describe simulations of the evolution of lattice
proteins, using two different models. In one model,
a single sequence performs a random walk in the
space of all suf®ciently stable sequences. In the
second model, we represent the evolution of a
population of lattice proteins as they undergo
random mutagenesis, reproduction, and death.
Proteins that emerge during the population simu-
lations have a robustness to sequence change simi-
lar to that found in biological proteins. In contrast,
proteins derived from the single-sequence random
walk, even with the same structure and stability,
are extremely fragile to sequence changes. The
evolution of robust sequences proceeds without
any explicit evolutionary pressure for robustness,
but rather results directly from the population
dynamics. In contrast, de novo sequence design
must deal with the more random set of possible
sequences. These results suggest that the observed
sequence plasticity of biological proteins may
occur because proteins have evolved to be robust
to these speci®c experiments. If so, we may need to
revise the conclusions based on these observations.
Results
We use a computational model of proteins
consisting of 25 residues con®ned to a maximally
compact, two-dimensional lattice. In addition to
studying the properties of protein sequences cho-
sen at random, we implement two different
dynamic models of sequence change. In the ®rst
model, we allow a single sequence to perform a
random walk in sequence space among all viable
sequences. Sequences are considered viable if the
native state of the protein remains ®xed and if it
remains suf®ciently stable, that is, with a free
energy of folding,
Gfolding, smaller than some
®xed parameter,
Gcrit. In the second model, we
consider a population of such proteins where the
sequences undergo random mutagenesis, death,
and random reproduction, again with the con-
straint that all proteins must fold into a constant
native state and remain suf®ciently stable in order
to survive to the next generation. Simulations are
performed for both models ®ve times for each of
®ve values of
Gcrit (0.0, ÿ0.5, ÿ1.0, ÿ1.5, ÿ2.0).
The robustness of the resulting sequences to
mutation is monitored by recording the depen-
dence of the change in stability (

Gmut) as a
function of the protein's stability prior to the
mutation (
Gwt).
Why Are Proteins So Robust To Site Mutations?
In Figure 1, we compare the destabilizing prob-
ability for a mutation (the probability that


Gmut > 0) made in a sequence chosen at random
with the destabilizing probability of a mutation in
a sequence resulting from population evolution
(with
Gcrit ˆ 0.0) as a function of
Gwt. As can be
seen, the sequences resulting from the population
evolution have a much smaller probability of
having a destabilizing mutation compared to
random sequences with identical initial stabilities.
Figure 2 shows the resulting distributions of


Gmut for the two different types of evolutionary
simulations for three different values of
Gcrit. In
the case of sequences performing a random walk,
most of the mutations lead to reduced stabilities
close to the average of the random sequence distri-
bution; only 0.04 % to 0.4 % of mutations resulting
in increased stability (depending upon the value of

Gcrit). Conversely, in the case of the population
trials, there is an appreciable probability (18 % to
28 %) of a mutation being stabilizing with the most
likely change in stability near zero, especially for
highly stable proteins with large negative values of

Gcrit.
Since our population evolution sequences are
mutated with a Poisson distribution, we have a
decreasing probability that there will be multiple
mutations of a particular sequence in the popu-
lation. Figure 3 presents the average probability
that a multiple mutation will be destabilizing com-
pared with single mutations, under the constraints
of a speci®ed
Gcrit. Note that even signi®cant
sequence changes (three out of 25 residues) in the
proteins resulting from population evolution have
a non-negligible chance of resulting in increased
stabilization.
Discussion
It is not surprising that most mutations in bio-
logical proteins result in decreased protein stab-
Figure 1. Probability of a destabilizing mutation
P(

Gmut > 0)) from sequences resulting from popu-
lation evolution with
Gcrit ˆ 0 (continuous line) is com-
pared with random sequences (broken line), as a
function of the original stability of the unmutated pro-
tein
Gwt. The destabilization probability for stable ran-
dom sequences (with
Gwt < 0) is close to unity.
Why Are Proteins So Robust To Site Mutations?
Figure 2. Density distribution of

Gmut from model
proteins undergoing population (continuous line) and
single sequence evolution (broken line), for various
values of
Gcrit.
ility. If we consider the fact that most random
sequences of amino acids do not have a stable
folded state, then any mutation in one of the few
viable sequences with a stable ground state would
most likely move the stability in the direction of
the more random sequences; that is, towards being
less stable.
The surprise is rather that experimental
mutations have a signi®cant probability in result-
ing in unchanged or increased stability. The exact
percentages of mutations that are stabilizing vary
according to the protein and the nature of the sub-
stitutions, ranging from approximately 8 % in
mutations of barnase11 and staphylococcal nucle-
ase12 ± 14 designed to eliminate speci®c interactions,
to 17 % in interior locations of myoglobin,15 20 % of
non-Ala locations in Arc repressor,16 and 29 % for
two speci®c solvent-exposed locations in phage T4
lysozyme.17 A more comprehensive set of 356 site-
directed mutations compiled from the literature by
Reddy and co-workers showed that 25 % of the
mutations increased protein stability.1 While the
speci®cs vary, these results are suf®ciently consist-
ent to conclude that robustness would seem to be a
general characteristic of systems that have come
into being through the Darwinian evolution of
populations. The range of these experimental
results is close to the 18 % to 28 % (depending
upon
Gcrit) we observed for our lattice proteins
evolving through population dynamics, but far
from the 0.04 % to 0.4 % observed for random-walk
sequences with comparable
Gcrit.
Note that this robustness occurs in the absence
of any selective pressure towards robustness. The
various sequences in the model with

Gfolding <
Gcrit have equal ®tness and equal
probability of contributing to the next generation.
Robustness towards mutations is just one of a
number of properties that emerge from neutral
evolution in sequence space, as has been empha-
sized by a number of authors.4,5,7,10,18 ± 24
481
Figure 3. Probability of destabilizing mutation from
model proteins undergoing population evolution,
according to the number of point mutations, as a func-
tion of
Gcrit (thin continuous line). The average rate for
all mutations (thick continuous line) and the high rate
of destabilizing mutations for single sequence evolution
(broken line) are included for comparison.
So how can evolution, where robustness is not
an explicit selection criterion, result in such unex-
pectedly robust proteins? Insight into this phenom-
enon comes from the pioneering work by Eigen.25
In analytical studies of RNA evolution, he found
that evolution selected for a network of genotypes,
what he called quasispecies. The relevant ®tness of
the quasispecies is a function of the ®tness of all of
the genotypes, so the population of any one geno-
type would be enhanced by being surrounded by
®t neighbors. This effect depends on the possibility
of back-mutations: if one genotype contributes to a
neighboring genotype in one generation, there is a
probability that the neighboring genotype will
return the favor in a future generation. Through
this mechanism, population dynamics result in an
evolutionary selection of genotypes biased by the
®tness of their neighbors; that is, on their robust-
ness to mutations. In the sense of ®tness land-
scapes, nature may choose broad ®tness plateaus
of well-connected neighbors even in the presence
of higher, yet poorly connected ®tness peaks. This
evolutionary heritage is encoded in the genotype,
resulting in a sequence plasticity that distinguishes
these sequences from random sequences chosen to
have the same phenotype. Bornberg-Bauer &
Chan, for instance, found that evolutionary
dynamics would result in a bias in the population
towards ``prototype'' sequences with the maximum
number of ``neutral neighbors''.6 The work
described here concentrates on protein stability,
but it should be true of any protein property that
is important for survival of the organism. This
evolutionary trend towards robustness may be a
general characteristic of biological systems.26,27
There are a number of important consequences
of this effect. Firstly, the lessons of sequence plas-
ticity in biological proteins may be inapplicable to
arti®cally designed proteins. It may be necessary to
482
have a de novo sequence exquisitely designed to
have properties similar to biological proteins. This
also suggests that taking advantage of the
observed robustness by modifying existing pro-
teins may be a more effective route. Alternatively,
in vitro evolution studies may provide proteins
with the same degree of sequence plasticity as
natural proteins. More optimistically, proteins may
have compromised possible interactions and prop-
erties in developing this robustness, which
suggests that more effective if less robust proteins
may be available.
In addition, these results suggest that the
observed sequence plasticity may have non-
obvious consequences for our understanding of
proteins and their evolution. For instance, Baker
and co-workers observed that sequence changes in
the IgG binding domain of protein L often resulted
in proteins that folded faster than the wild-type
protein, and concluded that this indicates that the
folding rate is not under strong selective pressure.2
The model presented here results in the opposite
conclusion, that properties of the protein under
stronger selective pressure are more likely to be
``buffered'' and thus robust to mutations. In other
words, robustness to site mutations would para-
doxically be an indication of stronger selective
pressure on these characteristics.
Finally, we note that there is growing interest in
the relationship between robustness and evolvabil-
ity; that is, between the ability to buffer genotypic
variations and the ability of an organism to modify
to new situations and environments.28 If so, the
tendency of population dynamics to increase
sequence plasticity might have had signi®cant
impact on the evolutionary process, including
the development of new functionalities of existent
proteins.
Methods
We consider a highly simpli®ed representation of
evolving proteins. Our model proteins consist of chains
of n ˆ 25 monomers, con®ned to a 5  5 two-dimen-
sional, maximally compact square lattice with each
monomer located at one lattice point. This provides us
with 1081 possible conformations represented by the
1081 self-avoiding walks on this lattice, neglecting struc-
tures related by rotation, re¯ection, or inversion. The
non-compact states were neglected in order to allow for
a reasonable number of stable sequences. Alternatively,
we would expect the non-compact states to be neglect-
ible as long as the contact energies were suf®ciently
attractive. The fact that most protein structures are
reasonably compact makes this assumption not too
unreasonable. There are important differences between
the two-dimensional and three-dimensional models,
especially in folding simulations where the two-dimen-
sional conformation space may not be ergodic.29,30 While
these limitations are critical in folding simulations, we
are more interested in the mapping of sequence to struc-
ture rather than how the sequence folds to that given
structure; the thermodynamic properties described
below involve sums over states and should be less
affected by the dimensionality of the model. We use the
Why Are Proteins So Robust To Site Mutations?
two-dimensional model to provide a more realistic ratio
of buried to exposed sites.
We assume that the energies of any sequence in con-
formation k is given by a simple contact energy of the
form:
X
Eˆ g…Ai Aj †Uijk …1†
i<j
Here, Ukij is equal to 1 if residues i and j are not cova-
lently connected but are on adjacent lattice sites in con-
formation k, and g …Ai Aj † is the contact energy between
amino acid Ai at location i and Aj at location j in the
sequence. We use the contact energies derived by Miya-
zawa & Jernigan based on a statistical analysis of the
database of known proteins that implicitly includes the
effect of interactions of the protein with the solvent.31 In
our simpli®ed proteins, there are 132 pairs of residues
that can possibly come into contact, with 16 of these con-
tacts present in any given compact structure.
Using equation (1) we can calculate the energy of a
given protein sequence in all 1081 possible confor-
mations. We make the assumption that the thermodyn-
amic hypothesis is obeyed and that the lowest-energy
structure is the native state;32 the other 1080 possible
structures represent the ensemble of unfolded states.
Not all possible protein sequences are viable. In gener-
al, a protein must ful®l a number of conditions relating
to stability, functionality, and foldability. Here, we con-
centrate on stability. For each sequence, we calculate the
free energy of folding:

Gfolding ˆ Ef ‡ kT ln…Z ÿ exp…ÿEf =kT†† …2†
where Z is the partition function. (For the Miyazawa-
Jernigan potential, we use kT ˆ 0.6.) We consider a
sequence as representing a viable protein as long as its

Gfolding is less than some speci®ed
Gcrit.
We implement two different dynamic models of
sequence change. In the ®rst model, we choose a
sequence at random and make point mutations until we
arrive at a suf®ciently stable protein sequence. Starting
with this initial stable sequence, residue positions are
randomly mutated with the number of mutations chosen
from a Poisson distribution with an average of 0.002
mutations per amino acid residue per generation. With
this low mutation rate, multiple mutations are rare (the
ratio of single mutants to multiple mutants is 200). We
calculate the stability of the new sequence; if
Gfolding is
larger than
Gcrit or the structure has changed, the
mutation is rejected and the original sequence retained.
Generations where no mutations occurred are not
counted. This allows the single sequence to diffuse ran-
domly over the range of acceptable sequences, analogous
to random-walk models in which a particle has average
zero velocity when a boundary is encountered. This is
done ®ve times for each of ®ve values of
Gcrit (0.0,
ÿ0.5, ÿ1.0, ÿ1.5, ÿ2.0). Sequences that arise during these
runs are probed for robustness to mutations. We make
mutations in the sequence with a Poisson distribution
with mean 0.002 mutation per amino acid residue, main-
taining a constant rare rate of multiple mutations. We
then calculate the probability that a mutation results in a
given change in stability (

Gmut) as a function of the
stability prior to the mutation (
Gwt).
For the second model, we simulate the effect of popu-
lation dynamics using an evolutionary scheme, using a
method described elsewhere.8 We construct a population
of N ˆ 3000 identical viable sequences. For each gener-
Why Are Proteins So Robust To Site Mutations?
ation, each residue in the protein population has a prob-
ability of 0.002 to be mutated to another random residue;
both the population size and mutation rate were chosen
to be comparable to previous analytical models of evol-
ution processes.33 ± 35 The stability of each protein in the
population is then calculated. We use truncation selec-
tion where the N0 sequences having
Gfolding <
Gcrit
and a conserved native state structure are considered
viable and capable of reproducing; the rest are removed
from the population. The next generation of N sequences
is chosen from the N0 surviving sequences randomly
with replacement, representing the stochastic process
of reproduction. The population is ®rst allowed to pre-
equilibrate for 30,000 generations. The evolutionary
simulations are then continued for an additional 30,000
generations. In the subsequent 30,000 generations, we
monitor the stability of the sequences (
Gwt) as well as
the changes in stability that occur with mutations
(

Gmut). We perform these calculations ®ve times for
the same
Gcrit constraints used for the single-sequence
trials.
Acknowledgments
We thank Lee Altenberg, Nicolas Buchler, Matthew
Dimmic, Walter Fontana, Luca Peliti, Kevin Plaxco,
David Pollock, and Peter Wolynes for insights and help-
ful comments, and Matthew Dimmic, Bin Qian, and
Todd Raeker for computational assistance. Financial sup-
port was provided by NIH grant numbers LM05770 and
GM08270, and NSF shared equipment grant number
BIR9512955.
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Edited by J. Thornton