Review articles
A biological cosmos of parallel
universes: does protein structural
plasticity facilitate evolution?
Sebastian Meier1* and Suat Özbek2
Summary
While Darwin pictured organismal evolution as ‘‘descent
with modification’’ more than 150 years ago, a detailed
reconstruction of the basic evolutionary transitions at the
molecular level is only emerging now. In particular, the
evolution of today’s protein structures and their concur-
rent functions has remained largely mysterious, as the
destruction of these structures by mutation seems far
easier than their construction. While the accumulation of
genomic and structural data has indicated that proteins
are related via common ancestors, naturally occurring
protein structures are often considered to be evolutiona-
rily robust, thus leaving open the question of how protein
structures can be remodelled while selective pressure
forces them to function. New information on the proteome,
however, increasingly explains the nature of local and
global conformational diversity in protein evolution, which
allows the acquisition of novel functions via molecular
transition forms containing ancestral and novel structures
in dynamic equilibrium. Such structural plasticity may
permit the evolution of new protein folds and help account
for both the origins of new biological functions and the
nature of molecular defects. BioEssays 29:1095–1104,
2007. ! 2007 Wiley Periodicals, Inc.
Introduction
Proteins have an outstanding functional capacity due to their
ability to fold into distinct structures.(1) The precise sequence
features that result in functional protein structures are there-
fore of seminal interest for the development of a protein folding
theory. The earliest structural studies on proteins had
previously indicated that protein structures are far more
conserved than their sequences.(2) In fact, similar structures
can be assumed by proteins without significant sequence
homology.(3) While the evolution of novel functions in a
conserved structural scaffold is well established,(4,5) the way
1
Institute of Molecular Biology and Physiology, August Krogh
Building, University of Copenhagen, Universitetsparken 13, DK-2100
Copenhagen, Denmark.
2
Institute of Zoology, Department of Molecular Evolution and Geno-
mics, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany.
*Correspondence to: Sebastian Meier, Sebastian Meier, Carlsberg
Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark.
E-mail: smeier@crc.dk
DOI 10.1002/bies.20661
Published online in Wiley InterScience (www.interscience.wiley.com).
BioEssays 29:1095–1104, ! 2007 Wiley Periodicals, Inc.
in which new structural frameworks evolve via simple muta-
tions has remained much more elusive. The retrospective
character of evolutionary studies inevitably requires specula-
tion, especially as historic transition forms at the molecular
level are absent due to the absence of molecular fossils. In fact,
the disclosure of missing links is among the central achieve-
ments of the Darwinian theory of evolution but reconstructing
them at the organismal or molecular levels remains a major
challenge.(6)
The vast number of possible sequences—a 100 amino acid
protein can occur in 20100 sequences, each of which has
100 ! 19 neighbours differing by one amino acid in ‘‘sequence
space’’—makes a comprehensive mapping of genotype–
phenotype relationships at the protein level impossible and
suggests that lessons can best be learned from evolutionary
sequence design as performed by nature. Until recently,
protein sequence–structure mapping could be considered
‘‘largely Terra incognita’’,(7) in contrast to insightful RNA-
folding models.(8) This latter work has supported the classic
notion that often unrelated sequences fold to the same
structure and are connected in ‘‘a continuous network which
can be traversed by single mutational steps without passing
through non-functional intermediates’’.(9) Models of structural
innovation based upon mutational walks on such phenotypi-
cally neutral networks have been proposed. These models,
however, crave experimental validation, especially in proteins.
In principle, structural plasticity could allow smooth evolu-
tionary transformations of structures and concomitant func-
tional innovation via intermediate states containing a structural
ensemble.(10,11) Here, we review an increasing body of largely
incidental observations from a variety of biological disciplines,
which strongly indicate that a wealth of protein folds is
transmutable via plastic intermediates.
Relationship between natural proteins
Structures evolve slowly compared to the underlying se-
quences and only a few thousand kinds of protein folds
account for today’s domain diversity. Clearly, the earliest
protein structures must have evolved de novo;(12) this process
has been retraced experimentally by in vitro evolution of a
known natural fold.(13) The total set of evolutionary relation-
ships between today’s protein domains by divergence from a
few such primordial protein structures however, has remained
BioEssays 29.11 1095
Review articles
much more controversial. Sequence similarity essentially
proves evolutionary relationships, whereas neither functional
nor structural similarity alone is taken as a valid indication of
evolutionary relationships:(14,15) the limited number of protein
folds makes fold convergence possible while functional
similarity can be the result of comparable selection pressures
or the limited chemical repertoire needed by cells and provided
by protein functional groups.(14) As a result, major innovations
of novel functional scaffolds by sequence divergence are
particularly hard to detect. The accumulation of structural data
over recent years suggests that the variety of today’s protein
domain folds results from evolutionary dynamics rather than
from de novo evolution and thus properties of fold space alone.
The combination of structural identity and functional similarity
indicate evolutionary relationships even when there is low
sequence similarity(16,17) and allow the construction of putative
evolutionary links. Hard evidence for the evolutionary relation-
ships of folds has come from the network topology depicted in
graphs of protein structural similarity.(18 –21) Networks of
structural similarity between protein domains show that the
number of connections across the network follows a power
(Zipf) law, indicative of a sequential evolutionary growth by the
addition of domains.(20,22) Phenomenological models of
duplication and divergence reproduce the structural relation-
ships of proteins in these networks by the divergent expansion
of the protein universe in what Shakhnovich has termed the
‘‘biological Big Bang’’.(20) Notably Shakhnovich’s model
produces a relatively large number of structural innovations
as compared to independent de novo evolution of folds.(20) The
detailed molecular mechanisms of how evolutionary transi-
tions in protein structural space are achieved, however,
remains unclear, as the destruction of protein folds and
functions is substantially easier than their construction.(23)
Functional and conformational plasticity
of proteins
Proteins can evolve new functions, for instance antibiotic
resistance, in a matter of months by a small number of
mutations.(24) As a result, closely related proteins can have
different functions, while distantly related proteins can perform
the same task. Such biological functionality has been linked to
local structural fluctuations and chemical adaptations in a
stable structural background,(25) while global structural
changes in proteins are rarely observed. The gradual evolution
of new functions has been associated with gradual changes of
populations in diverse protein ensembles.(26) Multispecificity
both in enzymes and antibodies,(27) for example, is permitted
by a pre-equilibrium between multiple conformers of nearly
identical energy. The conformational selection from a hetero-
geneous ensemble has recently been demonstrated in anti-
body maturation by a combined spectroscopic/molecular
dynamics approach, thus showing how cooperatively acting
mutations rigidify the variable regions in an antibody.(26)
1096 BioEssays 29.11
Conformational diversity in the immune system—a ‘‘micro-
cosm of protein evolution’’(27)—shows how unlimited func-
tional diversity can be provided by a limited number of
sequences.(27) Enzymes presumably evolve novel functions
by mutational ‘tinkering’ with a specialized protein sequence,
thus frequently enabling a specialized enzyme to adopt a novel
function via a ‘generalist’ intermediate sequence conducting
diverse functions at surprisingly high efficiency.(24,28)
Plasticity is the phenotypic diversity that generates various
selectable ground states in one genotype and thus may
facilitate major evolutionary changes. It has been implicated in
the course of evolutionary alterations in developmental
processes, in particular.(29,30) Proteins may also show plastic
behavior by sampling new functions, while retaining their
original functions with nearly uncompromised efficiency.(10,24)
A fairly large sampling of conformational space by plastic
biomolecular structures would facilitate the molecular evolu-
tion of novelty and the attainment of global fitness maxima
(Fig. 1). Such plasticity will increase phenotypic variation and
thus contribute to the capacity of biomolecules to undergo
change in form and function. In this way an evolving organism
could be ‘‘extensively remodeled while it is running’’.(29)
Divergent specialization of plastic biomolecular structures
could occur by minor genetic changes, for example by single
mutations following gene duplication (Fig. 1).(31)
In these scenarios, plasticity has a buffering effect that
allows the biomolecular structure to develop novel specialized
phenotypes without abandoning the original tasks. On the
other hand, structural diversity can be costly, as each
conformer occupies only a fraction of the total population. If
the fitness function is a composite over diverse conformations,
fractions of beneficial structures will increase by selection.
Thus, selection would act to reduce plasticity after an
innovation, potentially explaining the rarity of natural transition
forms.(32) The existence of plastic transition regions between
biomolecular structures has been demonstrated for RNA(8) but
has remained controversial for protein sequence space. There
is no doubt, however, that relatively simple changes in the
genotype suffice to evolve new protein topologies. For
example, recent in vitro evolution by the Tawfik group has
demonstrated the gradual transformation between circular
permutations of DNA methyltransferase via gene rearrange-
ments.(33) Most importantly, in this instance, evolution pro-
ceeds to naturally occurring topologies via functional
intermediates.
Divergence of structures
The mapping of sequences to structures is highly redundant
both for RNA and proteins in the sense that many sequences
can fold to the same structure. Models of sequence–structure
topology have been especially well explored for RNA with
powerful secondary structure prediction algorithms.(8,34,35)
Random mutational walks by phenotypically neutral point
 Review articles

Figure 1. Protein structure plasticity increases phenotypic variation and allows the exploration of novel functions, which can be selected
from to become the novel wild type. The different colours symbolize different functional phenotypes. Efficient exploration of phenotypic
space via functional intermediates (top right) aids the discovery of global adaptive maxima. Redundancy by gene duplication adds further
robustness to the system by freeing restraints on one of the duplicates. Darwinian evolution suggests that novel, better solutions are only
found, when intermediates are not too disadvantageous. A static picture of proteins would suggest a vast prevalence of loss of function upon
non-neutral mutations in an evolved sequence. Note that the dimensionality of adaptive landscapes is much larger than three, as genotypes
and phenotypes differ in a wealth of factors.

mutations span large regions of sequence space without
altering the RNA secondary structure, thus leading to
extensive neutral networks in sequence space. RNA studies
show that finding particular novel structures by mutation
and selection is straightforward due to the proximity in
sequence space of neutral networks belonging to different
RNA secondary structures. In vitro selection experiments
principally have validated the notion of molecular transitions by
evolutionary pathways for RNA.(7) Accordingly, two different
naturally occurring ribozymes have been linked by a series of
point mutations, where each of the intermediate sequences
retains activity. An evolutionary bridge sequence between both
ribozymes exhibits both folds and functionalities in a single
RNA sequence.(36) The degeneracy of structures in such
bridge states offers attractive features to allow the continuous
evolution of novel folds and functions via plastic intermediates,
which assume globally different structures in a single
sequence.
If this notion holds for real proteins, extensive walks by
single mutational steps on neutral networks can explore
sequence space by amino acid exchanges that leave the
foldability intact (Fig. 2). While the over-all fraction of neutral or
non-neutral mutations in proteins remains a matter of debate,
potential sequence changes in proteins draw from nineteen
alternatives rather than three in RNA, implying a wealth of
phenotypically neutral mutations along the chain. The ability of
many different sequences to fold into the same structure on a
neutral network implies mutational robustness, but also leads
to evolvability: the large covering of sequence space by folds
implies small sequence distances relative to the number of
amino acids both between folds and between a random amino
acid sequence and a particular target fold. As a result,
evolution of the earliest structures from unfolded sequences
and subsequent divergence by fold change is possible by a
limited number of mutations, where the sequence–structure
map extends across sequence space.
As we still lack an analytical folding theory for the prediction
of sequence foldability, the analysis of evolutionary sequence
selection to derive folded proteins from random ensembles is
particularly important. The structural starting ensemble of a
random sequence is very sensitive to minor external perturba-
tions and sequence changes.(37) Evolution acts to select a
native structure conferring the highest fitness, and mutational
changes to the sequence optimize consistent folding to that
structure.(38,39) In this way, thermodynamically and kinetically
foldable sequences evolve, where native-like contacts are on
average more stable than non-native interactions.(40) The
entropic loss upon folding is thus driven by specific enthalpic
contacts and the folding reaction is sculpted by evolution to
become cooperatively stabilized. Both thermodynamically and

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Review articles
Figure 2. Top: particular protein folds can be formed by
largely different sequences, which are not clustered in
sequence space. Sequences forming one fold are organized
in a net-like arrangement in so-called ‘neutral networks’, where
single neutral mutations connect the sequences forming the
same fold. Top: Rare evolutionary bridges between different
folds allow continuous evolutionary transitions. Bottom: In
such bridges, the notion that protein sequences fold to unique
states is not valid, as shown for cysteine-rich domains from
Hydra(45). Mutated residues are shown in magenta.
kinetically populated states can evolve in this way into unique
ground state structures.(41,42) For a detailed recent review on
folding theory and evolutionary sequence selection, see
Shakhnovich.(43)
Ascertaining the closest approach in sequence for different
folds is central to the question whether mutational pathways
can principally prompt divergence between biomolecular
structures ‘‘without passing through non-functional intermedi-
ates’’. Various simulations on protein lattice models raised the
possibility that neutral networks of different protein folds
approach to distances of a few amino acid exchanges or
overlap in sequence space.(7,44) Relative to RNAs the overlap
seems largely reduced due to the larger chemical diversity of
the 20 amino acids as compared to the four RNA nucleo-
tides.(36) The paucity of molecular intermediate forms and the
1098 BioEssays 29.11
mutational robustness of functional folds over evolutionary
time(42) indicate that protein structural overlap in sequence
space is rare. The very existence of such overlap regions with
tertiary structural plasticity in single sequences is at odds with
the common picture of proteins, but incidental discoveries
have accumulated that demonstrate such plasticity rather than
unique ground state structures (Table 1). Global structural
changes in proteins have been primarily linked to protein
misfolding diseases. In contrast, a functional interconversion
of naturally occurring cysteine-rich tertiary structures was
achieved via a bridge state sequence folding into both
domain structures by reconstructing evolutionary pathways
from sequence data.(45) Cysteine-rich domains (CRDs) of
Hydra nematocyst wall proteins have a conserved motif
CXXXCXXXCXXXCXXXCC and fold to two different struc-
tures with three different disulfide bridges and a globally
different topology. Single mutations to a natural CRD
sequence suffice to induce a bridge state with some 20% of
the original fold and a major fraction converted to the other
naturally occurring structure (Fig. 2). An additional mutation
can virtually complete the conversion between the two folds.
The different structures occur in the N- and C-terminal
domains of minicollagens as cross-linking elements of the
ultrastable nematocyst wall. The mutations introduced in vitro
reflect a natural design principle to introduce structural polarity
into minicollagens.(45) Naturally occurring protein folds thus
can overlap in sequence space and continuous mutational
paths both for RNAs and for proteins can allow adaptive walks
and structural innovation via functional intermediates due to
the non-unique ground states in evolutionary bridges (Fig. 3,
Table 1). Saddle regions between unique folds are formed by
sequences that cannot saturate interactions in either of the two
folds. The crossing of such bridge states by walks in the
hypothetical free energy landscapes depends on the function-
ality of these sequences, that is their foldability relative to
physiological requirements.(46) Furthermore, plasticity in
these saddle regions reduces the effective concentration of
each conformation and is thus costly. As a result, fitness
effects and the rarity of transition sequences lead to a
disparity between time scales for sequence and structure
evolution.(43)
Experimental limitations
The exploration of shortest mutational paths for structural
innovation in sequence space has largely remained sporadic.
While chance observations of protein tertiary structure
plasticity in transition sequences between domain structures
accumulate, it is still unclear whether energetically nearly
degenerate bridge states between folds are a common feature
of protein structures and how many natural sequences are
within one or a few point mutations of defined non-native
structures. To estimate whether a limited number of amino acid
changes will suffice to change a protein structure, the number
 Review articles

Table 1. Examples of protein structure plasticity

Protein Degenerate forms and structural changes Trigger

Arc repressor(11,78) helix-sheet switch in a bridge state degenerate structures in parallel folding
reactions
Rop(51) repacking in different helical bundle dimers due to topological frustration degenerate structures in parallel folding
reactions
IGF-1(53) degenerate structures with different disulfide patterns degenerate structures in parallel folding
reactions
huPrP(64,65) equilibrium between a/b structure in the terminal domain of the soluble form degenerate structures in parallel folding
reactions
cysteine-rich domains(45) bridge state between different natural folds upon single mutation degenerate structures in parallel folding
reactions
Mad2(52) two distinct structures N1-Mad2 and N2-Mad2 with a potential role degenerate structures in parallel folding
in signalling reactions
DNA-methyltransferase(33) new protein topologies (circular permutation) through directed evolution multistep gene rearrangements
designed switches(56,72,73,86) e.g. a $ b rich(87) pH, ion binding, redox state, temperature
parallel coiled coil $ antiparallel coiled coil(88)
coiled coild $ amyloid(89)
homeodomain $ zinc finger(86)
coiled coild $ zinc finger(72,90)
toxins(57,58) refolding between prepore- and pore-conformations pH
hemagglutinin(59) secondary and tertiary structure refolding pH
protein GB1(91) monomer $ intertwined tetramer 5 conservative mutations
Janus(50) protein GB1 fold $ Rop fold 20% sequence change
(92,93)
Amyloidogenic proteins secondary and tertiary structure switch, manifested by aggregation spontaneous, crowding, impaired
chaperoning, mutation, seeding
KH domains(17) different topologies terminal extensions
chameleon sequences(94,95) short sequences with different conformations in different tertiary backgrounds,tertiary background
includes helix-sheet transitions
Lysozyme(96,97) large fluctuations with less native-like contacts upon destruction of long-rangeW62G
interactions
CD2(98) monomer/dimer, strand exchange kinetic trapping
Functional prions(60,66,67,68,69) soluble/fibril: self-propagating secondary and tertiary structure switch protein-conformational chain reaction
implicated in memory formation, structural functions and evolvability.
>30% of the proteome(99) intrinsically unfolded or molten globule proteins fold upon binding: switch free enthalpy of binding
unfolded $ folded

of amino acids determining a fold would need to be known. The

Figure 3. Folding free energy in dependence of protein
sequence. The relative stability of protein structure varies
massively with sequence. Functional sequences have to fold to
reasonably stable structures in biologically relevant time.
Prototypical sequences of highest stability are no prerequisite
for protein function and less stable protein sequences occur
more frequently. The marginal stability of native protein folds
ensures that novel configurations are evolvable upon few
mutations via intermediate bridge sequences.
relative importance of each residue in the folding of a protein
sequence has however not been established yet. Some
examples like Arc(11,78) and CRDs(45) show nonetheless that
even fairly localized changes can suffice to change the native
state. The effect of exchanging one or two amino acids will be
even more pronounced for residues involved in cooperative
interactions, as in the formation of a hydrophobic core.(37)
Computational and kinetic analysis has indicated that very few
residues determine native protein topology in the transition
state ensemble.(47,48) The identification of these residues by
sequence comparison is, however, complicated by the fact that
sequence selection will not only preserve the folding core but
include additional factors like functional selection.(43) On the
other hand, it has long been considered a challenge to design
sequences with more than 50% identity folding into different
structures (the so-called Paracelsus challenge).(49) Such
conformational transitions between unrelated protein folds
depending upon only a few mutations had been predicted in
simulations based on potentials derived from structural
databases.(44) The structures of the mainly b sheet protein

BioEssays 29.11 1099

Review articles
GB1 and the four a-helix bundle protein Rop (repressor of
primer) were used to solve the challenge experimentally. In
fact, the structures can be interconverted between their fully
folded, stable states by changing less than 20% of the
sequence.(50)
Only degenerate structures with substantial populations of
the different states and sufficient kinetic stability will allow a
characterization or even isolation of the different structures. In
other words, experimental description will be greatly aided, if a
limited number of energetically nearly degenerate structures
exists (Fig. 4) with sufficient free energy barriers between the
folds. Such a situation is known for Rop itself on the quaternary
structure level, as Rop assumes two different dimers of four-
helix bundle in a double-funnelled folding landscape similar to
that of the CRD bridge state shown in Fig. 4.(51) Likewise, the
spindle checkpoint protein Mad2 adopts two distinct folds at
equilibrium, which may be critical for its in vivo functionality in
signalling.(52) Disulfide-linked proteins abound in the examples
of nearly degenerate tertiary structures (Table 1). Alternative
structures of similar energy have thus been described
for Hydra cysteine-rich domains,(45) the insulin-like growth
factor-1,(53) and superoxide dismutase.(54) Conformational
dynamics between different disulfide linkages depend on the
presence of redox catalyst, if disulfide bond reshuffling occurs
via reduced intermediate states. As a result, different shapes
can be trapped by the absence of redox catalyst or by low
pH during purification. While X-ray crystallography inherently
is a purification procedure, thus precluding the detection of
structural diversity, biomolecular NMR spectroscopy requires
a significant population of the diverse states due to the
relatively low sensitivity of the technique, in order to achieve an
atomic scale description of structures. An ensemble of struc-
tures in dynamic equilibrium may well give NMR spectra
reminiscent of unfolded proteins(23) and the number of confor-
mers in equilibrium may be smaller than anticipated.(55) Most
importantly, the detailed refolding equilibrium in soluble prion
protein forms has eluded atomic characterization to date. The
observation of large-scale refolding in silico is still impeded by
the restricted time accessible to all atom molecular dynamics
simulations of proteins. In consequence, exploring the true
diversity of protein conformations on all structural levels will
require further methodological development.
Protein conformational switching
The structural variability of proteins is applied as a molecular
switch in different cellular contexts. Minor changes in the
solution state, like changing redox conditions, pH or ion
concentrations suffice to introduce structural changes in
natural protein conformational switches.(56) Such natural
switching mostly occurs in the form of rearrangements within
a stable fold. Prominent exceptions are, however, the refolding
of various membrane-punctuating toxins(57,58) and of hemag-
glutinin upon lowering of the pH,(59) as well as the structural
1100 BioEssays 29.11
changes associated with refolding and fibril formation in
prions.
The detection of misfolding in amyloid diseases is
alleviated by the self-perpetuation and trapping through
protein-conformational chain reactions(60) in the formation of
morphologically detectable plaques. Single mutations(61,62)
suffice to induce pathogenesis in humans at a post-reproduc-
tive age, where selection acts less strongly.(60,63) Structural
plasticity between structure rich in a-helix or b-sheet in the N-
terminal domain (residues 127–164) of the cellular prion
protein indicates an underlying role of structural plasticity in the
refolding to the pathogenic scrapie form.(64,65) The detailed
structural equilibria have remained controversial as mentioned
above. While the occurrence of prions in mammals is linked to
disease and death, examples for the biological functionality of
evolutionarily conserved prions have accumulated in lower
organisms. The seeding behaviour in prions notably allows a
hereditable protein-based information flow.(60) Among others,
the switching to fibrils has been implicated in memory
formation,(66) structural functions and providing surfaces(67,68)
and in an increased phenotypic variation by reducing
transcriptional fidelity in adverse environments.(69) Thus,
proteins seem to employ global structural changes both in
soluble and in aggregating forms to vastly increase their
functional repertoire. The view that proteins are a direct and
simple incarnation of the information encoded in the DNA in
‘one gene–one protein–one function’ relationships can there-
fore be regarded as outdated. Rather, the variation of protein
structures produces novel—and often beneficial—pheno-
types, thus placing proteins at centre stage in responsive
developmental processes.(70)
The number of designed tertiary structure switches has
grown vastly over the last few years due to the relevance of
pathological misfolding and due to improved design methods
(for recent reviews see Ambroggio et al.(56) and Wright
et al.(71)). In cases, the generation of hybrids of the target fold
sequences suffices to create switches. A particularly promis-
ing design approach relies on an algorithm that optimizes the
amino acid sequence by using Monte Carlo search in
sequence space.(72) Protein switches between tertiary struc-
tures are sequences in bridge regions of the sequence–
structure map, which offer the advantage that their refolding is
triggered by an environmental stimulus. As a result, these
triggered structural switches are easier to characterize than
mutational switches in evolutionary bridge states. Conforma-
tional switches have been designed between unfolded and
folded states, between different secondary structures and
between different tertiary structures. The switching of folds
can be triggered by various changes to solution states and
results in refolding, e.g. between antiparallel and parallel coiled
coils, coiled coils and zinc fingers as well as zinc fingers and
homeodomain folds (Table 1).(73) This demonstrates that a
variety of natural protein folds overlap in sequence space,
 Review articles

Figure 4. Parallel reactions in the folding of bridge state proteins and of their point mutants. Even the modulation of single local
interactions suffices to evolve predominant populations. A detailed structural characterization of bridge state structures benefits from
sufficiently high populations (DDG < KBT) and sufficiently high energetic barriers between competing folds, thus explaining the dearth of
evolutionary transition forms in protein structures.

notably including upstream transcription factor domains.
Hence both mutation and environmental stimuli can induce
phenotypic variation on the protein structure level. Refinement
of existing computational methods for switch design could
extend sequence–structure mapping and identify transition
regions between folds.(72)
Protein evolution and the buffering of variation
The theory of protein folding has fostered a statistical view of
protein conformational transitions in the folding reaction.
Competing structures are not uncommon in the folding
pathways of natural proteins. Folding intermediates formed
early in the protein-folding pathway can have well-character-
ized non-native structures.(74–76) Protein sequences often
populate non-native structures or kinetically trapped misfolded
states, which can evolve to form new ground state structures
upon few mutations.(37,41,77) Proteins are evolved to saturate
favourable interactions while reducing the prevalence of
conflicting interactions. This concept has been termed ‘‘the
consistency principle’’ or ‘‘the principle of minimal frustration’’.
Conflicting interactions are not eliminated in this way, but are
normally weak enough to allow escape and folding to the native
state. These predictions are validated in the quantification of
bridge state populations in cysteine-rich domains and Arc
repressor, where the competing folding reactions to novel
structures predominate after the abolishing of a hydrogen
bond donor or the shifts of methyl groups. Removing a
hydrogen bond in CRD by a mutation K21P reduces the
population of fold I from >95% to 20%.(45) In the case of
secondary structure switching by the Arc repressor, an N11I
mutation results in >95% of the molecules adopting the native
antiparallel b-sheet form, whereas changing only the position
of the methyl group in a further mutation I11L results in a
majority of the molecules in artificial helical form.(78) In addition
to mutational studies, de novo designed proteins offer an
opportunity to distinguish inherent from evolved protein-like
properties in polypeptides. A recent study by the Baker lab
shows that the designed folded polypeptide Top7 is not
protein-like in lacking a cooperative folding transition to a
unique native structure. Rather, a multiphasic folding kinetics is
observed in this case and a non-native folded structure
coexists in thermodynamic equilibrium with the designed
target structure.(79) Thus evolutionary sequence selection
most likely accounts for the cooperative foldability of natural
proteins to unique structures, as previously proposed by a
physically realistic model.(80)

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Review articles
Robustness of native protein folds is conferred not only by
sequence selection but is aided by chaperones and by the
threshold stability of protein folds, which initially allow the
accumulation of detrimental mutations. The stabilization of
proteins against misfolding and unfolding introduces capaci-
tance with respect to phenotypic variation by buffering the
emergence of non-native structures.(81,82) This effectively
extends the neutral networks of protein folds and further
decreases the distance between different protein shapes in
sequence space. While all genes and gene networks that
preserve the status quo can act in this way,(83) chaperones are
especially attractive candidates for evolutionary capacitance
due to their sensitivity to environmental stress, indicative of
changing fitness landscapes. The high activity of chaperones
in cancer cells likewise seems to allow a large genetic variation
in a stressful cellular environment, thus driving oncogen-
esis.(84,85)
Conclusion
Natural entities inevitably raise questions about the range of
their possible states and their origins, transmutations and
potentials for future development. During evolution, change
towards fitter states requires that the intermediate forms are
not substantially less functional. The folding of proteins to
unique structures and their structural stability over evolution-
ary time has been a long standing paradigm attributed to the
chemical diversity of amino acids, intrinsic biophysical proper-
ties of protein structures and the sequence-fold map: chains
encoding different structures allegedly have to differ by a large
fraction of their sequence. This picture has been changing
despite experimental shortcomings in the characterization of
diverse conformational ensembles and proteins appear
increasingly as both robust and evolvable molecules. Plasticity
at the secondary, tertiary and quaternary structure level
illustrates an unanticipated adaptability of proteins (Table 1).
Currently, it remains a matter of personal judgement whether
the majority of natural protein folds are considered isolated
islands in sequence space or nets in close sequence proximity.
Due to the marginal stability of protein folds additional
structures in the conformational ensemble can appear as a
consequence of only few mutations. Where mapped in close
enough detail, sequence space thus appears to allow
structural innovation of domain folds by a fairly limited number
of amino acid exchanges along pathways, where the novel
structure is present from the beginning of the transition. This
allows transformations of protein structures at low fitness
costs resulting from the reduced time of the protein spent in its
most active conformation.(32) Accordingly, the earliest folds
may have evolved gradually from functional but intrinsically
unfolded proteins.(27) Further instances of fold overlap in
sequence space, however, hold great promise for additional
surprises at the crossroads of the biological and biophysical
disciplines. The emerging picture is that the proteome
1102 BioEssays 29.11
harbours a wealth of shapes and functions, which provides
the basis for a mutation and selection process with a
surprisingly integrative dynamics. Thus, the most-basic
genetic events can provide distinctive selective advantages
in the Darwinian evolution of complex features with surpris-
ingly high frequency.
Acknowledgments
We wish to thank A. Wilkins, P.R. Jensen and nameless
reviewers for critical reading of the manuscript and useful
comments.
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