Báo cáo khoa học: Thermodynamic stability and folding of proteins from hyperthermophilic organisms doc

MINIREVIEW
Thermodynamic stability and folding of proteins from
hyperthermophilic organisms
Kathryn A. Luke
1,2
, Catherine L. Higgins
3
and Pernilla Wittung-Stafshede
1,2,4
1 Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA
2 Keck Center for Structural and Computational Biology, Rice University, Houston, TX, USA
3 Section of Atherosclerosis and Vascular Medicine, Department of Medicine, Baylor College of Medicine, Houston, TX, USA
4 Department of Chemistry, Rice University, Houston, TX, USA
Introduction
Proteins from thermophilic (growth temperature  45–
75 °C) and hyperthermophilic (growth tempera-
ture ‡ 80 °C) organisms exhibit remarkable thermal
stability and resistance to chemical denaturants [1–3].
Despite decades of research in this field, a general con-
cept for how this stability is achieved remains elusive.
The necessary differences are subtle, because homolo-
gous proteins from thermophilic ⁄ hyperthermophilic
and mesophilic organisms have nearly identical
sequences and overall structures [4]. Thermostability
appears to be implemented by a variety of strategies,
using combinations of virtually all known structural
parameters: increased number of ionic interactions,
increased extent of hydrophobic-surface burial,
increased number of prolines, decreased number of
glutamines, improved core packing, greater rigidity,
extended secondary structure, shorter surface loops,

and higher states of oligomerization [4–11].
Some years ago, it was argued that proteins from
extreme thermophiles (growth temperature around
100 °C) are stabilized in different ways compared to
those from moderately thermophilic organisms [3].
Using a data set of 24 thermostable, five hyperthermo-
stable, and 64 mesostable protein structures in 25
Keywords
hyperthermostability; protein folding;
stability profile; unfolding kinetics
Correspondence
P. Wittung-Stafshede, Department of
Biochemistry and Cell Biology, 6100 Main
Street, Rice University, Houston, TX 77251,
USA
Fax: +1 713 348 5154
Tel: +1 713 348 4076
E-mail:
(Received 28 February 2007, accepted
18 April 2007)
doi:10.1111/j.1742-4658.2007.05955.x
Life grows almost everywhere on earth, including in extreme environments
and under harsh conditions. Organisms adapted to high temperatures are
called thermophiles (growth temperature 45–75 °C) and hyperthermophiles
(growth temperature ‡ 80 °C). Proteins from such organisms usually show
extreme thermal stability, despite having folded structures very similar to
their mesostable counterparts. Here, we summarize the current data on
thermodynamic and kinetic folding ⁄ unfolding behaviors of proteins from
hyperthermophilic microorganisms. In contrast to thermostable proteins,
rather few (i.e. less than 20) hyperthermostable proteins have been thor-

oughly characterized in terms of their in vitro folding processes and their
thermodynamic stability profiles. Examples that will be discussed include
co-chaperonin proteins, iron-sulfur-cluster proteins, and DNA-binding pro-
teins from hyperthermophilic bacteria (i.e. Aquifex and Theromotoga) and
archea (e.g. Pyrococcus, Thermococcus, Methanothermus and Sulfolobus).
Despite the small set of studied systems, it is clear that super-slow protein
unfolding is a dominant strategy to allow these proteins to function at
extreme temperatures.
Abbreviations
GuHCl, guanidine hydrochloride; T
M
, midpoint of thermally induced unfolding transition; DG
U
, change in free energy upon protein unfolding;
DC
p
, difference in heat capacity between folded and unfolded states; Fd, ferredoxin; [GuHCl]
1 ⁄ 2
, GuHCl concentration at midpoint of
equilibrium unfolding transition.
FEBS Journal 274 (2007) 4023–4033 ª 2007 The Authors Journal compilation ª 2007 FEBS 4023
structural families, Szilagyi and Zavodszky proposed
that hyperthermostable proteins have stronger ion
pairing, fewer cavities, and higher b-sheet contents as
compared to the thermostable proteins [3]. Hyper-
thermophilic microbes are found in the most basal
positions in the universal tree of life in both bacteria
and Archea domains [1]; these organisms may thus
bear similarities to ancient life forms. Whereas bacteria
only include two genera of hyperthermophilic organ-

isms (i.e. Aquifex and Thermotoga), there is consider-
able phylogenic diversity among the hyperthermophilic
Archaea (e.g. Pyrococcus, Thermococcus, Methanother-
mus and Sulfolobus) [2]. Notably, no hyperthermophilic
eukaryote has yet been discovered [1].
Comparisons of the thermodynamics and kinetics of
the folding of proteins from mesophilic and thermo-
philic ⁄ hyperthermophilic organisms can provide an
insight into the mechanisms of stabilization that can-
not be obtained from static structural and sequence
investigations. The thermodynamic stability of a pro-
tein is quantitatively defined by the Gibbs free-energy
change upon unfolding (DG
U
¼ –RTlnK
U
) deduced
from the equilibrium constant (K
U
). When postulated
as a simple reversible two-state transition [12], the
equilibrium constant (K
U
¼ k
f
⁄ k
u
) is characterized by
the rate constants of folding (k
f

) and unfolding (k
u
)
rates. The stability of a protein therefore involves both
equilibrium and kinetic aspects; increased protein sta-
bility may be reflected either as slower unfolding (k
u
),
faster folding (k
f
), or a combination of the two
(Fig. 1A). In vitro folding ⁄ unfolding experiments in
solution often involve chemical (i.e. urea or guanidine
hydrochloride, GuHCl) or thermal perturbations of
the protein; the progress of the reaction being moni-
tored by spectroscopic methods such as aromatic
fluorescence (tertiary interactions near fluorophores),
far-UV circular dichroism (secondary structure con-
tent), or visible absorption (cofactor environment). For
time-resolved folding investigations, stopped-flow mix-
ing instruments are often necessary, which have a mix-
ing dead time of 1–2 ms. Experimental analyses of the
kinetic and thermodynamic origin of protein thermo-
stability and hyperthermostbility, however, have often
been hampered by unfolding irreversibility of such pro-
teins in vitro [13–15].
Three thermodynamic models have been proposed
to explain the high stability of thermostable and hy-
perthermostable proteins [4,16] (Fig. 1B). In the first
model (Model 1), compared to a protein from a me-

sophilic organism, the thermostable protein would be
more thermodynamically stable throughout the tem-
perature range (i.e. have higher DG
U
at every temper-
ature, shifting the profile vertically upwards). A
second model (Model 2) implies that the free-energy
profile of the thermostable protein would be horizon-
tally displaced to higher temperatures. In this model,
TS
A
B
U
Reaction Coordinate
Free Energy
F
Temperature
Free Energy
Model 1
Model 2
Model 3
ΔG
‡
U
ΔG
U
ΔG
‡
F
Fig. 1. (A) Scheme linking protein-thermodynamic stability (DG

U
)to
folding (k
f
) and unfolding (k
u
) rate constants. U, unfolded; F, folded;
TS, transition state. For a two-state folding process, the difference in
equilibrium stability (i.e. DG
U
) is related to the difference in activation
parameters (i.e. DG
à
F
and DG
à
U
) as: DG
à
U
) DG
à
F
¼ )RT*ln(k
f
⁄ k
u
) ¼ DG
U
.

(B) Thermodynamic profiles (i.e. DG
U
versus temperature) illustrat-
ing the three models by which thermostability can be achieved. A
protein (black solid line) can achieve higher thermal stability by
increasing its free-energy at all temperatures (i.e. Model 1, dotted
line), by horizontally shifting its stability profile to higher temperatures
(i.e. Model 2, gray line), or by broadening the stability profile (i.e.
Model 3, dashed line) while keeping the temperature of maximum
DG
U
the same.
Thermodynamic stability and folding of proteins K. A. Luke et al.
4024 FEBS Journal 274 (2007) 4023–4033 ª 2007 The Authors Journal compilation ª 2007 FEBS
the maximum value for DG
U
would be equal for both
proteins, but the maxima would occur at different
temperatures. At high temperatures, the thermostable
protein would be more stable; at lower temperatures,
the protein from the mesophile would be more stable.
Finally, a third model (Model 3) indicates that the
free-energy profile for the thermostable protein would
be a flattened version of that for the protein from the
mesophile. Thus, the thermostable protein would have
a more shallow dependence of DG
U
on temperature,
corresponding to a lower specific heat capacity
change of unfolding (D C

p
). According to this model,
the maximal DG
U
value would again be equal for
both proteins and would occur at the same tempera-
ture. Support for all three models, and combinations
thereof, has been reported for different thermostable
proteins [17,18].
In this minireview, we look at protein hyperthermo-
stability from an energetic point of view; specifically,
we describe existing data on equilibrium stability and
kinetic folding ⁄ unfolding processes of proteins from
hyperthermophiles. To collect as many examples as
possible, the literature has been searched comprehen-
sively. In the following sections, we discuss biophysical
data for hyperthermostable: (a) co-chaperonin pro-
teins, (b) nonheme iron proteins, (c) DNA-binding
proteins, as well as (d) a few other proteins. Although
the number of characterized hyperthermostable pro-
teins is rather small (Table 1, Fig. 2), some common
themes are evident and will be discussed in the final
section.
Co-chaperonin proteins
Co-chaperonin protein 10 (cpn10) works in conjunc-
tion with cpn60 to fold substrate proteins in most
organisms in nature [19–21]. The tertiary and quater-
nary structures of cpn10 proteins appear conserved;
seven irregular b-barrels assemble into a ring-shaped
heptameric structure [22]. Cpn10 from hyperthermo-

philic Aquifex aeolicus (Aacpn10) is unique among
cpn10 proteins in that each monomer contains a
25-residue C-terminal extension [23]. The sequence of
the C-terminal tail shows no significant similarity
with any known protein domain; its orientation in
the heptamer is yet unknown. Comparative biophysi-
cal studies using a truncated version of Aacpn10
where the tail has been removed, Aacpn10del-25,
demonstrated that the tail protects against cpn10
aggregation at high temperatures and at high protein
concentrations [24]. The tail, however, is not neces-
sary for protein folding, heptamer assembly, co-chap-
eronin function, or protein hyperthermostability
[24,25].
By contrast to many other oligomeric proteins, the
unfolding and disassembly of Aacpn10 and Aacpn10-
del-25 are fully reversible reactions in vitro [23]. We
have therefore been able to characterize, in detail, the
equilibrium and kinetic unfolding ⁄ dissociation and
folding ⁄ assembly behaviors of Aacpn10 and Aacpn10-
del-25 [24,26]. The results have been compared to the
corresponding data for the mesostable human mito-
chondrial cpn10 (hmcpn10) [27] and Escherichia coli
Table 1. List of hyperthermostable proteins for which chemical ⁄ thermal stability and ⁄ or folding ⁄ unfolding dynamic parameters (Table 2) have
been reported in the literature. For each protein, the source organism, its maximum growth temperature, the fold of the protein, the pres-
ence of cofactors, the oligomeric state, and the protein databank accession code (PDB ID) (if known) are provided.
Protein Organism T
max
(growth) Fold Cofactor Oligomer PDB ID
Co-chaperonin protein 10 Aquifex aeolicus

a
93 b – Heptamer –
Ferredoxin 1 and 5 Aquifex aeolicus
a
93 a ⁄ b 2Fe)2S Monomer 1F37
Ferredoxin Acidianus ambivalens
b
95 a ⁄ b 7Fe)8S Monomer –
Ferredoxin Thermotoga maritima
a
90 a ⁄ b 4Fe)4S Monomer 1VJW
Rubredoxin Pyrococcus furiosus
b
103 b Fe Monomer 1ZRP
Sac7d Sulfolobus acidocaldarius
b
85 a ⁄ b – Monomer 1WD0
ORF56 Sulfolobus islandicus
b
85 a ⁄ b – Dimer –
Cold shock protein Thermotoga maritima
a
90 b – Monomer 1G6P
Histone Methanothermus fervidus
b
97 a – Dimer 1HTA
Histone Pyrococcus strain GB-3a
b
95 a – Dimer –
HU Thermotoga maritima

a
90 a ⁄ b – Dimer 1B8Z
Methylguanine methyltransferase Thermococcus kodakaraensis
b
95 a ⁄ b – Monomer –
Dihydrofolate reductase Thermotoga maritima
a
90 a ⁄ b – Dimer 1CZ3
Pyrrolidone carboxyl peptidase Pyrococcus furiosus
b
103 a ⁄ b – Tetramer 1IOF
Ribonuclease HII Thermococcus kodakaraensis
b
95 a ⁄ b – Monomer 1X1P
CheY Thermotoga maritima
a
90 a ⁄ b – Monomer 1TMY
a
Bacteria.
b
Archaea.
K. A. Luke et al. Thermodynamic stability and folding of proteins
FEBS Journal 274 (2007) 4023–4033 ª 2007 The Authors Journal compilation ª 2007 FEBS 4025
cpn10 (GroES) [26] homologs. Whereas Aacpn10 is
much more resistant to thermal perturbation (T
M
¼
119, 73, 72 °C for Aacpn10, GroES, and hmcpn10,
respectively; 50 lm protein, pH 7.5), the equilibrium
unfolding mechanism is similar for all three cpn10

proteins [24,26,27]. In GuHCl, and upon heating,
Aacpn10, Aacpn10del-25, hmcpn10, and GroES exhibit
apparent two-state equilibrium transitions, in which
unfolding and dissociation steps are coupled
[22,24,26,27]. Thermodynamic analysis revealed that
the increased stability of the Aacpn10 heptamer arises
due to more stable monomers and not to increased
subunit–subunit affinity. Whereas the stability is
approximately 2–3 kJÆ mol
)1
for GroES and hmcpn10
monomers, it is greater than 5 kJÆmol
)1
for the
Aacpn10 monomer (pH 7, 20 °C) [24,26,28]. Nonethe-
less, over 85% of the overall heptamer stability comes
from the interface interactions in both the mesostable
and hyperthermostable variants of cpn10 [26–28]. This
property may be a functional requirement to assure a
heptameric state of cpn10 when it cycles on and off of
the cpn60 complex in vivo.
Cpn10 unfolds ⁄ dissociates in a biphasic reaction in
GuHCl that involves protein unfolding prior to hept-
amer dissociation [29]. When comparing the data for
the two bacterial cpn10 variants, both unfolding and
dissociation of GroES are much faster than for
Aacpn10 [26,30]. By contrast to unfolding ⁄ dissociation,
the time-resolved refolding ⁄ reassembly pathways show
notable variations among the three cpn10 homologs.
Refolding and reassembly of hmcpn10 follow along

two, apparent two-state parallel pathways. Most of the
molecules (approximately 75%) fold before assembling
into the heptamer, whereas the rest assemble prior to
protein folding [29,30]. GroES refolding ⁄ reassembly,
by contrast, follows a single sequential pathway, with
monomer folding preceding a much slower heptamer
assembly step [26]. The kinetic refolding ⁄ reassembly
path for Aacpn10 is similar to that of GroES but more
complex [30]. Upon triggering refolding ⁄ reassembly,
Aacpn10 molecules first populate a misfolded mono-
meric species. This unproductive intermediate then
unwinds, and a productive intermediate species forms.
Finally, the productive intermediates assemble into the
A
FG
IJ
LK
H
BCDE
Fig. 2. Structural models of the hyperthermostable proteins in Table 1 for which high-resolution structures have been reported. (red, a-helix;
yellow, b-sheet; green, loop). (A) AaFd. (B) TmFd. (C) PfRu. (D) Sac7d from Sulfolobus acidocaldarius. (E) TmCsp. (F) MfrH. (G HU from Ther-
motoga maritima. (H) TmDHFR. (I) PfPCP. (J) TkRNase. (K) TmCheY HII. (L) Aacpn10del-25 (model based on 1WE3).
Thermodynamic stability and folding of proteins K. A. Luke et al.
4026 FEBS Journal 274 (2007) 4023–4033 ª 2007 The Authors Journal compilation ª 2007 FEBS
heptamer, and final folding takes place [30]. The high
thermodynamic stability of the folded Aacpn10 mono-
mer [24] can explain why transient intermediates are
populated only for the hyperthermostable variant.
Stability profiles for Aacpn10 and GroES have been
derived using equilibrium unfolding ⁄ dissociation data

at a range of temperatures [26]. Comparison reveals
that the hyperthermostable cpn10 uses a combination
of all three thermodynamic models described in the
Introduction to increase the heptamer stability at high
temperatures. Careful inspection demonstrates that
Models 1 and 2 are most important for the stabilizing
effect [26].
Nonheme iron proteins
Iron-sulfur (Fe–S) clusters are common cofactors in
nature that facilitate electron transport in many pro-
teins (e.g. ferredoxins; Fds) [31]. Aquifex aeolicus is the
only hyperthermophile known to contain so-called
plant- and mammalian-type [2Fe)2S] Fds: AaFd1 and
AaFd5 [32,33]. Fd unfolding in vitro is irreversible due
to cluster degradation and cysteine oxidation in the
unfolded state [34–37]. Using linear extrapolations of
thermal midpoints in the presence of different GuHCl
concentrations, AaFd1 and AaFd5 were found to exhi-
bit midpoints well above 100 °C at pH 7 in buffer
(Table 2). At pH 2.5, both AaFd5 and AaFd1 are less
stable than at neutral pH, indicating that electrostatic
interactions are important for the high thermal stabil-
ity at physiological pH [32,33]. AaFd1 and AaFd5
unfold extremely slowly at pH 7 (20 °C), and polypep-
tide unfolding and Fe–S cluster degradation processes
appear kinetically coupled. Extrapolation of kinetic
data in the presence of denaturants suggests that
unfolding of the hyperthermostable Fds at pH 7 in
buffer (20 °C) requires hundreds of years [35]. For the
homologous [2Fe)2S] Fd from mesophilic Spinacea

oleracea (SpFd), only a few hours are required for
complete unfolding at the same experimental condi-
tions [34].
The role of the disulfide bond in AaFd1 was
assessed using the variant AaFd1-C87A (i.e.
Cys87Ala), in which one of the disulfide bond-forming
cysteines is eliminated [33]. We found AaFd1-C87A
to be less stable than the wild-type protein towards
thermal [T
M
(wt) ) T
M
(C87A)  8 °C] and chemical
([GuHCl]
1 ⁄ 2
(wt) ) [GuHCl]
1 ⁄ 2
(C87A)  0.9 M) pertur-
bations. AaFd1 is therefore a rare case of a Fd that is
stabilized by a disulfide bond [33]. Disulfide bonds are
not thought to be a method to achieve protein thermo-
stability [5]. In general, hyperthermostable proteins
contain lower fractions of cysteines and are poorer in
disulfide bonds than their thermostable and mesostable
Table 2. Thermal midpoints (T
M
), thermodynamic stability (DG
U
), and kinetic folding ⁄ unfolding parameters (k
f

and k
u
) for hyperthermostable
proteins. If not otherwise stated, T
M
and DG
U
refer to pH 7, and k
f
⁄ k
u
to pH 7 and 20–25 °C, conditions. In the last column, the thermo-
dynamic models used to increase thermal stability are given (see Introduction for definitions).
Protein Organism T
M
(°C) DG
U
(kJÆmol
)1
) k
f
(s
)1
) k
u
(s
)1
)
Thermodynamic
model used

Aacpn10 [24] Aquifex aeolicus 119
a
266 (30 °C)
a,f
0.0041
d
5.5 · 10
)5d
1, 2 (+ 3)
Aacpn10del-25 [24] Aquifex aeolicus 111
a
279 (30 °C)
a,f
0.0033
d
2.7 · 10
)4d
1, 2 (+ 3)
AaFd1 [33–35] Aquifex aeolicus 121 – – 2 · 10
)12
–
AaFd5 [32] Aquifex aeolicus 106 – – 2 · 10
)12
–
AmFd [15,35] Aquifex ambivalens 122 79 (20 °C) – 2 · 10
)4
(pH 10) –
TmFd [38] Thermotoga maritima 125 40 (50 °C) – – 1, 2, 3
PfRu [40,41] Pyrococcus furiosus 176–195 63 (100 °C) – 2 · 10
)10

(pH 2) 1, 2
Sac7d [42] Sulfolobus acidocaldarius 91 31 (25 °C) – – 3
ORF56 [43] Sulfolobus islandicus 107.5
c
85 (25 °C) 7 · 10
7
(M
)1
Æs
)1
)
e
1.8 · 10
)7
1
TmCsp [44] Thermotoga maritima 85 26 (25 °C) 565
e
0.018 –
MfrH [46] Methanothermus fervidus 101–109 65 (35 °C) – – 1
PyArH [46] Pyrococcus strain GB-3a 110 72 (44 °C) – – 1
HU [47] Thermotoga maritima 78 (pH 4)
b
29 (pH 4, 25 °C) – – 1, 3
TkMGMT [48,51] Thermococcus kodakaraensis 95 62 (31 °C) – 1.5 · 10
)7
1, 2, 3
TmDHFR [52] Thermotoga maritima – 142 (15 °C) – 4.6 · 10
)12
1, 2
PfPCP [53,62] Pyrococcus furiosus 104 79 (25 °C) 0.093

e
1.6 · 10
)15
1, 3
TkRNase HII [55] Thermococcus kodakaraensis 83 44 (50 °C) 0.78 (50 °C)
e
5 · 10
)8
(50 °C) 1, 2
TmCheY [56] Thermotoga maritima 99 40 (29 °C) – – 1, 3
a
50 lM monomer.
b
120 lM monomer.
c
5 lM monomer.
d
Final folding ⁄ unfolding step (processes not two-state).
e
Two-state process.
f
Coupled unfolding ⁄ dissociation.
K. A. Luke et al. Thermodynamic stability and folding of proteins
FEBS Journal 274 (2007) 4023–4033 ª 2007 The Authors Journal compilation ª 2007 FEBS 4027
counterparts [34]. Because the variant is still much
more stable than SpFd, it was concluded that electro-
static interactions also contribute to the high stability
of AaFd1.
Like the A. aeolicus Fd proteins, the [4Fe)4S] Fd
from the hyperthermophile, Thermotoga maritima

(TmFd) and the di-cluster [3Fe)4S] ⁄ [4Fe)4S] Fd from
hyperthermophilic Acidianus ambivalens (AmFd), dis-
play irreversible unfolding reactions in vitro [15,38].
The time-resolved reactions appear to be two-state,
suggesting that unfolding and cluster degradation are
also coupled steps for these Fd proteins [36]. The ther-
mal unfolding midpoints are 125 °C and 122 °C
(pH 7) for TmFd and AmFd, respectively [38]. At
pH 2.5, however, the unfolding midpoint for AmFd
decreased to 64 °C [15,36]. Also, the apparent DG
U
value for AmFd is strongly pH dependent; at 20 °C, it
decreases from 79 to 20 kJÆmol
)1
when the pH drops
from 7 to 2.5 [15]. Analysis of a structural model of
AmFd suggests that a combination of additional sur-
face ion pairs, the zinc cofactor, and an efficiently
packed core govern the high stability of this protein
[36]. According to the crystal structure, TmFd also
contains an increased number of hydrogen bonds
between charged residues as compared to thermolabile
Fd proteins [38].
Rubredoxin from the hyperthermophile, Pyrococcus
furiosus (PfRu) is another hyperthermostable nonheme
iron protein (a single iron bound by four cysteines)
that has been well characterized with respect to its
unfolding features in vitro. It was found that the ther-
mal unfolding midpoint of PfRu is 42 °C higher at
pH 7 than at pH 2 [39]. In addition, the unfolding

rates for PfRu increase dramatically upon decreasing
the pH from 7 to 2 [40]. Compared with rubredoxin
from mesophilic Clostridium pastureianum (CpRu),
PfRu unfolds much more slowly at all experimental
conditions. Electrostatic-energy calculations suggest
that ion pairs placed at key surface positions play a
kinetic role by ‘clamping’ the hyperthermostable vari-
ant [13]. Based on hydrogen-exchange experiments, a
thermodynamic stability profile was constructed for
PfRu, which displayed a maximum DG
U
of 63 kJÆ
mol
)1
at 100 °C (pH 7) and an extrapolated T
M
(but
probably not realistic) close to 200 °C (pH 7) [41].
DNA-binding proteins
One of the first hyperthermostable proteins studied
with respect to folding was the Sac7d DNA-binding
protein from Sulfolobus acidocaldarius. Sac7d is an
attractive model protein because it is a small, 66-resi-
due monomeric protein that unfolds in a two-state
reversible process in vitro [42]. Sac7d is highly resistant
to thermal (T
M
of 91 °C at pH 7 and 63 °C at pH 0),
chemical ([GuHCl]
1 ⁄ 2

¼ 2.8 m GuHCl, pH 7, 20 °C)
and acidic (remains folded in the pH range 0–10)
perturbations. The thermodynamic stability of Sac7d,
however, is similar to that of many mesostable pro-
teins; at pH 7 and 20 °C, DG
U
is only 22 kJÆmol
)1
[42].
A comparison of the stability profile for Sac7d to
those for mesostable proteins of similar sizes reveals
that the curve for Sac7d is flattened compared to the
others. Thus, Sac7d employs Model 3 to increase its
stability. Accordingly, calorimetric experiments pro-
vided a DC
p
value for Sac7d unfolding of 0.5 kcalÆ
molK
)1
, which is significantly lower than DC
p
values
for unfolding of mesostable proteins of similar sizes
[42]. It was hypothesized that Sac7d survives with a
low free energy in vivo due to post-translational modi-
fications as well as interactions with compatible osmo-
lytes, and by binding to DNA [42].
Like Sac7d, ORF56 from Sulfolobus islandicus is a
DNA-binding protein that appears to be stabilized by
interactions with DNA [43]. ORF56 is also a small

protein (56 residues). It forms a tetramer when bound
to DNA and exists as a dimer in the absence of DNA.
Equilibrium unfolding of the ORF56 dimer in vitro is
an apparent two-state reversible reaction, in which
unfolding and dissociation are coupled processes [43].
The thermal unfolding midpoint for the ORF56 dimer
in the absence of DNA is 107.5 °C (pH 7). The stabil-
ity profile constructed from GuHCl-induced unfold-
ing ⁄ dissociation data at different temperatures suggests
that ORF56 uses the first thermodynamic model
(Model 1) to increase dimer stability at high tempera-
tures; the stability maximum remains at 30 °C and
DC
p
is equal to that for a mesostable protein of the
same size [43]. The kinetic unfolding ⁄ dissociation and
refolding ⁄ reassembly reactions for ORF56 have been
characterized; they are also two-state processes.
Because the rate constants of refolding ⁄ reassembly are
dependent on the protein concentration, association
appears to be the rate-limiting step [43]. The lack of
an initial monomer-folding phase suggests that the
assembly takes place between unfolded monomers.
Several DNA-binding proteins act by protecting
DNA from adopting unwanted secondary structures
[44]. The family of cold shock proteins has this func-
tion and is a good model system for proteins with all
b-sheet structures. The folding reactions of the cold
shock proteins from hyperthermophilic T. maritima
(TmCsp) and mesophilic Bacillus subtilis (BsCsp) have

been extensively studied in vitro [44]. Both equilibrium
and time-resolved folding ⁄ unfolding processes are two-
state. Interestingly, the rate constants of refolding are
Thermodynamic stability and folding of proteins K. A. Luke et al.
4028 FEBS Journal 274 (2007) 4023–4033 ª 2007 The Authors Journal compilation ª 2007 FEBS
similar for the two homologs and the processes occur
within milliseconds, although their native fold is all
b-sheet (pH 7, 20 °C). Comparing BsCsp and TmCsp,
all sequence variations map to the protein surface [44].
This agrees with the rate-limiting step in folding being
hydrophobic collapse of the protein core, which is
identical in both proteins. TmCsp, however, has signifi-
cantly greater thermal and chemical stability (T
M
of
85 °C, pH 7; DG
U
of 26 kJÆmol
)1
,pH7,20°C) than
BsCsp (T
M
of 50 °C, pH 7; DG
U
of 11 kJÆmol
)1
,pH7,
20 °C) [44]. This difference in thermodynamic stability
correlates with two orders of magnitude slower unfold-
ing of TmCsp as compared to unfolding of BsCsp [44].

Charged surface interactions unique to TmCsp appear
to increase the entropic barrier to unfolding and
thereby slow down the reaction [45].
In contrast to many other hyperthermostable pro-
teins, histone proteins do not use surface charges
to achieve thermostability. The archaeal histones
from the hyperthermophilic Methanothermus fervidus
(MfrH) and Pyrococcus strain GB-3a (PyArH) were
found to have significant increases in bulky, aromatic
residues in their cores compared to mesostable histones
[46]. As a result of more tightly packed protein interi-
ors, DG
U
is 65 (pH 7, 35 °C) and 72 kJÆmol
)1
(pH 7,
44 °C) for MfrH and PyArH, respectively, compared
to 28 kJÆmol
)1
(pH 7, 43 °C) for a mesostable histone
from Methanobacterium formicicum (ForH). The DC
p
of unfolding for the hyperthermostable and mesostable
histone homologs is approximately the same. Instead,
the stability profiles for MfrH and PyArH are shifted
vertically upwards, in line with the first thermody-
namic model [46]. We note that the histone-like HU
protein from T. maritima differs from MfrH and
PyArH in that it remains folded at high temperatures
using a combination of Models 1 and 3 [47]. More-

over, this protein is thought to be stabilized by a high
percentage of charged residues scattered throughout
the structure [47].
One of the more complete studies of protein hyper-
thermostability focuses on the small, monomeric O
6
-
methyl-guanine-DNA methyltransferase from hyper-
thermophilic Thermococcus kodakaraensis (TkMGMT)
and the C-terminal domain of the Ada protein from
E. coli (EcAdaC) [48–51]. GuHCl-induced equilibrium
unfolding experiments show that both proteins display
two-state, reversible transitions, with TkMGMT being
significantly more stable than EcAdaC ([GuHCl]
1 ⁄ 2
¼
5.2 and 1.6 m GuHCl for TkMGMT and EcAdaC,
respectively, pH 8.0, 20 °C) [49]. Inspection of their
stability profiles reveals that both proteins have the
same free energy of unfolding at their respective organ-
ism’s growth temperature. It appears that TkMGMT
uses a combination of all three thermodynamic models
to generate its high stability [50,51]. Time-resolved
unfolding experiments in GuHCl indicated that
EcAdaC will unfold in < 1 s, whereas the unfolding
time for TkMGMT is 4.5 · 10
6
s (approximately
2 months) when the data are extrapolated to buffer
conditions (pH 8, 20 °C) [48]. Disruption of internal

ion pairs through residue-specific mutations was found
to increase the unfolding-rate constant of TkMGMT
[50]. This finding supports that charged interactions
are of importance for governing TkMGMT hyperther-
mostability.
Other proteins
In addition to the described groups of proteins, only
a few other hyperthermostable proteins (i.e. DHFR,
PCP, RNase, CheY) have been characterized with
respect to folding and stability in vitro. Dihydro-
folate reductase from hyperthermophilic T. maritima
(TmDHFR) is a very stable dimeric protein [52].
Folded monomers have not been detected at any equi-
librium solvent condition or during TmDHFR unfold-
ing in vitro. Denaturant-induced equilibrium unfolding
is an apparent two-state process, involving only folded
dimers and unfolded monomers: DG
U
is 142 kJÆmol
)1
at pH 7, 15 °C [52]. The stability profile for TmDHFR
is shifted upwards and to the right compared to that
for DHFR from E. coli. Like most other hyperthermo-
stable proteins for which kinetics have been reported,
the unfolding reaction for TmDHFR is several orders
of magnitude slower than for the mesostable homolog
at corresponding conditions [52].
Pyrrolidone carboxyl peptidase from P. furiosus
(PfPCP) and from Bacillus amyloliquefaciens (BaPCP)
is another set of hyperthermostable ⁄ mesostable homo-

logs for which equilibrium and kinetic folding data
have been collected at different pH values [53]. A vari-
ant substituted with serines at Cys142 and Cys188
(PfPCP-142 ⁄ 188S) was prepared to eliminate complex-
ity due to sulfur oxidation [53]. GuHCl-induced
unfolding reactions of PfPCP-142 ⁄ 188S and BaPCP
are reversible for both proteins, but the DG
U
values
differ dramatically: DG
U
is 57 kJÆmol
)1
(pH 7, 60 °C)
and 8 kJÆmol
)1
(pH 7, 40 °C) for PfPCP-142 ⁄ 188S and
BaPCP, respectively. Unfolding-rate constants for
PfPCP-142 ⁄ 188S and BaPCP are also drastically dif-
ferent (1.6 · 10
)15
Æs
)1
and 1.5 · 10
)8
Æs
)1
, respectively;
pH 7, 25 °C), whereas the refolding rate constants are
similar (9.3 · 10

)2
Æs
)1
and 3.6 · 10
)1
Æs
)1
, respectively)
[53]. Also, at pH 2.3, where PCP exists in monomeric
form, unfolding of PfPCP-142 ⁄ 188S is much slower
than BaPCP unfolding [54].
K. A. Luke et al. Thermodynamic stability and folding of proteins
FEBS Journal 274 (2007) 4023–4033 ª 2007 The Authors Journal compilation ª 2007 FEBS 4029
Ribonuclease HII from hyperthermophilic Thermo-
coccus kodakaraensis (TkRNase HII) has also been the
subject of equilibrium and kinetic folding studies [55].
Both GuHCl- and heat-induced unfolding reactions
are reversible, albeit the very slow unfolding process
prohibited acquisition of equilibrium unfolding curves
at temperatures below 40 °C (pH 7) [55]. At 50 °C,
unfolding reactions attained their equilibrium values
after 2 weeks of incubation, and a DG
U
value of
approximately 44 kJÆmol
)1
(pH 7) could be calculated.
The unfolding-rate constant for TkRNase HII is much
lower than those for RNase HI from E. coli and RN-
ase HII from thermophilic Thermus thermophilus (Tt),

whereas the refolding speeds for all three proteins are
similar [55]. The stability profiles of TkRNase HII and
TtRNase HII are similar, although TkRNase HII
exhibits a higher temperature of maximum stability
and is folded in a smaller range of temperatures. The
DC
p
for TkRNase HII is higher than that for TtRN-
ase HII, explaining the more narrow range of tempera-
tures where the hyperthermostable protein remains
folded as compared to the thermostable homolog.
Both TkRNase HII and TtRNase HII have higher
temperatures of maximum stability compared to the
mesostable EcRNase HI [55].
Finally, the thermodynamic parameters for two
CheY homologs, one from hyperthermophilic T. mari-
tima (TmCheY) and the other from mesophilic B. sub-
tilis (BsCheY) have been compared. Based on
denaturant-induced unfolding studies TmCheY dis-
plays increased T
M
(98 °C versus 55 °C, pH 7) and
DG
U
(40 kJÆmol
)1
versus 13 kJÆmol
)1
;pH7,50°C)
values as well as a decreased DC

p
for unfolding (1.2
versus 2.3 kcalÆmolK
)1
, pH 7) compared to BsCheY
[56].
Conclusions
We have summarized the in vitro data that exist on
thermodynamic stability and folding ⁄ unfolding reac-
tions of proteins from hyperthermophilic organisms.
The number of proteins that have been characterized
to date is low (i.e. less than 20; Table 1). Clearly, addi-
tional studies are needed to make general conclusions
for how thermodynamic parameters correlate with
hyperthermostability. Nonetheless, some common
themes are evident when analyzing the present data.
First, most of the hyperthermostable proteins in
Table 2 have high T
M
and DG
U
values, at least around
neutral pH (Fig. 3). To achieve high stability, the three
thermodynamic models (Fig. 1B) are used in different
combinations by these proteins (Table 2, final column).
In our data set, Model 1 (vertical shift of DG
U
to
higher values) is clearly the most prevalent mechanism,
and most often it is combined with Model 2 (horizon-

tal shift of the profile to higher temperatures). This
trend differs from previous reports, which have con-
cluded that a decrease in DC
p
(i.e. Model 3, either
alone or in combination with Model 1) is the most
common method for proteins to achieve high thermal
stability [4,17,18,57]. Notably, in the earlier com-
parisons, no separation between thermostable and
hyperthermostable proteins was made, and few hyper-
thermostable proteins were included. Perhaps proteins
from hyperthermophilic organisms most often use
Models 1 and 2, whereas thermostable proteins are
more likely to use Models 1 and 3. It was recently pro-
posed that the choice of structural strategy for thermal
stabilization of hyperthermostable proteins depends on
the evolutionary history of the organism [58].
Second, because stability and ⁄ or T
M
is much
reduced at low pH for most of the hyperthermostable
proteins, electrostatic interactions and ⁄ or specific ion
pairing appear to be an important way for these pro-
teins to govern high stability at neutral pH. This is
reasonable because charge–charge interactions become
stronger, whereas the importance of the hydrophobic
effect decreases, at higher temperatures [5].
Third, for all hyperthermostable proteins with
reported unfolding kinetics (Table 2), the unfolding
speed is always dramatically slower (up to eight orders

of magnitude!) for the hyperthermostable protein than
for the mesostable homolog (at room temperature).
Still, in the five cases tested (i.e. Aacpn10, TmCsp,
PfPCP, TkRNase HII and ORF56), protein refolding
0
20
40
60
80
100
40 60 80 100 120 140
G
U
(kJ/mol)
T
M
(deg C)
Fig. 3. T
M
versus DG
U
values for hyperthermostable proteins in
Table 2 (filled circles, those for which both values are known;
cpn10 proteins excluded) along with their mesophilic counterparts
(open circles, data mentioned in the text). The plot shows that the
two parameters are correlated (solid line) for both sets of proteins.
Thermodynamic stability and folding of proteins K. A. Luke et al.
4030 FEBS Journal 274 (2007) 4023–4033 ª 2007 The Authors Journal compilation ª 2007 FEBS
kinetics are similar for the hyperthermostable and mes-
ostable variants. This suggests that protein hyperther-

mostability is linked directly to kinetic resistance to
unfolding. There may have been evolutionary pressure
in hyperthermophiles to select proteins with reduced
unfolding rates, rather than with very high folding
rates, because the rates of irreversible modification
depend on the protein-unfolding speed [59]. One may
speculate that an increase in favorable surface interac-
tions, such as extra ion pairs, creates an entropic bar-
rier towards unfolding of hyperthermostable proteins.
Despite this apparent structural rigidity, some hyper-
thermostable proteins (i.e. HU and PfRu) were found
to have unexpectedly high flexibility in their native
states [11,47,60]. An important future task is to probe
folding ⁄ unfolding kinetics as a function of tempera-
ture: most importantly, at temperatures closer to the
hyperthermophilic organisms’ growth temperatures. In
the only study of this [44], it was found that TmCsp,
as compared to BsCsp, indeed had slower unfolding
rate constants in a wide temperature range.
Despite the general theme of super-slow unfolding,
it appears that evolution can (and does) make use of
everything that works and therefore we will never find
an overarching chemical ⁄ biophysical ⁄ energetic expla-
nation of protein hyperthermostability. In other words,
‘There’s more than one way to skin a cat’ [61].
Acknowledgements
This work was funded by Grants from NIH
(GM059663) and the Robert A. Welch Foundation
(C-1588). KAL is supported by the Houston Area
Molecular Biophysics Program (GM08280). CLH is

supported by NIH Training Grant (T32 HL007812;
TTGA).
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