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: pernilla@rice.edu (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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The number of proteins that have. [1]. Comparisons of the thermodynamics and kinetics of the folding of proteins from mesophilic and thermo- philic ⁄ hyperthermophilic organisms can provide