INTRODUCTION

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Hyperthermophiles grow optimally at temperatures between 80 and 110°C. Only represented by bacterial and archaeal species, these organisms have been isolated from all types of terrestrial and marine hot environments, including natural and man-made environments. Enzymes from these organisms (or hyperthermophilic enzymes) developed unique structure-function properties of high thermostability and optimal activity at temperatures above 70°C. Some of these enzymes are active at temperatures as high as 110°C and above (349). Thermophilic organisms grow optimally between 50 and 80°C. Their enzymes (thermophilic enzymes) show thermostability properties which fall between those of hyperthermophilic and mesophilic enzymes. These thermophilic enzymes are usually optimally active between 60 and 80°C. Active at high temperatures, thermophilic and hyperthermophilic enzymes typically do not function well below 40°C.

Current theory and circumstancial evidence suggest that hyperthermophiles were the first life-forms to have arisen on Earth (318). Hyperthermophilic enzymes can therefore serve as model systems for use by biologists, chemists, and physicists interested in understanding enzyme evolution, molecular mechanisms for protein thermostability, and the upper temperature limit for enzyme function. This knowledge can lead to the development of new and/or more efficient protein engineering strategies and a wide range of biotechnological applications.

This review will encompass the sources and uses of thermophilic and hyperthermophilic enzymes, as well as the molecular determinants for protein stability. Emphasis will be placed on hyperthermophilic enzymes, because most current research is focused on these enzymes and on hyperthermophiles. What is the upper temperature for life? Back in 1969, when T. D. Brock and colleagues discovered Thermus aquaticusnow known for its Taq polymerase in PCR techniquesT. aquaticus was considered an extreme thermophile since it grew optimally at 75°C (41). Today, of course, hyperthermophiles such as Pyrolobus fumarii, which grows at up to 113°C (28), are considered extreme.

Thermophilic and hyperthermophilic enzymes (also called thermozymes [see reference 349]) are part of another enzyme category called extremozymes, which evolved in extremophiles. Extremozymes can function at high salt levels (halozymes), under highly alkaline conditions (alkalozymes), and under other extreme conditions (pressure, acidity, etc.) (see references 4, 144, 223, and 371). Intrinsically stable and active at high temperatures, thermophilic and hyperthermophilic enzymes offer major biotechnological advantages over mesophilic enzymes. (i.e., enzymes optimally active at 25 to 50°C) or psychrophilic enzymes (i.e., enzymes optimally active at 5 to 25°C): (i) once expressed in mesophilic hosts, thermophilic and hyperthermophilic enzymes are easier to purify by heat treatment, (ii) their thermostability is associated with a higher resistance to chemical denaturants (such as a solvent or guanidinium hydrochloride), and (iii) performing enzymatic reactions at high temperatures allows higher substrate concentrations, lower viscosity, fewer risks of microbial contaminations, and often higher reaction rates.

Already the object of extensive reviews (140, 317, 319, 320), hyperthermophiles are only briefly described here. No exhaustive description of all the enzymes isolated and characterized from thermophiles and hyperthermophiles is presented, since that information is available elsewhere (2, 3, 139, 263, 349). Instead, we will focus on the latest findings that explain the molecular determinants of extreme protein thermostability and on the thermophilic and hyperthermophilic enzymes with the highest commercial relevance.

The interest shown by the scientific community in hyperthermophiles has constantly increased over the last 30 years. This growing interest is demonstrated by the increasing number of hyperthermophilic species that have been described (from 2 in 1972 [40, 372] to more than 70 at the end of 1999 [140, 320]), by the exponentially growing number of publications on the subject, and by the major central place occupied by hyperthermophiles in worldwide genome-sequencing projects (six completed genome sequences, and at least four genome-sequencing projects in progress) (see Table 1 and http://www.tigr.org) Studies of environmental 16S rRNA sequences (18, 19) in samples originating from a single continental hot spring (Obsidian Pool at Yellowstone National Park) and environmental lipid analysis (128) suggest that known hyperthermophiles represent only a fraction of hyperthermophilic species diversity.

Now that we are able to collect samples almost routinely from deep-sea floors, access to hyperthermophilic biotopes is not the limiting factor in studying hyperthermophile diversity. Isolating and growing pure cultures of new hyperthermophiles has beenand remainsa challenge. A striking example of this difficulty is the bacterium Thermocrinis ruber (147). This pink-filament-forming bacterium was described as early as 1967 by Brock (39), but it took more than 25 years to successfully cultivate this organism (147). A major task for scientists in the near future will be to develop new isolation techniques for microorganisms with different, unforeseen metabolic requirements. Huber et al. (145) took the lead by cloning a new archaeal hyperthermophile by using optical tweezers.

Hyperthermophiles have been isolated almost exclusively from environments with temperatures in the range of 80 to 115°C. Hot natural environments include continental solfataras, deep geothermally heated oil-containing stratifications, shallow marine and deep-sea hot sediments, and hydrothermal vents located as far as 4,000 m below sea level (Table 1). Hyperthermophiles have also been isolated from hot industrial environments (e.g., the outflow of geothermal power plants and sewage sludge systems). Deep-sea hyperthermophiles thrive in environments with hydrostatic pressures ranging from 200 to 360 atm. Some of these species are barotolerant (281) or even barophilic (95, 233, 257). The most thermophilic organism known, P. fumarii, grows in the temperature range of 90 to 113°C. The upper temperature at which life is possible is still unknown, but it is probably not much above 113°C. Above 110°C, molecules such as amino acids and metabolites become highly unstable (ATP is spontaneously hydrolyzed in aqueous solution at temperatures below 140°C) and hydrophobic interactions weaken significantly (163).

Of the more than 70 species, 29 genera, and 10 orders of hyperthermophiles that have been described (320), most are archaea. Thermotogales and Aquificales are the only bacteria (Table 1). Thermotogales and Aquificales are the deepest branches in the bacterial genealogy, and for this reason they represent an obvious interest in evolutionary studies (1). One of the most striking findings extracted from the complete Thermotoga maritima genome sequence (258) is the abundance of evidence supporting lateral gene transfer between archaea and bacteria: (i) 24% of the T. maritima open reading frames (versus 16% in Aquifex aeolicus) encode proteins that are more similar to archaeal than to bacterial proteins; (ii) these archaea-like genes are not uniformly distributed among the biological categories; (iii) 81 of these genes are clustered in 15 4- to 20-kb regions, in which the gene order can be the same as in archaea; and (iv) The T. maritima genome sequence does not have a homogeneous G+C contentamong the 51 regions having significantly different G+C contents, 42 contain "archaea-like" genes.

The archaeal domain is composed so far of two branches: the Crenarchaeota and the Euryarchaeota. A 16S rRNA isolated from a hyperthermophilic environment was recently sequenced that is not related to any other archaeal rRNA. This new rRNA species suggests the existence of a third branch in the archaeal domain, the Korarcheota, that branches deeper in the archaeal tree than the Crenarchaeota and the Euryarchaeota (18). Hyperthermophiles are represented in the Crenarchaeota and Euryarchaeota, and they systematically represent the deepest and shortest lineages in these two branches (see references 140 and 320 for phylogenetic trees). In addition to thermoacidophiles, Crenarchaeota include halophiles. Among the Euryarchaeota, methanogens have mesophilic relatives.

Hyperthermophile communities are complex systems of primary producers and decomposers of organic matter. All hyperthermophilic primary producers are chemolithoautotrophs (i.e., sulfur oxidizers, sulfur reducers, and methanogens) (104, 223). In relation to the high sulfur content of most hot natural biotopes, most hyperthermophiles are facultative or obligate chemolithotrophs: they either reduce S⁰ with H₂ to produce H₂S (the anaerobes) or oxidize S⁰ with O₂ to produce sulfuric acid (the aerobes). Extremely acidophilic hyperthermophiles belong to the order Sulfolobales. They are all strict aerobes (e.g., Sulfolobus) or facultative aerobes (e.g., Acidianus), and they have been isolated almost exclusively from continental solfataras (Table 1). While most heterotrophs are obligate sulfur reducers, all members of the Thermotogales and most members of the Pyrococcales and Thermococcales can grow independently of S⁰, obtaining their energy from fermentations (Table 1). Because of the extremely low organic matter content of their submarine environments, hyperthermophilic heterotrophs typically obtain their energy and carbon from complex mixtures of peptides derived from the decomposition of primary producers. A few species are able to use polysaccharides (e.g., starch, pectin, glycogen, and chitin); to date, Archeoglobus profundus is the only known species that uses organic acids.

BIOCHEMICAL AND MOLECULAR PROPERTIES OF HYPERTHERMOPHILIC ENZYMES

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Thermal and Catalytic Properties

Most enzymes characterized from hyperthermophiles are optimally active at temperatures close to the host organism's optimal growth temperature, usually 70 to 125°C (see references 139 and 349 for lists of purified hyperthermophilic enzymes and their properties). Extracellular and cell-bound hyperthermophilic enzymes (i.e., saccharidases and proteases) are optimally active at temperatures abovesometimes far abovethe host organism's optimum growth temperature and are, as a rule, highly stable. For example, Thermococcus litoralis amylopullulanase is optimally active at 117°C, which is 29°C above the organism's optimum growth temperature of 88°C (43). While they are usually less thermophilic than extracellular enzymes purified from the same host, intracellular enzymes (such as xylose isomerases) are usually optimally active at the organism's optimal growth temperature. Only a few enzymes have been described that are optimally active at 10 to 20°C below the organism's optimum growth temperature (108, 197, 278). While most hyperthermophilic enzymes are intrinsically very stable, some intracellular enzymes get their high thermostability from intracellular factors such as salts, high protein concentrations, coenzymes, substrates, activators, or general stabilizers such as thermamine.

Arrhenius plots for hyperthermophilic and mesophilic enzymes are typically linear (20, 29, 62), suggesting that mesophilic and hyperthermophilic enzyme functional conformations remain unchanged throughout their respective temperature ranges. If enzyme structures changed in a catalytically significant manner with increasing temperature, one would expect to find (i) nonlinear Arrhenius plots for most enzymes and (ii) different types of plots for different enzyme classes. Biphasic Arrhenius plots reported for a number of hyperthermophilic enzymes (58, 98, 101, 133, 366) represent an important exception to the typical Arrhenius-like behavior. Biphasic Arrhenius plots can often be correlated with functionally significant conformational changes, detected by spectroscopic methods (101, 133, 222). Although not much information is typically available on the effect of temperature on the activity of mesophilic enzymes, a few examples exist of mesophilic enzymes showing bent Arrhenius plots (110), suggesting that such discontinuities are not a specific trait of hyperthermophilic enzymes.

With the exception of phylogenetic variations, what differentiates hyperthermophilic and mesophilic enzymes is only the temperature ranges in which they are stable and active. Otherwise, hyperthermophilic and mesophilic enzymes are highly similar: (i) the sequences of homologous hyperthermophilic and mesophilic proteins are typically 40 to 85% similar (79, 350); (ii) their three-dimensional structures are superposable (16, 63, 143, 160, 227, 284, 327); and (iii) they have the same catalytic mechanisms (22, 350, 386).

More than 100 genes from hyperthermophiles have been cloned and expressed in mesophiles. Most of this work has been done in the last 5 years. Only a small fraction of them have been isolated by direct expression and activity screening (i.e., by complementation of growth or activity assay) of a genomic library in Escherichia coli (Table 2). Most other genes from hyperthermophiles have been isolated by hybridization or have been directly cloned after PCR amplification. Since archaeal transcription systems (including promoter sequences) are more closely related to eucaryal than to bacterial systems, it is not surprising that most archaeal genes are expressed in E. coli only when they are cloned under the control of strong promoters (plac, ptac, or T7 RNA polymerase promoter). Pyrococcal intergenic regions are particularly AT- rich, and E. coli consensus promoter-like sequences can be found that explain why some P. furiosus genes are directly expressed in E. coli (85, 86, 343). Another difficulty encountered in expressing archaeal genes in E. coli can be low expression due to a significantly different codon usage in the expressed gene. This difficulty is often alleviated by the expression in E. coli of rare tRNA genes together with the target gene (344). A few genes from hyperthermophilic archaea have been successfully expressed in yeast systems (77). They are able to complement yeast mutations (90, 275, 282).

When the properties of the native and recombinant hyperthermophilic enzymes are compared, the majority of hyperthermophilic enzymes expressed in E. coli retain all of the native enzyme's biochemical properties, including proper folding (121), thermostability, and optimal activity at high temperatures (8, 14, 115, 338, 350). Thus, while a few proteins from hyperthermophiles might require extrinsic factors (e.g., salts or polyamines), or posttranslational modifications (e.g., glycosylation) to be fully thermostable, most proteins from hyperthermophiles are intrinsically thermostable, and they can fold properly even at temperatures 60°C below their physiological conditions. The fact that most hyperthermophilic enzymes are properly expressed and folded in E. coli has greatly facilitated their study, since they can be purified from E. coli rather than from an often hard-to-grow hyperthermophilic organism. Additional indirect evidence for the correct folding of recombinant hyperthermophilic proteins is the fact that crystal structures of recombinant hyperthermophilic proteins are typically similar to that of their mesophilic homologues (160, 183, 227, 284, 327, 368). The idea that recombinant and native hyperthermophilic protein structures are identical has become so widely accepted that in some studies both the native and recombinant enzymes are used indifferently in crystallization studies (5).

It is unclear whether all hyperthermophilic proteins can be expressed in a mesophilic environment, since unsuccessful experiments are typically not reported. So far, fewer than 10% of all the hyperthermophilic enzymes expressed in E. coli have been reported to have stability, catalytic, or structural properties different from those of the enzyme purified from the native organism (51, 239). The recombinant P. furiosus ornithine carbamoyltransferase was as stable as the native enzyme when it was expressed in Saccharomyces cerevisiae but was less stable when expressed in E. coli. When expressed in E. coli, the Sulfolobus solfataricus 5'-methylthioadenosine phosphorylase (a hexameric enzyme containing six intersubunit disulfide bridges) forms incorrect disulfide bridges and is less stable and less thermophilic than the native enzyme (51). The recombinant P. furiosus glutamate dehydrogenase (GDH) is a partially active hexamer that can be fully activated upon incubation at 90°C but remains less stable than the native P. furiosus GDH (202). Such hyperthermophilic enzymes might require posttranslational modifications (e.g., glycosylation) or specific chaperones to reach their fully functional and stable folded state.

A current working hypothesis is that hyperthermophilic enzymes are more rigid than their mesophilic homologues at mesophilic temperatures and that rigidity is a prerequisite for high protein thermostability. This hypothesis is supported by a growing body of experimental data that includes frequency domain fluorometry and anisotropy decay (229), hydrogen-deuterium exchange (35, 164, 370), and tryptophan phosphorescence (114) experiments. Figure 1 illustrates one of the hydrogen-deuterium exchange experiments. At 20°C a much smaller fraction of the amide protons in Sulfolobus acidocaldarius adenylate kinase (53%) are exchanged than in the porcine cytosolic enzyme (83%), indicating that considerable more amide protons are involved in stable hydrogen bonds in the thermophilic enzyme. Temperatures of 80 to 90°C are needed before S. acidocaldarius adenylate kinase can show an exchange level comparable to that of the catalytically active mesophilic enzyme (35). In protein structure determination, atomic temperature factors provide an adequate representation of local flexibility. In a 1987 study, Vihinen (351) calculated protein flexibility indexes for mesophilic and thermophilic proteins, starting from normalized atomic temperature factors. His results showed that flexibility decreased as thermostability increased. This study needs to be updated since Vihinen's sample was small and did not include data on hyperthermophilic proteins. A computer simulation showed that a mesophilic rubredoxin was more flexible, on the picosecond timescale, than its P. furiosus homologue at room temperature (201).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Hydrogen-deuterium exchange recorded in S. acidocaldarius and porcine muscle cytosol adenylate kinases during a temperature gradient experiment. Fractions of unexchanged protons as a function of temperature were calculated from the normalized amide II intensities at 1,546 cm1 (S. acidocaldarius enzyme) and 1,542 cm1 (porcine enzyme). The exchange was completed at 56 and 97°C for the porcine and S. acidocaldarius enzymes, respectively. Reprinted from reference 35 with permission of the publisher. (Note that the two enzymes are not directly comparable because the pig enzyme is a monomer whereas the Sulfolobus enzyme is a trimer.)

While most flexibility comparisons in mesophilic and hyperthermophilic proteins have reached the same conclusion that hyperthermophiliic proteins are more rigid enzymes, one recent study (134) does not support this conclusion. Using amide hydrogen exchange data, Hernández et al. show that (i) all the hydrogen bonding involving the amide hydrogens of P. furiosus rubredoxin are disrupted in less than 1 s at temperatures close to P. furiosus rubredoxin's temperature of maximal thermodynamic stability; (ii) conformational opening for solvent access takes place in the millisecond range for the entire protein; and (iii) at alkaline pHs, the maximum enthalpy contributed by hydrogen-bonded amides accounts for less than 5% of the total activation enthalpy normally associated with protein unfolding. These results suggest that the most stable protein characterized so far shows a degree of conformational flexibility comparable to that of mesophilic proteins.

Lazaridis et al. (201) argue that there is no single measure of flexibility (a protein can be rigid on a nanosecond scale but flexible on a millisecond scale) and that there is no fundamental reason for stability and rigidity to be correlated. Flexibility implies increased conformational entropy of the folded state, and it should therefore be favorable to thermodynamic stability. More studies on hyperthermophilic enzyme flexibility at various temperatures are needed before we can get a better understanding of the role of conformational rigidity in protein stability.

It has also been proposed that excessive rigidity explains why hyperthermophilic enzymes are often inactive at low temperatures (i.e., around 20 to 37°C). One set of evidence that tends to support this hypothesis is that denaturants (e.g., guanidinium hydrochloride and urea) (23, 195, 364), detergents (e.g., Triton X-100 and sodium dodecyl sulfate) (82, 283, 290), and solvents (78, 195) often activate hyperthermophilic enzymes at suboptimal temperatures. This activation tends to disappear as the temperature gets closer to the enzyme's temperature of maximal activity (T_opt) (23). At that temperature, the enzyme is flexible enough in the absence of a denaturant to show full activity. Recent findings that show increasing levels of hydrogen tunneling with increasing temperature in a thermophilic alcohol dehydrogenase provide additional evidence for the role of thermally induced protein motions in modulating enzyme activity (190). A few hyperthermophilic enzymes have been characterized that are more active than their mesophilic counterparts, even at 37°C (156, 246, 315). Since they are thermostable, these enzymes are expected to be quite rigid at mesophilic temperatures. Their high catalytic activity at mesophilic temperatures suggests that these enzymes combine local flexibility in their active site (which is responsible for their activity at low temperatures) with high overall rigidity (which is responsible for their thermostability). The existence of such enzymes (and of highly stable, engineered mesophilic enzymes [116, 374]) also suggests that thermostability is not incompatible with high activity at moderate temperatures. Hyperthermophiles probably only need enzymes with activities at their optimal temperatures comparable to that of their mesophilic homologues. While there is probably no evolutionary pressure for an organism to have more efficient enzymes, this does not mean that more efficient thermostable enzymes cannot be engineered in the laboratory.

The free energy of stabilization (G_stab, where G_stab = H_stab  TS_stab) of a protein is the difference between the free energies of the folded and the unfolded states of that protein. It directly measures the thermodynamic stability of the folded protein. H_stab (the stabilization enthalpy) and S_stab (the stabilization entropy) are large numbers that vary almost linearly with temperature in the temperature range of the activities of most enzymes. Also a function of temperature, G_stab is usually small (83, 162) (Table 3). The G_stab of globular mesophilic proteins is typically between 5 and 15 kcal/mol at 25°C (Table 3). Not many proteins have been studied to determine the free energies of stabilization. Such studies are hindered by the fact that the thermal denaturation of most proteins is irreversible: complete denaturation is often almost immediately followed by aggregation and precipitation (see below). Thus, most G_stab data are for small monomeric proteins (277) (Table 3). G_stab calculations are made even more difficult for hyperthermophilic proteins, since their denaturation transitions take place outside the temperature range of most calorimeters (141, 274). To overcome this difficulty, most thermodynamic studies of hyperthermophilic protein stability are performed in the presence of guanidinium hydrochloride (168) or at pHs outside the physiological conditions (241). These various conditions allow the temperature of the denaturation transition to become accessible to physical measurement, and in some cases they allow the enzyme to unfold reversibly. In one case, the stability parameters of a hyperthermophilic protein were determined under native conditions using hydrogen exchange to measure the reversible cycling between the native and unfolded proteins (141). Table 3 shows that in most cases the difference in G_stab values of hyperthermophilic and mesophilic proteins is small, usually in the range of 5 to 20 kcal/mol. Stability studies of enzyme mutants (173, 261), showing that differences in G_stab as small as 3 to 6.5 kcal/mol can account for thermostability increases of up to 12°C, are in complete agreement with the stability data listed in Table 3.

View this table: [in this window] [in a new window]   TABLE 3.   Comparison of the Gstab-versus-T curves for some mesophile, thermophile, and hyperthermophile proteins

As a consequence of the enthalpic and/or entropic stabilizations occurring in a hyperthermophilic protein, the G_stab-versus-T curve of this protein will be different from that of its mesophilic counterpart. Figure 2 illustrates the three theoretical ways by which increased protein thermodynamic stability can be achieved (265): (a) the G_stab-versus-T curve of a hyperthermophilic protein can be shifted toward higher G_stab values, (b) it can be shifted toward higher temperatures, or (c) it can be flattened (due to a smaller difference in partial molar heat capacity between the protein's folded and unfolded states [C_p]). As seen in Table 3, a majority of thermophilic and hyperthermophilic proteins use various combinations of these three mechanisms to reach their superior thermodynamic stabilities. For example, the G_stab-versus-T curve of the P. furiosus histone is shifted by approximately 12°C toward higher temperatures and by 10 kcal/mol toward higher G_stab values, compared to the G_stab-versus-T curve of the Methanobacterium formicicum histone. The most common stabilization mechanism among both thermophilic and hyperthermophilic proteins is the shift of their G_stab-versus-T curves toward higher G_stab values.

View larger version (8K): [in this window] [in a new window]   FIG. 2.   Comparison of theoretical Gstab-versus-T curves for mesophilic and hyperthermophilic proteins. M, theoretical Gstab-versus-T curve for a mesophilic protein. (a), (b), and (c), theoretical Gstab-versus-T curves for a hyperthermophilic protein. In curve (a), the hyperthermophilic protein has the same temperature of maximal stability (Ts) as the mesophilic protein, and the Gstab-versus-T curve of the hyperthermophilic protein is shifted upward to higher Gstab values. In curve (b), hyperthermophilic and mesophilic protein have same Ts values and the same Gstab values at Ts. The Gstab-versus-T curve of the hyperthermophilic protein is flatter. In curve (c), hyperthermophilic and mesophilic proteins have different Ts values but have the same Gstab at their respective Ts. The Gstab-versus-T curve of the hyperthermophilic protein is shifted toward higher temperatures.

MECHANISMS OF PROTEIN INACTIVATION

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Unfolding, Formation of Scrambled Structures, and Aggregation

Native, active proteins are held together by a delicate balance of noncovalent forces (e.g., H bonds, ion pairs, and hydrophobic and Van der Waals interactions). When high temperatures disrupt these noncovalent interactions, proteins unfold. Protein unfolding can be observed by different techniques, including differential scanning calorimetry, fluorescence, circular dichroism spectroscopy, viscosity, and migration patterns. The T_m, as determined by calorimetry and spectroscopic techniques, is typically the same (216). Numerous studies have shown that inactivation becomes significant only a few degrees below the T_m. In most cases, the loss of secondary and tertiary structures is concomitant with enzyme inactivation at high temperature. Small monomeric proteins commonly unfold via a two-state transition (i.e., unfolding intermediates are barely detectable or not detectable). Some proteins might regain their native, active conformation upon cooling. This unfolding is called thermodynamically reversible unfolding, and the thermodynamic parameters describing the folded and unfolded states can be determined (it is most easily done using calorimetry data) (17, 277).

Most mesophilic proteins, however, unfold irreversibly. They unfold into inactive but kinetically stable structures (scrambled structures), and they often form aggregates (intermolecular mechanism). During aggregation, the hydrophobic residues that are normally buried in the native protein become exposed to the solvent and interact with hydrophobic residues from other unfolding protein molecules to minimize their exposure to the solvent (354). Such irreversible unfolding usually follows the general model proposed by Tomazic and Klibanov (334):
This model is consistent with an intramolecular rate-determining step in thermal inactivation. The natural logarithm of the residual activity is a linear function of the inactivation time:
where k is the inactivation rate and t is the inactivation time. In this model, the inactivation rate constant is independent of the initial protein concentrations.

Hyperthermophilic proteins that denature reversibly are probably as rare as reversibly denaturing mesophilic proteins. High E_a values for inactivation of hyperthermophilic enzymes (above 100 kcal/mol) suggest that the limiting step in their inactivation is still unfolding (55, 268, 352). These different observations suggest that chemical modifications (e.g., deamidation, cysteine oxidation, and peptide bond hydrolysis) take place only once the protein is unfolded. Accelerated at elevated temperatures, chemical modifications are another process that make denaturation irreversible.

While there have been numerous studies of mesophilic enzymes affected by deamidation in vivo (reference 367 and references therein), it is still unclear whether some hyperthermophilic proteins are inactivated via covalent mechanisms. Studies performed with a few enzymes (e.g., hen egg white lysozyme, RNase A, and Bacillus -amylases) at temperatures neighboring or even above their melting temperatures clearly showed that elevated temperatures trigger chemical modifications that irreversibly inactivate reversibly denatured proteins (6, 334, 335, 369).

Deamidation. Two deamidation mechanisms are known for Asn and Gln residues (367), but it not often known which mechanism is responsible for an enzyme deamidation. In the general acid-base mechanism, a general acid (HA) protonates the Asn (or Gln) amido (---NH) group. A general base (A or OH) attacks the carbonyl carbon of the amido group or activates another nucleophile (Fig. 3). The transition state is supposed to be an oxyanion tetrahedral intermediate. The order of the acid and base attacks varies with pH. In the -aspartyl shift mechanism, the Asn side chain amide group is attacked by the n + 1 peptide nitrogen (acting as a nucleophile). The succinimide intermediate then breaks down to yield an -linked (Asp) or -linked (isoAsp) residue, typically in the ratio 1:3 (Fig. 3). In this mechanism, Gly, Ser, and Ala are favored in n + 1 because their small side chains do not obstruct the cyclization into the succinimide intermediate. In both deamidation mechanisms, conformation and rigidity seem to be instrumental in limiting the extent of deamidation. Conformation probably also explains the approximately 10-fold-higher propensity of Asn to deamidate than Gln.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Mechanisms of protein degradation involving Asn residues.

Hydrolysis of peptide bonds. Hydrolysis of peptide bonds happens most often at the C-terminal side of Asp residues, with the Asp-Pro bond being the most labile of all (354). Two factors seem to be responsible for this lability. The proline nitrogen is more basic than that of other residues, and Asp has an increased propensity for - isomerization when linked on the N side of a proline. Peptide chain cleavage can also occur at Asn-Xaa linkages in a -aspartyl shift-like mechanism (367). In this reaction the Asn amido (---NH₂) group acts as the nucleophile, attacking its own main-chain carboxyl carbon (Fig. 3) (132). Such cleavage occurs in five positions in the M. fervidus GAPDH when conditions favor unfolding (i.e., temperatures above 85°C and low salt concentrations). Less susceptible to hydrolysis, the more thermostable P. woesei GAPDH contains substitutions in three of these cleavage positions. Cleavage at the two remaining Asn-Xaa locations is probably inhibited by the higher conformational rigidity of the P. woesei enzyme.

-Elimination of disulfide bridges. Destruction of disulfide bridges under alkaline conditions is known to occur via a -elimination reaction, yielding dehydroalanine and thiocysteine. Dehydroalanine then reacts with nucleophilic groupsespecially the -amino group of lysineto form lysinoalanine. The fate of thiocysteine is not completely understood (354). The -elimination reaction produces free thiols that can catalyze disulfide interchange and further inactivate the enzyme (369).

Cysteine oxidation. Cysteines are the most reactive amino acids in proteins. Their autooxidation, usually catalyzed by metal cations (especially copper), leads to the formation of intramolecular and intermolecular disulfide bridges or to the formation of sulfenic acid (354). Cysteines can also catalyze disulfide interchange, causing disulfide bond reshuffling as well as important structural variations. The recombinant S. solfataricus 5'-methylthioadenosine phosphorylase forms incorrect intersubunit disulfide bridges that make it less stable and less thermophilic than the native enzyme (51).

Other reactions. Aside from the above commonly observed degradative reactions, other, less frequent chemical inactivation mechanisms have been identified (354). Methionine can be oxidized to its sulfoxide counterpart, and some residues (Asp and Ser, in particular) can be racemized to their D-form. Lysine can react with reducing sugars via the Maillard reaction (279). Last, thermolysin-like neutral proteases are susceptible to autolysis. Local unfolding of one of their surface loops determines their inactivation by autolysis (93).

The hydrophobic effect is considered to be the major driving force of protein folding (83). Hydrophobicity drives the protein to a collapsed structure from which the native structure is defined by the contribution of all types of forces (e.g., H bonds, ion pairs, and Van der Waals interactions). Dill (83) reviewed the evidences supporting this theory: (i) nonpolar solvents denature proteins; (ii) hydrophobic residues are typically sequestered into a core, where they largely avoid contact with water; (iii) residues and hydrophobicity in the protein core are more strongly conserved and related to structure than any other type of residue (replacements of core hydrophobic residues are generally more disruptive than other types of substitutions); and (iv) protein unfolding involves a large increase in heat capacity. Given the central role of the hydrophobic effect in protein folding, it was easy to assume that the hydrophobic effect is also the major force responsible for protein stability. The sequencing, structure, and mutagenesis information accumulated in the last 20 years confirm that hydrophobicity is, indeed, a main force in protein stability. Two observations suggest that mesophilic and hyperthermophilic homologues have a common basic stability afforded by the conserved protein core: (i) hydrophobic interactions and core residues involved in secondary structures are better conserved than surface area features, and (ii) numerous stabilizing substitutions are found in solvent-exposed areas (as observed in mesophilic and hyperthermophilic protein structures comparisons and in protein directed-evolution experiments, see below). The high level of similarity encountered in the core of mesophilic and hyperthermophilic protein homologues suggests that even mesophilic proteins are packed almost as efficiently as possible and that there is not much room left for stabilization inside the protein core. Stabilizing interactions in hyperthermophilic proteins are often found in the less conserved areas of the protein. As illustrated below, factors such as surface ion pairs, decrease in solvent-exposed hydrophobic surface, and anchoring of "loose ends" (i.e., the N and C termini and loops) to the protein surface seem to be instrumental in hyperthermophilic protein thermostability.

Enough experimental evidence (e.g., sequence, mutagenesis, structure, and thermodynamics) has been accumulated on hyperthermophilic proteins in recent years to conclude that no single mechanism is responsible for the remarkable stability of hyperthermophilic proteins. Increased thermostability must be found, instead, in a small number of highly specific mutations that often do not obey any obvious traffic rules.

Protein amino acid composition has long been thought to be correlated to its thermostability. The first statistical analyses comparing amino acid compositions in mesophilic and thermophilic proteins indicated trends toward substitutions such as GlyAla and LysArg. A higher alanine content in thermophilic proteins was supposed to reflect the fact that Ala was the best helix-forming residue (10). As more experimental data accumulate (in particular, complete genome sequences), it is becoming obvious that "traffic rules of thermophilic adaptation cannot be defined in terms of significant differences in the amino acid composition" (31). The comparison of residue contents in hyperthermophilic and mesophilic proteins based on the genome sequences of eight mesophilic and seven hyperthermophilic organisms shows only minor trends (Table 4). More charged residues are found in hyperthermophilic proteins (+3.24%) than in mesophilic proteins, mostly at the expense of uncharged polar residues (4.98%; in particular Gln, 2.21%). Hyperthermophilic proteins also contain slightly more hydrophobic and aromatic residues than mesophilic proteins do. These data obtained from genome sequencing cannot be generalized, since large variations exist among hyperthermophile genomes themselves: the Aeropyrum pernix protein pool actually contains fewer charged residues (23.64%), fewer large hydrophobic residues (27.29%), and fewer aromatic residues (7.42%) than do the mesophiles listed in Table 4. Instead, A. pernix proteins contain more Ala, Gly, Pro, Ser, and Thr residues. Thus, a bias in a hyperthermophilic protein amino acid composition might often be evolutionarily relevant, rather than an indication of its adaptation to high temperatures. Probably more relevant to thermostability than amino acid composition are the distribution of the residues and their interactions in the protein. The two homologous proteases Bacillus amyloliquefaciens subtilisin BPN' and Thermoactinomyces vulgaris thermitase contain the same number of charged residues, but the thermophilic enzyme thermitase contains eight more ion pairs (331).

In relation to the idea that protein stability was determined by the stability and tight packing of its core, the propensity of the individual residues to participate in helical or strand structures was studied as a potential stability mechanism. In their comparison of mesophilic and thermophilic protein structures, Facchiano et al. (99) observed that helices of thermophilic proteins are generally more stable than those of mesophilic proteins. The only trend they detected was a decreasing content in -branched residues (Val, Ile, and Thr) in the helices of thermophilic proteins (-branched residues are not as well tolerated in helices as linear residues are) (99). A number of examples exist in which this trend is not followed. The P. furiosus and T. litoralis GDHs contain a larger number of isoleucines. If Leu and Ile residues are compared, these two residues have the highest (and equivalent) partial specific volumes. In proteins, the Leu side chain is most often found in one of two rotamer conformations (1 of 180° and 300°) but not in the one with 1 = 60°. The Ile side chain frequently adopts four different rotamer conformations, and the three 1 values are found. With this conformational flexibility, Ile might be better able to fill various voids that can occur during protein core packing (38). Dill (83) also noted that context effects (e.g., salt bridge formation, aromatic interactions, burial of hydrophobic surface, and cavity filling) could be as important as the intrinsic helical propensity. In many cases, secondary structures found in protein structures do not correspond to the secondary structures predicted by intrinsic propensity, suggesting that intrinsic propensity is not enough to account for the stability of -helices in proteins (83).

Several properties of Arg residues suggest that they would be better adapted to high temperatures than Lys residues: the Arg -guanido moiety has a reduced chemical reactivity due to its high pK_a and its resonance stabilization. The -guanido moiety provides more surface area for charged interactions than the Lys amino group does. Figure 4 illustrates the ability of Arg to participate in multiple noncovalent interactions. Because the Arg side chain contains one fewer methylene group than Lys, it has the potential to develop less unfavorable contacts with the solvent. Last, because its pK_a (approximately 12) is 1 unit above that of Lys (11.1), Arg more easily maintains ion pairs and a net positive charge at elevated temperatures (pK_a values drop as the temperature increases) (252, 354). The average Arg/Lys ratios in the protein pools of the mesophiles and hyperthermophiles listed in Table 4 (0.73 ± 0.37 and 0.87 ± 0.60, respectively) are associated with large standard deviations. (Among hyperthermophiles, Arg/Lys ratios vary from 0.52 in Aquifex aeolicus proteins to 2.19 in Aeropyrum pernix proteins.) These results suggest that if an increased Arg content is indeed stabilizing, this mechanism is not universally used among hyperthermophiles.

View larger version (75K): [in this window] [in a new window]   FIG. 4.   Stereo view of the ion pair between Arg19 and Asp111 in S. solfataricus indole-3-glycerol phosphate synthase. The Arg19 guanidinium group also forms a cation- interaction with the Tyr93  system and two H bonds with Thr84. Reprinted from reference 185 with permission of the publisher.

An indirect indication that deamidation affects hyperthermophilic proteins (156) is the high activity of T. maritima L-isoaspartyl methyltransferase. This enzyme methylates L-isoAsp residues that result from Asn deamidation or from Asp isomerization. Its high activity suggests that it has been adapted for the high load of protein damage that could occur at high temperatures. Resistance to deamidation seems to result from at least three adaptation mechanisms. (i) Some hyperthermophilic enzymes contain less Asn than their mesophilic homologues do. P. woesei 3-phosphoglycerate kinase (PGK) contains less Asn than the Methanobacterium bryantii enzyme does. In both Asn-Ala and two of the three Asn-Gly sequences present in M. bryantii PGK, the Asn residue is substituted in the P. woesei enzyme (136). The only conserved Asn-Gly sequence is conserved in all PGKs. It is possible that the four nonconserved sequences would have been susceptible to deamidation at high temperatures and that they have been selected against in the hyperthermophilic PGK. A direct correlation was also shown between the Asn+Gln content in type II D-xylose isomerases and their respective temperatures of maximal activity (ranging from 55 to 95°C) (350). (ii) Other hyperthermophilic enzymes contain as many Asn residues, but these residues are in locations and in conformations in which they are not susceptible to deamidation. The resistance of P. woesei GAPDH to deamidation and peptide bond hydrolysis was shown to be related to the enzyme's higher conformational stability (132). S. solfataricus 5'-methylthioadenosine phosphorylase is optimally active at 120°C, and its T_m is 132°C. It is not inactivated after 2 h at 100°C (52). It is interesting that it contains twice as many Asn as a related enzyme from E. coli, including one Asn in the sequence Asn-Gly, a sequence normally highly susceptible to deamidation.

The Asn and Gln contents listed in Table 4 suggest that hyperthermophilic proteins do not acquire their resistance to deamidation only through a decreased Asn content. Instead, it is curious that the seven hyperthermophiles show the same significant decrease in Gln residues in their proteins.

Cysteine's high sensitivity to oxidation at high temperature suggests that hyperthermophilic enzymes contain fewer cysteines than their mesophilic counterparts do. While Table 4 indicates that hyperthermophilic proteins in average contain fewer cysteines than mesophilic proteins do, large variations exist among species. Archaeoglobus fulgidus and Methanococcus jannaschii proteins contain more cysteines (1.17 and 1.27%, respectively), in fact, than an average mesophile protein pool does (1.10%). From the seven hyperthermophilic organisms included in Table 4, A. aeolicus and A. pernix are microaerophilic and aerophilic organisms, respectively, whereas the others are strict anaerobes. Interestingly, A. aeolicus and A. pernix proteins contain more cysteines (0.79 and 0.93%, respectively) than Pyrococcus abyssi, P. horikoshii, and T. maritima proteins do (0.55, 0.63, and 0.71%, respectively). One would expect a high selection pressure against the presence of cysteines in proteins from aerobic hyperthermophiles (and the absence of such selection pressure in anaerobic hyperthermophiles). Cysteines that are present in proteins from aerobic hyperthermophiles are often involved in specific stabilizing interactions (e.g., disulfide bridges and metal liganding) and/or are inaccessible to the solvent. Drastic denaturing conditions are required (2 h at 70°C in the presence of 6 M guanidinium HCl) for 10 mM dithiothreitol to reduce most of the six intersubunit disulfide bridges in native S. solfataricus 5'-methylthioadenosine phosphorylase (51). In contrast, the GAPDH from the anaerobe T. maritima contains three Cys residues, one of them essential in the active site and two others described by Schultes et al. as "unnecessary" (299).

Disulfide bridges are believed to stabilize proteins mostly through an entropic effect, by decreasing the entropy of the protein's unfolded state (237). The entropic effect of the disulfide bridge increases in proportion to the logarithm of the number of residues separating the two cysteines bridged.

Because of the susceptibility of cysteines and disulfide bridges to destruction at high temperatures, 100°C was believed to be the upper limit for the stability of proteins containing disulfide bridges (353). This notion was based on the fact that early studies characterizing protein inactivation mechanisms were performed with the only enzymes available at that time: mesophilic enzymes. These studies determined that all proteins studied that contained disulfide bridges had the same rate of -elimination at 100°C. This rate was independent of the protein structure and was higher at pH 8.0 (t_1/2 of 1 h) than at pH 6.0 (t_1/2 of 12.4 h). The limitation of these studies was that at 100°C all the proteins studied were in the unfolded state. The recent characterization of disulfide bridge-containing proteins that are optimally active and stable at temperatures above 100°C suggests that disulfide bridges can be a stabilization strategy above 100°C and that conformational environment and solvent accessibility are determining factors in the protection of disulfide bridges against destruction. When expressed in E. coli, S. solfataricus 5'-methylthioadenosine phosphorylase forms incorrect, destabilizing disulfide bridges. This observation indirectly suggests that the disulfide bridges present in the native enzyme are stabilizing (52). An Aquifex pyrophilus serine protease was recently described that contains eight cysteines (none are present in subtilisin BPN') (64). A dithiothreitol treatment reduced its t_1/2 at 85°C from 90 h to less than 2 h. This destabilization by dithiothreitol at high temperature suggests that this enzyme indeed contains disulfide bridges and that they are highly inaccessible. The enzyme's 6-h t_1/2 at 105°C and pH 9.0, which is much longer than the t_1/2 calculated for disulfide bridges in unfolded proteins at pH 8.0 (1 h), suggests that this enzyme's disulfide bridges are protected from destruction by their inaccessibility in the protein. Thus, not all disulfide bridges have equal susceptibility to thermal destruction.

As suggested in Table 4 and illustrated in Table 5, hydrophobic interactions are a stabilization mechanism in hyperthermophilic proteins. An average increase in stability of 1.3 (± 0.5) kcal/mol was calculated for each additional methyl group buried in protein folding (269) (based on cavity-creating mutations in which a large aliphatic residue was replaced with a smaller aliphatic residue). Mutations attempting to fill cavities are often less stabilizing when they create unfavorable Van der Waals interactions that need local rearrangements (158). While Table 5 gives crystallographic evidence for the potential role of hydrophobic interactions in thermostability, not much direct, experimental evidence is available to confirm the stabilizing role of hydrophobic interactions in hyperthermophilic proteins. The stability properties of an enzyme chimera constructed between the Methanococcus voltae and M. jannaschii adenylate kinases indicated that a larger and more hydrophobic enzyme core (which is due to an increase in aliphatic residue content and in aliphatic side chain volume) may be responsible for M. jannaschii adenylate kinase's thermostability (124). The 3-isopropylmalate dehydrogenase from the thermophile Thermus thermophilus contains intersubunit hydrophobic interactions that do not exist in the E. coli enzyme. Thermus 3-isopropylmalate dehydrogenase Leu246Glu/Val249Met and E. coli Glu256Leu/Met259Val mutant derivatives were constructed that destabilized and stabilized the Thermus and E. coli enzymes, respectively. Polyacrylamide gel electrophoresis of the mutant and wild-type enzymes in the presence of urea showed that the hydrophobic interactions made the dimer more resistant to dissociation (180).

Aromatic-aromatic interactions (aromatic pairs) are defined by a distance of less than 7.0 Å between the phenyl ring centroids. The following characteristics of aromatic pairs were extracted from the analysis of 272 aromatic pairs in 34 high-resolution structures of mesophilic proteins: in two-thirds of the pairs, the interacting rings are not far from perpendicular; most are involved in a network; most link distinct secondary structural elements (i.e., nonlocal interactions); most are energetically favorable (80% have potential energies between 0 and 2 kcal/mol); and most take place between buried or partially buried residues (50). Among the hyperthermophilic proteins whose structures have been solved (Table 5), at least one might be stabilized by extra aromatic interactions. P. furiosus -amylase also contains 5% more aromatic residues than the homologue from Bacillus licheniformis, but is it unknown whether these additional residues are involved in stabilizing interactions (85). A few examples also exist among thermophilic proteins. Thermitase, the serine proteinase produced by Thermoactinomyces vulgaris, contains 16 aromatic residues involved in aromatic pairs; the mesophilic homologue Bacillus amyloliquefaciens subtilisin BPN' contains only 6 aromatic pairs (331). Two clusters of aromatic interactions also exist in the Thermus RNase H that are not present in the E. coli enzyme (159). The solvent-exposed aromatic pair, Tyr13-Tyr17, in B. amyloliquefaciens RNase was replaced with Ala or Phe residues (single and double mutations). Both Tyr-Tyr and Phe-Phe pairs contributed approximately -1.3 kcal/mol toward thermodynamic stabilization (303).

Another type of interaction involving aromatic residues exists in proteins, but it has not been studied in relation to thermostability. In cation- interactions, positive charges (most often metal cations but possibly cationic side chains of Arg and Lys) typically interact with the center of the aromatic ring (an example is shown in Fig. 4). The stabilization energy of the cation- interaction does not decrease as a function of 1/r³ but, rather, exhibits a 1/rⁿ dependence with n < 2, which resembles more a Coulombic (1/r) than a hydrophobic interaction. The low dependence of the cation- interaction on distanceand the fact that Phe, Tyr, and Trp do not have high desolvation energies and can easily be accommodated in hydrophobic environmentsmakes these interactions a potential stabilization mechanism (88).

H bonds are typically defined by a distance of less than 3 Å between the H donor and the H acceptor and by donor-hydrogen-acceptor angle below 90°. The effect of hydrogen bonds on RNase T1 stability has been carefully studied (307). RNase T1 contains 86 H bonds with an average length of 2.95 Å. Their contribution to RNase T1 stability (approximately 110 kcal/mol, as determined by mutagenesis and unfolding experiments) was found to be comparable to the contribution of hydrophobic interactions; individual H bonds contributed an average of 1.3 kcal/mol to the stabilization (307). Because the identification of H bonds is highly dependent on the distance cutoff and because a number of hyperthermophilic protein structures have not been refined to sufficiently high resolutions, studying the role of H bonds in thermostability by structure analysis has not provided clear-cut answers.

One study done by Tanner et al. showed a strong correlation between GAPDH thermostability and the number of charged-neutral H bonds (i.e., between a side chain atom of a charged residue and either a main chain atom of any residue or a side chain atom of a neutral residue) (330). Tanner et al. list two reasons why this type of H bond might be particularly thermodynamically stabilizing: (i) the desolvation penalty associated with burying such H bonds is less than the desolvation penalty for burying an ion pair (that involves two charged residues), and (ii) the enthalpic reward of a charged-neutral H bond is greater than that of a neutral-neutral H bond because of the charge-dipole interaction. This correlation between charged-neutral H bonds and GAPDH stability suggests that the role of charged residues in protein stabilization may not be limited to forming ion pairs. An increased number of charged-neutral H bonds was also found in the T. maritima ferredoxin (Table 5). These H bonds either stabilize the structure of turns or anchor turns to one another.

Because ion pairs are usually present in small numbers in proteins and because they are not highly conserved, they are not a driving force in protein folding (83). Earlier work by Perutz (272) had suggested, however, that electrostatic interactions represent a significant stabilizing force in folded proteins. He stated that ion pairs are stronger in proteins than in solvents because they are formed between fixed charges. (In bulk water, solvation makes the stability of opposite charges almost independent of distance.) A single ion pair was calculated to be responsible for a 3 to 5-kcal/mol stabilization of T4 lysozyme (7). The desolvation contribution [G(desolvation)] to the free energy of folding associated with bringing oppositely charged side chains together is large and unfavorable. It has been suggested that ion pairs are destabilizing in proteins, because this G(desolvation) is not sufficiently compensated by the electrostatic energy provided by the ion pair. This unfavorable G(desolvation), however, decreases at high temperatures, partially because of a decrease in the water dielectric constant. This reduction is almost entirely electrostatic, primarily affecting the surface charged residues (the water molecules are less ordered and, on average, farther away from charged residues at high temperatures). Thus, charged residues tend to rearrange their conformations to improve their direct electrostatic interactions among each other, and the loss in solvation free energy is almost exactly compensated by a gain in interaction energy with other charged residues in the protein (80, 94). While ion pairing might not be the optimum stabilizing mechanismor might even be destabilizing for mesophilic proteinsit can represent a strong stabilizing mechanism for hyperthermophilic proteins, as illustrated in Table 5. P. furiosus GDH is 34% identical to the Clostridium symbiosum enzyme. The main difference between these two enzyme structures is found in their ion pair contents (Table 6). A higher percentage of charged residues participate in ion pairs, in particular Arg (90% of all the Arg residues in the P. furiosus GDH form ion pairs). The P. furiosus enzyme contains 0.11 ion pairs per residue against 0.06 in the C. symbiosum GDH (the average for mesophilic enzymes is approximately 0.04). Arg residues form ion pairs plus H bonds with the carboxylic acids. The ion pairs form large networks that crisscross the protein surface and the subunit interfaces. P. furiosus GDH's largest ion pair network (Fig. 5) is composed of 24 residues (belonging to four different subunits) connected by 18 ion pairs. Ion pair networks are energetically more favorable than an equivalent number of isolated ion pairs, because for each new pair the burial cost is cut in half: only one additional residue must be desolvated and immobilized (368).

The stabilizing potential of buried ion pairs has been investigated, but it remains controversial because of the large G(desolvation) associated with burying two charged residues. In a recent study using continuum electrostatic calculations, an average G(desolvation) of +12.9 ±5.6 kcal/mol was calculated for buried ion pairs. The large G(desolvation) was compensated for by the large Coulombic energy created by the ion pair. (Buried ion pairs are in a low dielectric-constant environment and thus are not exposed to a large screening.) The study's conclusion was that salt bridges with favorable geometry were likely to be stabilizing anywhere in the protein (196). Four additional buried ion pairs between -helices have been suggested as a stabilization mechanism in P. kodakaraensis O⁶-methylguanine-DNA methyltransferase. For these four pairs, distances are short: between 2.74 and 3.02 &Arin