Different unfolding pathways for mesophilic and thermophilic homologues of serine hydroxymethyltransferase

To determine how much information can be transferred from folding and unfolding studies of one protein to another member of the same family or between the mesophilic and thermophilic homologues of a protein, we have characterized the equilibrium unfolding process of the dimeric enzyme serine hydroxymethyltransferase (SHMT) from two sources, Bacillus subtilis (bsSHMT) and Bacillus stearothermophilus (bstSHMT). Although the sequences of the two enzymes are highly identical ( approximately 77%) and homologous (89%), bstSHMT shows a significantly higher stability against both thermal and urea denaturation than bsSHMT. The GdmCl-induced unfolding of bsSHMT was found to be a two-step process with dissociation of the native dimer, resulting in stabilization of a monomeric species, followed by the unfolding of the monomeric species. A similar unfolding pathway has been reported for Escherichia coli aspartate aminotransferase, a member of the type I fold family of PLP binding enzymes such as SHMT, the sequence of which is only slightly identical ( approximately 14%) with that of SHMT. In contrast, for bstSHMT, a highly cooperative unfolding without stabilization of any monomeric intermediate was observed. These studies suggest that mesophilic proteins of the same structural family even sharing a low level of sequence identity may follow a common unfolding mechanism, whereas the mesophilic and thermophilic homologues of the same protein despite having a high degree of sequence identity may follow significantly different unfolding mechanisms.     

Stability and immunological cross-reactivity of malate dehydrogenases from mesophilic and thermophilic sources

The thermostability in vitro of dimeric and tetrameric malate dehydrogenases [S)-malate:NAD+ oxidoreductase, EC 1.1.1.37) from mesophilic and thermophilic bacteria shows a good correlation to the growth temperature of the source organism but no consistent relationship to enzyme subunit structure. The thermophile malate dehydrogenases are, in general, more resistant to the surfactants, sodium dodecyl sulphate (SDS) and hexadecyltrimethylammonium bromide, and to the denaturants, guanidinium chloride and urea, than their mesophilic counterparts, with the dimer in each thermal class being more resistant to the chemical perturbants than the tetramer. Sedimentation analysis suggests that denaturation of the malate dehydrogenases by acid-periodate or SDS produces discrete subunits, whereas denaturation by guanidinium chloride followed by carboxymethylation yields ill-defined protein species. SDS and acid-periodate were therefore preferred to generate denatured malate dehydrogenases for use as immunogens and antigens. The native malate dehydrogenases exhibit immunological cross-reactivity only when they are in the same oligomeric form and derived from closely related species, which may, however, be from different thermal classes. Taking immunological cross-reactivity as an indicator of structural similarity, this supports the idea that the thermophilic trait evolved independently within each phyletic line. With denatured malate dehydrogenases as immunogens and antigens, cross-reactivity is manifested between all the malate dehydrogenases examined. This suggests that appreciable primary structural homology exists between the malate dehydrogenases, whether dimeric or tetrameric, from thermophiles and mesophiles and from various taxa.     

Electrostatic contributions to the stability of a thermophilic cold shock protein

The thermophilic Bacillus caldolyticus cold shock protein (Bc-Csp) differs from the mesophilic Bacillus subtilis cold shock protein B (Bs-CspB) in 11 of the 66 residues. Stability measurements of Schmid and co-workers have implicated contributions of electrostatic interactions to the thermostability. To further elucidate the physical basis of the difference in stability, previously developed theoretical methods that treat electrostatic effects in both the folded and the unfolded states were used in this paper to study the effects of mutations, ionic strength, and temperature. For 27 mutations that narrow the difference in sequence between Bc-Csp and Bs-CspB, calculated changes in unfolding free energy (Delta G) and experimental results have a correlation coefficient of 0.98. Bc-Csp appears to use destabilization of the unfolded state by unfavorable charge-charge interactions as a mechanism for increasing stability. Accounting for the effects of ionic strength and temperature on the electrostatic free energies in both the folded and the unfolded states, explanations for two important experimental observations are presented. The disparate ionic strength dependences of Delta G for Bc-Csp and Bs-CspB were attributed to the difference in the total charges (-2e and -6e, respectively). A main contribution to the much higher unfolding entropy of Bs-CspB was found to come from the less favorable electrostatic interactions in the folded state. These results should provide insight for understanding the thermostability of other thermophilic proteins.     

Interface Matters: The Stiffness Route to Stability of a Thermophilic Tetrameric Malate Dehydrogenase

In this work we investigate by computational means the behavior of two orthologous bacterial proteins, a mesophilic and a thermophilic tetrameric malate dehydrogenase (MalDH), at different temperatures. Namely, we quantify how protein mechanical rigidity at different length- and time-scales correlates to protein thermophilicity as commonly believed. In particular by using a clustering analysis strategy to explore the conformational space of the folded proteins, we show that at ambient conditions and at the molecular length-scale the thermophilic variant is indeed more rigid that the mesophilic one. This rigidification is the result of more efficient inter-domain interactions, the strength of which is further quantified via ad hoc free energy calculations. When considered isolated, the thermophilic domain is indeed more flexible than the respective mesophilic one. Upon oligomerization, the induced stiffening of the thermophilic protein propagates from the interface to the active site where the loop, controlling the access to the catalytic pocket, anchors down via an extended network of ion-pairs. On the contrary in the mesophilic tetramer the loop is highly mobile. Simulations at high temperature, could not re-activate the mobility of the loop in the thermophile. This finding opens questions on the similarities of the binding processes for these two homologues at their optimal working temperature and suggests for the thermophilic variant a possible cooperative role of cofactor/substrate.     

Comparison of the folding processes of T. thermophilus and E. coli ribonucleases H

In order to examine how the stabilization of thermophilic proteins affects their folding, we have characterized the folding process of Thermus thermophilus ribonuclease H using circular dichroism, fluorescence, and pulse-labeling hydrogen exchange. Like its homolog from Escherichia coli, this thermophilic protein populates a partially folded kinetic intermediate within the first few milliseconds of folding. The structure of this intermediate is similar to that of E.coli RNase H and corresponds remarkably well to a partially folded form that is populated at low levels in the native state of the protein. Proline isomerization appears to partly limit the folding of the thermophilic but not the mesophilic protein. Lastly, unlike other thermophilic proteins, which unfold much more slowly than their mesophilic counterparts, T.thermophilus RNase H folds and unfolds with overall rates similar to those of E.coli RNase H.     

A simple strategy for the purification of large thermophilic proteins overexpressed in mesophilic microorganisms: application to multimeric enzymes from Thermus sp. strain T2 expressed in Escherichia coli

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an alpha-galactosidase and a beta-galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.     

The thermostability of DNA-binding protein HU from mesophilic, thermophilic, and extreme thermophilic bacteria

Based on primary structure comparison between four highly homologous DNA-binding proteins (HUs) displaying differential thermostability, we have employed in vitro site-directed mutagenesis to decipher their thermostability mechanism at the molecular level. The contribution of the 11 amino acids that differ between the thermophilic HUBst from Bacillus stearothermophilus (Tm = 61.6 degrees C) and the mesophilic HUBsu from Bacillus subtilis (Tm = 39.7 degrees C) was evaluated by replacing these amino acids in HUBst with their mesophilic counterparts. Among 11 amino acids, three residues, Gly-15, Glu-34, and Val-42, which are highly conserved in the thermophilic HUs, have been found to be responsible for the thermostability of HUBst. These amino acids in combination (HUBst-G15E/E34D/V42I) reduce the thermostability of the protein (Tm = 45.1 degrees C) at the level of its mesophilic homologue HUBsu. By replacing these amino acids in HUBsu with their thermophilic counterparts, the HUBsu-E15G/D34E/142V mutant was generated with thermostability (Tm = 57.8 degrees C) at the level of thermophilic HUBst. Employing the same strategy, we generated several mutants in the extremely thermophilic HUTmar from Thermotoga maritima (Tm = 80.5 degrees C), and obtained data consistent with the previous results. The triplet mutant HUTmar-G15E/E34D/V421 (Tm = 35.9 degrees C) converted the extremely thermophilic protein HUTmar to mesophilic. The various forms of HU proteins were overproduced in Escherichia coli, highly purified, and the thermostability of the mutants confirmed by circular dichroism spectroscopy. The results presented here were elucidated on the basis of the X-ray structure of HUBst and HUTmar (our unpublished results), and their mechanism was proposed at the molecular level. The results clearly show that three individual local interactions located at the helix-turn-helix part of the protein are responsible for the stability of HU proteins by acting cooperatively in a common mechanism for thermostability.     

An enhanced thermostability in thermophilic 5-S ribonucleic acids under physiological salt conditions.

The secondary structure of 5-S rRNAs of Thermus aquaticus (an extreme thermophile), Bacillus stearothermophilus (a moderate thermophile) and Escherichia coli (a mesophile) was compared using thermal denaturation techniques under varying ionic conditions. At a low ionic strength (10 mM K+), the Tm of T. aquaticus 5-S RNA differed by only 1 degrees C from that of E. coli RNA and the molecule was fully denatured well below the optimum growth temperature of the thermophile. The internal Na+, K+ and Mg2+ concentrations of T. aquaticus cells were determined to be 91 mM, 130 mM and 59 mM, respectively. Under these salt conditions, T. aquaticus 5-S RNA was significantly more stable than E. coli RNA and the 5-S RNA from B. stearothermophilus was intermediate as is its optimum growth temperature. The results suggest that the thermostability of macromolecules from thermophilic organisms may be specially dependent on the internal salt concentration. Furthermore, under these salt conditions, most of the secondary structure of the RNA remained stable at the optimum growth temperatures suggesting that ribosomal RNAs of thermophilic organisms contribute more to the thermostability of the ribosome than previously thought.     

Comparison of the molten globule states of thermophilic and mesophilic alpha-amylases

In recent years great interest has been generated in the process of protein folding, and the formation of intermediates during the folding process has been proven with new experimental strategies. In the present work, we have examined the molten globule state of Bacillus licheniformis alpha-amylase (BLA) by intrinsic fluorescence and circular dichroism spectra, 1-anilino naphthalene-8-sulfonate (ANS) binding and proteolytic digestion by pepsin, for comparison to its mesophilic counterpart, Bacillus amyloliquefaciens alpha-amylase (BAA). At pH 4.0, both enzymes acquire partially folded state which show characteristics of molten globule state. They unfold in such a way that their hydrophobic surfaces are exposed to a greater extent compared to the native forms. Chemical denaturation studies by guanidine hydrochloride and proteolytic digestion with pepsin show that molten globule state of BLA is more stable than from BAA. Results from gel filtration indicate that BAA has the same compactness at pH 4.0 and 7.5. However, molten globule state of BLA is less compact than its native state. The effects of polyols such as trehalose, sorbitol and glycerol on refolding of enzymes from molten globule to native state were also studied. These polyols are effective on refolding of mesophilic alpha-amylase but only slightly effect on BLA refolding. In addition, the folding pathway and stability of intermediate state of the thermophilic and the mesophilic alpha-amylases are discussed.     

Monomeric malate synthase from a thermophilic Bacillus. Molecular and kinetic characteristics

The molecular weight of malate synthase purified from a thermophilic Bacillus was determined to be 62,000 by sedimentation equilibrium methods, confirming the value obtained earlier by the gel filtration technique. This enzyme and its homologs from other bacteria, which are all monomeric proteins with molecular weights of approximately 60,000. therefore differ from the considerably larger and multimeric malate synthases from yeast, Neurospora crassa, and other eucaryotic microorganisms and plants. Amino acid analysis reveals the thermophile synthase to be relatively rich in glutamic acid and to have a higher content of arginine in comparison with the yeast enzyme. The Bacillus enzyme is an acidic protein with an isoelectric pH of 4.6 and has two sulfhydryl groups titratable with 5,5′-dithiobis(2-nitrobenzoic acid). Its parameters indicative of its overall hydrophobicity and of levels of helicity and turn, which were deduced from the amino acid composition, lie well within the range recorded for a number of mesophile and thermophile enzymes. However, the level of β-sheet structure is considerably lower than that calculated for the yeast synthase; this supports a trend recently observed for certain other thermophile proteins. The synthase isolated from the thermophilic Bacillus appears to be homogeneous by several criteria, although upon electrophoresis in the native state in polyacrylamide it yields two protein bands that are both enzymatically active. Several kinetic characteristics of this enzyme are also reported.     

Patterns of temperature adaptation in proteins from the bacteria Deinococcus radiodurans and Thermus thermophilus

Asymmetrical patterns of amino acid substitution in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are generally taken to indicate selection favoring different amino acids at different temperatures due to their biochemical properties. If that were the case, comparisons of different pairs of mesophilic and thermophilic taxa would exhibit similar patterns of substitutional asymmetry. A previous comparison of mesophilic versus thermophilic Methanococcus with mesophilic versus thermophilic Bacillus revealed several pairs of amino acids for which one amino acid was favored in thermophilic Bacillus and the other was favored in thermophilic Methanococcus. Most of this could be explained by the higher G+C content of the DNA of thermophilic Bacillus, a phenomenon not seen in the Methanococcus comparison. Here, I compared the mesophilic bacterium Deinococcus radiodurans and its thermophilic relative Thermus thermophilus, which are similar in G+C content. Of the 190 pairs of amino acids, 83 exhibited significant substitutional asymmetry, consistent with the pervasive effects of selection. Most of these significantly asymmetrical pairs of amino acids were asymmetrical in the direction predicted from the Methanococcus data, consistent with thermal adaptation resulting from universal biochemical properties of the amino acids. However, 12 pairs of amino acids exhibited asymmetry significantly different from and in the opposite direction of that found in the Methanococcus comparison, and 21 pairs of amino acids exhibited asymmetry that was significantly different from that found in the Bacillus comparison and could not be explained by the greater G+C content in thermophilic Bacillus. This suggests that selection due to universal biochemical properties of the amino acids and differences in G+C content are not the only causes of substitutional asymmetry between mesophiles and thermophiles. Instead, selection on taxon-specific properties of amino acids, such as their metabolic cost, may play a role in causing asymmetrical patterns of substitution.     

Different packing of external residues can explain differences in the thermostability of proteins from thermophilic and mesophilic organisms

MOTIVATION: Understanding the basis of protein stability in thermophilic organisms raises a general question: what structural properties of proteins are responsible for the higher thermostability of proteins from thermophilic organisms compared to proteins from mesophilic organisms? RESULTS: A unique database of 373 structurally well-aligned protein pairs from thermophilic and mesophilic organisms is constructed. Comparison of proteins from thermophilic and mesophilic organisms has shown that the external, water-accessible residues of the first group are more closely packed than those of the second. Packing of interior parts of proteins (residues inaccessible to water molecules) is the same in both cases. The analysis of amino acid composition of external residues of proteins from thermophilic organisms revealed an increased fraction of such amino acids as Lys, Arg and Glu, and a decreased fraction of Ala, Asp, Asn, Gln, Thr, Ser and His. Our theoretical investigation of folding/unfolding behavior confirms the experimental observations that the interactions that differ in thermophilic and mesophilic proteins form only after the passing of the transition state during folding. Thus, different packing of external residues can explain differences in thermostability of proteins from thermophilic and mesophilic organisms. AVAILABILITY: The database of 373 structurally well-aligned protein pairs is available at http://phys.protres.ru/resources/termo_meso_base.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Cation-induced thermostability of yeast and Escherichia coli pyrophosphatases

Inorganic pyrophosphatases (PPiases) from both yeast and Escherichia coli were found to be stable against heat denaturation in the presence of Mg2+, as previously observed with the enzymes from thermophilic bacteria. No loss of activity was observed after 1 h of incubation at 50 degrees C and pHs between 6 and 9 in the yeast enzyme, and at 60 degrees C and pHs between 7.2 and 9.2 in the E. coli enzyme. Such an induced thermostability of the E. coli enzyme was detected when Mn2+, Co2+, Ca2+, Cd2+, and Zn2+ were added in place of Mg2+. On the other hand, the degree of induced thermostability of the yeast enzyme was dependent upon the divalent cations used, and Ni2+ and Cu2+ accelerated the heat inactivation. On adding the divalent cations, the difference spectra of the E. coli enzyme always showed negative peaks in the ultraviolet region, but those of the yeast enzyme changed again depending upon the divalent cations. The circular dichroism spectra in the near ultraviolet region of both enzymes greatly differed from each other, but both were not affected so much by adding the divalent cations unlike the thermophilic enzymes from Bacillus stearothermophilus and thermophilic bacterium PS-3. Yeast and E. coli PPiases did not cross-link with the anti-immunoglobulin G's from the thermophilic enzymes, but the thermophilic enzymes did with each other's antisera. The results in the present study indicated that the conformation of PPiase, in which the aromatic amino acid residues were buried in the interior of the protein molecule, was very important for the thermostability and also that the protein structures of PPiases from B. stearothermophilus and thermophilic bacterium PS-3 were very similar to each other, but were very different from those of the mesophilic enzymes.     

A proposed mechanism of thermophily in facultative thermophiles

Glyceraldehyde-3-phosphate dehydrogenase in crude extracts of $\text{Bacillus coagulans}$ KU, a facultative thermophile, showed marked thermolability whether the cells were grown at mesophilic or thermophilic temperature. When extracts were brought to a given ionic strength by the addition of salt and then heat treated, it was possible to confer heat resistance well in excess of the thermophilic growth temperature. Disc electrophoresis is indicative that portions of the profiles are quite different between extracts of cells grown at 37° and 55°. Based on the data, a mechanism of thermophily is postulated that is different from any thus far proposed for thermophilic microorganisms.     

The N-terminal region is crucial for the thermostability of the G-domain of Bacillus stearothermophilus EF-Tu

Bacterial elongation factor Tu (EF-Tu) is a model monomeric G protein composed of three covalently linked domains. Previously, we evaluated the contributions of individual domains to the thermostability of EF-Tu from the thermophilic bacterium Bacillus stearothermophilus. We showed that domain 1 (G-domain) sets up the basal level of thermostability for the whole protein. Here we chose to locate the thermostability determinants distinguishing the thermophilic domain 1 from a mesophilic domain 1. By an approach of systematically swapping protein regions differing between G-domains from mesophilic Bacillus subtilis and thermophilic B. stearothermophilus, we demonstrate that a small portion of the protein, the N-terminal 12 amino acid residues, plays a key role in the thermostability of this domain. We suggest that the thermostabilizing effect of the N-terminal region could be mediated by stabilizing the functionally important effector region. Finally, we demonstrate that the effect of the N-terminal region is significant also for the thermostability of the full-length EF-Tu.     

Isocitrate Lyase from a Thermophilic Bacillus: Effect of Salts on Enzyme Activity

The isocitrate lyase from a thermophilic Bacillus is activated about threefold by a variety of salts. Such strong stimulation of activity is not seen with isocitrate lyase from the mesophiles, Bacillus licheniformis, Bacillus megaterium, Escherichia coli, and Aspergillus nidulans. The salt activation is markedly pH-dependent. At pH values above 8.6, salt (KCl) indeed inhibits the enzyme activity. Potassium chloride also causes a significant shift of the pH optimum of the enzyme towards the acid side. As the temperature of the enzyme reaction is raised, activation becomes progressively weaker. Potassium chloride also affords considerable protection against enzyme denaturation at 55 C. The activation and the stabilization, however, appear to be independent effects. Of six other enzymes in the thermophile that were examined, isocitrate dehydrogenase was equally strongly activated by KCl and malate synthase was less strongly, but significantly, activated; citrate synthase, malate dehydrogenase, glutamate dehydrogenase, and lactate dehydrogenase were unaffected or slightly inhibited by KCl. The property of being strongly activated by salt appears to be a peculiar characteristic of the thermophile isocitrate lyase and possibly evolved concomitantly with its thermostability.     

Bacterial elongation factor Ts: isolation and reactivity with elongation factor Tu.

An improved method for the purification of bacterial polypeptide elongation factor Ts (EF-Ts) from one mesophile (Escherichia coli) and two thermophiles (Bacillus stearothermophilus and PS3) is described. The improvements are both in the facility of isolation and in increased yields. The purified factors were used for cross-reactivity studies with elongation factor Tu (EF-Tu) obtained from the same bacterial strains. In all combinations studied, the efficiency of EF-Ts in catalyzing the exchange of EF-Tu-bound GDP was proportional to the strength of the protein-protein complex. Whereas the factors from the two thermophiles were interchangeable, the mesophilic EF-Ts formed a very weak complex with thermophilic EF-Tu; however, thermophilic EF-Ts formed very strong complexes with mesophilic EF-Tu. Thus, e.g., EF-Tu from E. coli formed a complex with EF-Ts from B. stearothermophilus which was 10 times more stable than the corresponding homologous complex.     

Alkaline pH-dependent differential unfolding characteristics of mesophilic and thermophilic homologs of dimeric serine hydroxymethyltransferase

Environmental variables such as pH can significantly influence the folding and stability of a protein molecule. In the present investigation, we compared the alkaline pH-induced unfolding of two homologous serine hydroxymethyltransferase from mesophilic Bacillus subtilis (bsSHMT) and thermophilic Bacillus stearothermophilus (bstSHMT) using various biophysical techniques. The thermophilic enzyme bstSHMT was found to be more resistant to alkaline denaturation compared to its mesophilic counterpart, bsSHMT. Unfolding studies using domain-swapped chimera, constructed by swapping the C-terminal domain of these two wild-type proteins, revealed that C-terminal domain plays a pivotal role in the folding, stability and subunit interaction of these proteins. Primary amino acid sequence analysis of the proteins showed that bsSHMT has six unconserved lysine residues in C-terminal domain, which are absent in bstSHMT. Chemical modification of lysine side chains resulted in stabilization of monomers, only in case of bsSHMT. Moreover, comparison between homology model of bsSHMT with the crystal structure of bstSHMT revealed that a small stretch of 11 amino acids at the end of C-terminal domain was found protruding outside the molecule as a flexible coiled structure in bsSHMT. Taken together these findings suggest that possibly the presence of these non-identical lysine moieties and a small extension of C-terminal domain may be responsible for low stability of bsSHMT under alkaline pH condition.     

Isolation of thermophilic mutants of Bacillus subtilis and Bacillus pumilus and transformation of the thermophilic trait to mesophilic strains

Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10 degrees C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 degrees C and 70 degrees C at a frequency of less than 10(-10). The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10(-3) to 10(-2) for single markers and about 10(-7) for two unlinked markers. With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10(-7), suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.