Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity.

Mutations in aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase, which selectively inhibit phosphatase activity have been isolated. These mutations yield amino acid substitutions within a 113-residue region of this 578-residue protein. These mutations may define a regulatory domain of this protein.     

Structural and thermodynamic studies on a salt-bridge triad in the NADP-binding domain of glutamate dehydrogenase from Thermotoga maritima: cooperativity and electrostatic contribution to stability

Cooperative interactions within ion-pair networks of hyperthermostable proteins are thought to be a major determinant for extreme protein stability. While the favorable thermodynamic contributions of optimized electrostatics in general as well as those of pairwise interactions have been documented, cooperativity between pairwise interactions has not yet been studied thermodynamically in proteins from hyperthermophiles. In this study we use the isolated cofactor binding domain of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima to analyze pairwise and cooperative interactions within the salt-bridge triad Arg190-Glu231-Lys193. The X-ray structure of the domain was solved at 1.43 A and reveals the salt-bridge network with surrounding solvent molecules in detail. All three participating charges in the network were mutated to alanine in all combinations. The X-ray structure of the variant lacking all three charges reveals that the removal of the side chains has no effect on the overall conformation of the protein. Using solvent denaturation and thermodynamic cycles, the interaction energies between each pair of residues in the network were determined in the presence and in the absence of the third residue. Both the Arg190-Glu231 ion pair and the Lys193-Glu231 salt bridge in the absence of the third residue, contribute favorably to the free energy for unfolding of the domain in urea. Using guanidinium chloride as denaturant reveals a strong cooperativity between the two ion-pair interactions, the presence of the second ion pair converts the first interaction from destabilizing into stabilizing by as much as 1.09 kcal/mol. The different energetics of the salt-bridge triad in urea and GdmCl are discussed with reference to the observed anion binding in the crystal structure at high ionic strength and their possible role in a highly charged, high-temperature environment such as the cytoplasm of hyperthermophiles.     

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.     

Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media.

Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN', also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueous-solvent-compatible enzymes.     

The consensus concept for thermostability engineering of proteins: further proof of concept

Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.     

Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease

Marble brain disease (MBD) also known as Guibaud-Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded beta-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619-633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.     

Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein

Twenty-five different temperature-sensitive point mutations at 20 sites in the lysozyme gene of bacteriophage T4 have been identified. All of the mutations alter amino acid side chains that have lower than average crystallographic thermal factors and reduced solvent accessibility in the folded protein. This suggests that the amino acids with well-defined conformations can form specific intramolecular interactions that make relatively large contributions to the thermal stability of the protein. Residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, suggesting that, in general, such amino acids contribute less to protein stability. The pattern of the sites of ts substitutions observed in the folded conformation of T4 lysozyme suggests that severe destabilizing mutations that primarily affect the free energy of the unfolded state are rare. These results indicate that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.     

Properties and gene structure of the Thermotoga maritima alpha-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium.

Thermotoga maritima MSB8 has a chromosomal alpha-amylase gene, designated amyA, that is predicted to code for a 553-amino-acid preprotein with significant amino acid sequence similarity to the 4-alpha-glucanotransferase of the same strain and to alpha-amylase primary structures of other organisms. Upstream of the amylase gene, a divergently oriented open reading frame which can be translated into a polypeptide with similarity to the maltose-binding protein MalE of Escherichia coli was found. The T. maritima alpha-amylase appears to be the first known example of a lipoprotein alpha-amylase. This is in agreement with observations pointing to the membrane localization of this enzyme in T. maritima. Following the signal peptide, a 25-residue putative linker sequence rich in serine and threonine was found. The amylase gene was expressed in E. coli, and the recombinant enzyme was purified and characterized. The molecular mass of the recombinant enzyme was estimated at 61 kDa by denaturing gel electrophoresis (63 kDa by gel permeation chromatography). In a 10-min assay at the optimum pH of 7.0, the optimum temperature of amylase activity was 85 to 90 degrees C. Like the alpha-amylases of many other organisms, the activity of the T. maritima alpha-amylase was dependent on Ca2+. The final products of hydrolysis of soluble starch and amylose were mainly glucose and maltose. The extraordinarily high specific activity of the T. maritima alpha-amylase (about 5.6 x 10(3) U/mg of protein at 80 degrees C, pH 7, with amylose as the substrate) together with its extreme thermal stability makes this enzyme an interesting candidate for biotechnological applications in the starch processing industry.     

Hyperthermostabilization of Bacillus licheniformis alpha-amylase and modulation of its stability over a 50 degrees C temperature range

Bacillus licheniformis alpha-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories. For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability. This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA. Remarkably, the Q264S-N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function. The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23 degrees C higher than the wild-type enzyme. In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25 degrees C in the half-inactivation temperature of the double mutant enzyme compared with wild-type. These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant. Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50 degrees C through a few point mutations.     

Stability and reconstitution of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima.

The molecular basis of thermal stability of globular proteins is a highly significant yet unsolved problem. The most promising approach to its solution is the investigation of the structure-function relationship of homologous enzymes from mesophilic and thermophilic sources. In this context, D-glyceraldehyde-3-phosphate dehydrogenase has been the most extensively studied model system. In the present study, the most thermostable homolog isolated so far is described with special emphasis on the stability of the enzyme under varying solvent conditions. D-Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima is an intrinsically thermostable enzyme with a thermal transition temperature around 110 degrees C. The amino acid sequence, electrophoresis, and sedimentation analysis prove the enzyme to be a homotetramer with a gross structure similar to its mesophilic counterparts. The enzyme in the absence and in the presence of its coenzyme, NAD+, exhibits no drastic structural differences except for a 3% change in sedimentation velocity reflecting slight alterations in the quaternary structure of the enzyme. At low temperature, in the absence of denaturants, neither "cold denaturation" nor subunit dissociation are detectable. Guanidinium chloride and pH-dependent deactivation precede the decrease in fluorescence emission and ellipticity, suggesting a complex denaturation mechanism. An up to 3-fold activation of the enzyme at low guanidinium concentration may be interpreted in terms of a compensation of the tight packing of the thermophilic enzyme at low temperature. Under destabilizing conditions, e.g. moderate concentrations of chaotropic agents, low temperature favors denaturation. The effect becomes important in reconstitution experiments after preceding guanidinium denaturation; the reactivation yield at low temperature drops to zero, whereas between 35 and 80 degrees C reactivation exceeds 80%. Shifting the temperature from approximately 0 degrees C to greater than or equal to 30 degrees C releases a trapped tetrameric intermediate in a fast reaction. Concentration-dependent reactivation experiments prove renaturation of the enzyme to involve consecutive folding and association steps. Reconstitution at room temperature yields the native protein, in spite of the fact that the temperature of the processes in vitro and in vivo differ by more than 60 degrees C.     

Structure determination of the glutamate dehydrogenase from the hyperthermophile Thermococcus litoralis and its comparison with that from Pyrococcus furiosus.

Glutamate dehydrogenase catalyses the oxidative deamination of glutamate to 2-oxoglutarate with concomitant reduction of NAD(P)(+), and has been shown to be widely distributed in nature across species ranging from psychrophiles to hyperthermophiles. Extensive characterisation of this enzyme isolated from hyperthermophilic organisms has led to its adoption as a model system for analysing the determinants of thermal stability. The crystal structure of the extremely thermostable glutamate dehydrogenase from Thermococcus litoralis has been determined at 2.5 A resolution, and has been compared to that from the hyperthermophile Pyrococcus furiosus. The two enzymes are 87 % identical in sequence, yet differ 16-fold in their half-lives at 104 degrees C. This is the first reported comparative analysis of the structures of a multisubunit enzyme from two closely related yet distinct hyperthermophilies. The less stable T. litoralis enzyme has a decreased number of ion pair interactions; modified patterns of hydrogen bonding resulting from isosteric sequence changes; substitutions that decrease packing efficiency; and substitutions which give rise to subtle but distinct shifts in both main-chain and side-chain elements of the structure. This analysis provides a rational basis to test ideas on the factors that confer thermal stability in proteins through a combination of mutagenesis, calorimetry, and structural studies.     

Extremely thermostable L(+)-lactate dehydrogenase from Thermotoga maritima: cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state.

L(+)-lactate dehydrogenase (LDH; E.C.1.1.1.27) from the hyperthermophilic bacterium Thermotoga maritima has been shown to represent the most stable LDH isolated so far (Wrba A, Jaenicke R, Huber R, Stetter KO, 1990, Eur J Biochem 188:195-201). In order to obtain the enzyme in amounts sufficient for physical characterization, and to analyze the molecular basis of its intrinsic stability, the gene was cloned and expressed functionally in Escherichia coli. Growth of the cells and purification of the enzyme were performed aerobically at 26 degrees C, i.e., ca. 60 degrees below the optimal growth temperature of Thermotoga. Two enzyme species with LDH activity were purified to homogeneity. Crystals of the enzyme obtained at 4 degrees C show satisfactory diffraction suitable for X-ray analysis up to a resolution of 2.8 A. As shown by gel-permeation chromatography, chemical crosslinking, light scattering, analytical ultracentrifugation, and electron microscopy, the two LDH species represent homotetramers and homooctamers (i.e., dimers of tetramers), with a common subunit molecular mass of 35 kDa. The spectroscopic characteristics (UV absorption, fluorescence emission, near- and far-UV CD) of the two species are indistinguishable. The calculated alpha-helix content is 45%, in accordance with the result of homology modeling. Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas KM is unalatered. The extreme intrinsic stability of the protein is reflected by its unaltered catalytic activity over 4 h at 85 degrees C; irreversible thermal denaturation becomes significant at approximately 95 degrees C. The anomalous resistance toward chemical denaturation using guanidinium chloride and urea confirms this observation. Both the high optimal temperature and the pH optimum of the catalytic activity correspond to the growth conditions of T. maritima in its natural habitat.     

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme.The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.     

iPTREE-STAB: interpretable decision tree based method for predicting protein stability changes upon mutations

We have developed a web server, iPTREE-STAB for discriminating the stability of proteins (stabilizing or destabilizing) and predicting their stability changes (delta deltaG) upon single amino acid substitutions from amino acid sequence. The discrimination and prediction are mainly based on decision tree coupled with adaptive boosting algorithm, and classification and regression tree, respectively, using three neighboring residues of the mutant site along N- and C-terminals. Our method showed an accuracy of 82% for discriminating the stabilizing and destabilizing mutants, and a correlation of 0.70 for predicting protein stability changes upon mutations. AVAILABILITY: http://bioinformatics.myweb.hinet.net/iptree.htm. SUPPLEMENTARY INFORMATION: Dataset and other details are given.     

Structural and energetic differences between insertions and substitutions in staphylococcal nuclease.

In a previous study, the small protein staphylococcal nuclease was shown to readily accommodate single alanine and glycine insertions, with average losses in stability comparable to substitutions at the same sites (PROT. 7:299-305, 1990). To more fully explore this unexpected adaptability to changes in residue spacing, 2 double amino acid insertions (alanyl-glycine, glycyl-glycine) and 3 additional single amino acid insertions with dissimilar side chains (proline, leucine, and glutamine) were constructed at 10 of the sites previously studied. At 8 of these sites, the type of amino acid side chain on the inserted residue significantly influenced the stability of the mutant protein. However, at 9 of the 10 sites, the double insertions were found to be no more destabilizing than the single alanine or glycine insertions. In contrast, double substitution mutations of staphylococcal nuclease, which replace two adjacent residues with alanine, do not show this striking degree of non-additivity. A comparison of the effects of single glutamine and single glycine insertions with alanyl-glycine insertions indicates that insertion of alanine into the peptide backbone is, on average, less destabilizing than appending the equivalent atoms onto the side chain of a glycine insertion. To explain their very different energetic effects, we propose that, unlike most substitutions, the inserted residue(s) must induce lateral displacements of the polypeptide chain, forcing the folded conformation away from that of wild type. The resulting obligatory shifts in the positioning of residues flanking the insertion generate a large number of degrees of freedom around which the mutant structure can relax.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disease-associated mutations in the coil 2B domain of human lamin A/C affect structural properties that mediate dimerization and intermediate filament formation

The lamin proteins are essential components of the nuclear lamina of eukaryotic cells, that are involved in a complex association mechanism to attain a functional supermolecular structure. Mutations of the lamin A/C gene are associated with several different neuromuscular diseases, and the detailed effect of disease-associated amino acid substitutions on the structure and stability of human lamin dimers is yet unknown. Here we present a structural and thermodynamic characterization by means of molecular dynamics simulations of the effect of pathological mutations (S326T, R331P, R331Q, E347K, E358K, M371K, and R377H) on the association of the coil 2B domains that mediate lamin A/C oligomerization. The structures attained during the simulations, along with the quantification of the contribution of each residue to the dimerization energies, support a lamin association mechanism mediated by homophilic intermolecular interactions promoted by dissociative conformational changes at distinct positions in the coiled coil. The pathogenic mutations can both increase or decrease the stability of lamin A/C dimers, and a possible correlation between the effect of the amino acid substitutions and disease onset and severity is presented.     

Stabilization of E. coli Ribonuclease HI by the 'stability profile of mutant protein' (SPMP)-inspired random and non-random mutagenesis

The change in the structural stability of Escherichia coli ribonuclease HI (RNase HI) due to single amino acid substitutions has been estimated computationally by the stability profile of mutant protein (SPMP) [Ota, M., Kanaya, S. Nishikawa, K., 1995. Desk-top analysis of the structural stability of various point mutations introduced into ribonuclease H. J. Mol. Biol. 248, 733-738]. As well, an effective strategy using random mutagenesis and genetic selection has been developed to obtain E. coli RNase HI mutants with enhanced thermostability [Haruki, M., Noguchi, E., Akasako, A., Oobatake, M., Itaya, M., Kanaya, S., 1994. A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions. J. Biol. Chem. 269, 26904-26911]. In this study, both methods were combined: random mutations were individually introduced to Lys99-Val101 on the N-terminus of the alpha-helix IV and the preceding beta-turn, where substitutions of other amino acid residues were expected to significantly increase the stability from SPMP, and then followed by genetic selection. Val101 to Ala, Gln, and Arg mutations were selected by genetic selection. The Val101-->Ala mutation increased the thermal stability of E. coli RNase HI by 2.0 degrees C in Tm at pH 5.5, whereas the Val101-->Gln and Val101-->Arg mutations decreased the thermostability. Separately, the Lys99-->Pro and Asn100-->Gly mutations were also introduced directly. The Lys99-->Pro mutation increased the thermostability of E. coli RNase HI by 1.8 degrees C in Tm at pH 5.5, whereas the Asn100-->Gly mutation decreased the thermostability by 17 degrees C. In addition, the Lys99-->Pro mutation altered the dependence of the enzymatic activity on divalent metal ions.