Stabilization of halophilic malate dehydrogenase

Malate dehydrogenase from the extreme halophile, Halobacterium marismortui, is stable only in highly concentrated solutions of certain salts. Previous work has established that its physiological environment is saturated in KCl; it remains soluble is saturated NaCl or KCl solutions; also it unfolds in solutions containing less than 2.5 M-NaCl or -KCl, salt concentrations which are still relatively high. New data show that the structure of this enzyme can be stabilized in a range of high concentrations of Mg2+ or other "salting-in" ions, also with exceptional protein-solvent interactions. "Salting-in" ions, contrary to stabilizing protein structure, usually favour unfolding. These, and most other results concerning the structure, stability and solvent interactions of the protein cannot be understood in terms of the usual effects of salts on protein structure. In this paper, a novel stabilization model is proposed for halophilic malate dehydrogenase that can account for all observations so far. The model results from experiments on the protein in salt solutions chosen for their different effects on protein stability (potassium phosphate, a strongly "salting-out" agent, and MgCl2, which is "salting-in"), and previously published data from NaCl and KCl solutions (mildly "salting-out"). Enzymic activity and stability measurements were combined with neutron scattering, ultracentrifugation and quasi-elastic light-scattering experiments. The analysis showed that the structure of the protein in solution as well as the dominant stabilization mechanisms were different in different salt solutions in which this enzyme is active. Thus, in molar concentrations of phosphate ions, stabilization and hydration are similar to those of non-halophilic soluble proteins, in which the hydrophobic effect dominates. In high concentrations of KCl, NaCl or MgCl2, on the other hand, solution particles are formed in which the protein dimer interacts with large numbers of salt and water molecules (the mass of solvent molecules involved depends on the nature of the salt but it is approximately equivalent to the protein mass). It is proposed that, under these conditions, the hydrophobicity of the protein core is too weak to stabilize the folded structure and the main stabilization mechanism is the formation of co-operative hydrate bonds between the protein and hydrated salt ions. Model predictions are in agreement with all experimental results, such as the different numbers of solvent molecules found in the solution particles formed with different salts, the loss of the exceptional solvent interactions concomitant with unfolding at non-physiological salt concentrations, and the different temperature denaturation curves observed for different salt solutions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polypeptide elongation factor Tu from Halobacterium marismortui

A GDP-binding protein of 60 kDa from Halobacterium marismortui has been purified to homogeneity. The purification has been carried out in high-salt buffers or in 50% glycerol buffers to protect the halophilic protein from denaturation. Evidence that this protein is the halophilic elongation factor Tu (hEF-Tu) is provided by the high homology of its N terminus with the corresponding sequences of other EF-Tus, and by immunological studies. Like some other EF-Tus the native protein can be cleaved with trypsin without concomitant loss of GDP-binding ability. The molecular mass of this hEF-Tu is higher than that for the corresponding factors from other sources including the halobacterium Halobacterium cutirubrum. The protein possesses typical halophilic characteristics, in that it is stable and active in 3 M KCl or 2 M (NH4)2SO4. Some other properties, like autofragmentation under sample treatment before SDS-PAGE, are described.     

Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber,an extremely halophilic bacterium

The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine. We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations. Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low. We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration. NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt. NaCl was stimulatory with optimum activity at 3-3.5 M. However, no activity was found above 2.5 M KCl. Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably. The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.     

The Oligomeric states of Haloarcula marismortui malate dehydrogenase are modulated by solvent components as shown by crystallographic and biochemical studies

The three-dimensional crystal structure of the (R207S, R292S) mutant of malate dehydrogenase from Haloarcula marismortui was solved at 1.95A resolution in order to determine the role of salt bridges and solvent ions in halophilic adaptation and quaternary structure stability. The mutations, located at the dimer-dimer interface, disrupt two inter-dimeric salt bridge clusters that are essential for wild-type tetramer stabilisation. Previous experiments in solution, performed on the double mutant, had shown a tetrameric structure in 4M NaCl, which dissociated into active dimers in 2M NaCl. In order to establish if the active dimeric form is a product of the mutation, or if it also exists in the wild-type protein, complementary studies were performed on the wild-type enzyme by analytical centrifugation and small angle neutron scattering experiments. They showed the existence of active dimers in NaF, KF, Na(2)SO(4), even in the absence of NADH, and in the presence of NADH at concentrations of NaCl below 0.3M. The crystal structure shows a tetramer that, in the absence of the salt bridge clusters, appears to be stabilized by a network of ordered water molecules and by Cl(-) binding at the dimer-dimer interface. The double mutant and wild-type dimer folds are essentially identical (the r.m.s. deviation between equivalent C(alpha) positions is 0.39A). Chloride ions are also observed at the monomer-monomer interfaces of the mutant, contributing to the stability of each dimer against low salt dissociation. Our results support the hypothesis that extensive binding of water and salt is an important feature of adaptation to a halophilic environment.     

Solution structure of glyceraldehyde-3-phosphate dehydrogenase from Haloarcula vallismortis

The subunit molecular mass of glyceraldehyde-3-phosphate dehydrogenase from the extreme halophile Haloarcula vallismortis (hGAPDH) was determined by mass spectrometry to be 35990 +/- 80 daltons, similar to other GAPDHs. Complementary density, sedimentation and light scattering experiments showed the protein to be a tetramer that binds 0.18 +/- 0.10 gram of water and 0.07 +/- 0.02 gram of KCl per gram of protein, in multimolar KCl solutions. At low salt (below 1 M), the tetramer dissociated into unfolded monomers. This is the third halophilic protein for which solvent interactions were measured. The extent of these interactions depends on the protein, but all form an invariant particle, in multimolar NaCl or KCl solutions, that binds a high proportion of salt when compared to non-halophilic proteins.     

Instability of waves formed by motile bacteria

Abstract Cell-free enzyme preparations of the obligately anaerobic halophilic eubacterium Haloanaerobium praevalens synthesize fatty acids from malonyl-CoA. The reaction is stimulated by NaCl and KCl at a concentration of 1 M, and only slightly inhibited by salt concentrations as high as 3 M. Thus, the fatty acid synthetase of H. praevalens is expected to the fully active at the high intracellular salt concentrations present, and it is the first fatty acid synthetase reported to be active in the presence of high salt concentrations.     

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids

Proteins of halophilic organisms, which accumulate molar concentrations of KCl in their cytoplasm, have a much higher content in acidic amino acids than proteins of mesophilic organisms. It has been proposed that this excess is necessary to maintain proteins hydrated in an environment with low water activity, either via direct interactions between water and the carboxylate groups of acidic amino acids or via cooperative interactions between acidic amino acids and hydrated cations. Our simulation study of five halophilic proteins and five mesophilic counterparts does not support either possibility. The simulations use the AMBER ff14SB force field with newly optimized Lennard-Jones parameters for the interactions between carboxylate groups and potassium ions. We find that proteins with a larger fraction of acidic amino acids indeed have higher hydration levels, as measured by the concentration of water in their hydration shell and the number of water/protein hydrogen bonds. However, the hydration level of each protein is identical at low (b_KCl = 0.15 mol/kg) and high (b_KCl = 2 mol/kg) KCl concentrations; excess acidic amino acids are clearly not necessary to maintain proteins hydrated at high salt concentration. It has also been proposed that cooperative interactions between acidic amino acids in halophilic proteins and hydrated cations stabilize the folded protein structure and would lead to slower dynamics of the solvation shell. We find that the translational dynamics of the solvation shell is barely distinguishable between halophilic and mesophilic proteins; if such a cooperative effect exists, it does not have that entropic signature.     

Structural stability of halophilic proteins

An examination of halobacterial amino acids exchanges as they appear in the known Spirulina platensis [2Fe-2S] ferredoxin tertiary structure indicated that most of the additional acidic residues of the halophiles occurred on the external surface of the alga structure; however, further negative changes were not placed in the ferredoxin active site region. A statistical investigation of the amino acid compositions of seven halophile and nonhalophile protein counterparts indicated that the bulkiness of amino acids used by halophiles is considerably reduced and that the overall hydrophobicity of halophilic and non halophilic molecules was essentially the same. It is suggested that the principal mode of structural stabilization for halophilic proteins is effective competition with the cytoplasmic salt for water through utilization of many external carboxyl groups of glutamic and aspartic acids. A reduction is residue bulkiness would prevent inactivation in the presence of the high molarity, antichaotropic KCl. Halophilic functionality is preserved through avoidance of additional negative charge at the active site surface.     

Structure of halophilic malate dehydrogenase in multimolar KCl solutions from neutron scattering and ultracentrifugation

The structure and solvent interactions of malate dehydrogenase from Halobacterium marismortui in multimolar KCl solvents are found to be similar to those in multimolar NACl solvents reported previously (G. Zaccai, E. Wachtel and H. Eisenberg, J. Mol. Biol. 190 (1986) 97). KCl rather than NaCl is predominant in physiological medium. At salt concentrations up to about 3.0 M, the protein (a dimer of M 87000 g/mol) can be considered to occupy an invariant volume in which it is associated with about 4100 molecules of water and about 520 molecules of salt. At very low resolution, the enzyme particle appears to have a compact protein core and protruding protein parts in interaction with the water and salt components, structural features that are not observed in non-halophilic mitochondrial malate dehydrogenase. The above conclusions were drawn from the analysis of neutron scattering and ultracentrifugation data, and the complementarity of these approaches is discussed extensively.     

Development of Salt-Resistant Active Transport in a Moderately Halophilic Bacterium

The moderately halophilic bacterium Vibrio costicola accumulates α-aminoisobutyric acid (AIB) by active transport. Substantial amounts of Na⁺ ions are needed for this transport. This is not due to an ionic requirement for respiration; cells respire as well as KCl as in NaCl but do not transport AIB in KCl. In cells grown in the presence of 1.0 or 2.0 M NaCl, AIB transport took place in higher NaCl concentrations than in cells grown in the presence of 0.5 M NaCl. The latter cells developed salt-resistant transport when they were exposed to 1.0 M NaCl in the presence of chloramphenicol and other antibiotics that inhibit protein synthesis. Two levels of salt-resistant transport were observed. One level (resistance to 3.0 M NaCl) developed in 1.0 M NaCl without the addition of nutrients, did not seem to require an increase in internal solute concentration, and was not lost when cells grown in 1.0 M NaCl were suspended in 0.5 M NaCl. The second level (resistance to 4.0 M NaCl) developed in 1.0 M NaCl only when nutrients were added, may have required an increased internal solute concentration, and was lost when 1.0 M NaCl-grown cells were suspended in 0.5 M NaCl or KCl. Among the substances that stimulated the development of salt-resistant AIB transport, betaine was especially active. Furthermore, direct addition of betaine permitted cells to transport AIB at higher NaCl concentrations. High salt concentrations inhibited endogenous respiration to a lesser extent than AIB transport, especially in 0.5 M NaCl-grown cells. Thus, these concentrations of salt did not inhibit AIB transport by inhibiting respiration. However, oxidation of glucose and oxidation of succinate were at least as sensitive to high salt concentrations as AIB transport, suggesting that a salt-sensitive transport step(s) is involved in the oxidation of these substrates.     

Characterization of nucleoside diphosphate kinase from moderately halophilic eubacteria.

Nucleoside diphosphate kinase was purified to apparent homogeneity from naturally isolated moderately halophilic eubacteria by ATP-agarose and phenyl-5PW column chromatographies. The molecular mass of this enzyme was 15 kDa by time-of-flight mass-spectrometry. This protein showed anomalous mobility on SDS-PAGE which is typical of a halophilic protein. It was stable and active over a wide range of salt concentrations, from 0 to 4.0 M NaCl.     

Lipid membranes from halophilic and alkali-halophilic Archaea have a low H+ and Na+ permeability at high salt concentration

The influence of pH and the salt concentration on the proton and sodium ion permeability of liposomes formed from lipids of the halophile Halobacterium salinarum and the haloalkaliphile Halorubrum vacuolatum were studied. In contrast with liposomes formed from Escherichia coli lipids, liposomes formed from halophilic lipids remained stable up to 4 M of NaCl and KCl. The proton permeability of the liposomes from lipids of halophiles was independent of the salt concentration and was essentially constant between pH 7 and pH 9. The sodium ion permeability increased with the salt concentration but was 10- to 100 fold lower than the proton permeability. It is concluded that the membranes of halophiles are stable over a wide range of salt concentrations and at elevated pH values and are well adapted to the halophilic conditions. Received: February 25, 1999 / Accepted: June 11, 1999     

Structure of phenylalanine-accepting transfer ribonucleic acid and of its environment in aqueous solvents with different salts

Yeast tRNA(Phe) was studied in different salt-containing solvents by UV absorbance and small-angle neutron scattering (SANS). This extends results obtained previously in NaCl and KCl solutions [Li, Z.-Q., Giegé, R., Jacrot, B., Oberthür, R., Thierry, J. C., & Zaccai, G. (1983) Biochemistry 22, 4380-4388]. As expected, at low concentrations of all salts studied, the tRNA molecule is unfolded. The importance of specific counterion interactions and the flexibility of the macromolecule are emphasized by the observation that it cannot take up its folded structure in N(CH3)4Cl solvents, even when that salt concentration is increased to 1 M, in the absence of Mg ions. In CsCl solvents, on the other hand, the folded conformation is obtained in salt concentrations above about 0.2 M, similar to NaCl or KCl. By a comparison of SANS results in CsCl H2O and CsCl 2H2O solvents with the data from NaCl and KCl solvents, thermodynamic and structural parameters were derived for the solvated macromolecule. All the data are accounted for, quantitatively, by a model for the particle in NaCl, KCl, or CsCl solution made up of tRNA76-, closely associated with 76 positive hydrated counterions, surrounded by an aqueous solvent layer that excludes salt (and, therefore, of density different from that of bulk solvent). The mass of water in that layer depends on salt concentration, and the values found are consistent with those predicted by the Donnan effect.     

Reverse micelles in organic solvents: a medium for the biotechnological use of extreme halophilic enzymes at low salt concentration

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeobacterium Halobacterium salinarum (previously halobium) was solubilized at low salt concentration in reverse micelles of hexadecyltrimethyl-ammoniumbromide in cyclohexane with 1-butanol as co-surfactant. The enzyme maintained its catalytic properties under these conditions. The thermodynamic "solvation-stabilization hypothesis" has been used to explain the bell-shaped dependence of pNPPase activity on the water content of reverse micelles, in terms of protein-solvent interactions. According to this model, the stability of the folded protein depends on a network of hydrated ions associated with acidic residues at the protein surface. At low salt concentration and low water content (the ratio of water concentration to surfactant concentration; w0), the network of hydrated ions within the reverse micelles may involve the cationic heads of the surfactant. The bell-shaped profile of the relationship between enzyme activity and w0 varied depending on the concentrations of NaCl and Mn2+.     

Osmometric and microscopic studies on bilayers of polar lipids from the extreme halophile, halobacterium cutirubrum

The major membrane polar lipid components in Halobacterium cutirubrum are the diphytanyl ether analogues of phosphatidylglycerol phosphate, glycolipid sulfate, phosphatidylglycerol and phosphatidylglycerol sulfate. Dispersions of total polar lipids in water formed large birefringent liposomes showing concentric lipid bilayers in the elctron microscope; they behaved as ideal osmometers in KCl or NaCl solutions in the concentration range 0.005–0.2 M. At concentrations above 0.2 M KCl the liposomes shrank to spherical particles which were much less birefringent, showed no distinct bilayer structures by electron microscopy, and no longer behaved as ideal osmometers. Dispersions of phosphatidylglycerol phosphate, phosphatidylglycerol or phosphatidylglycerol sulfate alone did not behave as osmometers at any concentration of KCl or NaCl, but glycolipid sulfate alone or mixed with phosphatidylglycerol phosphate or phosphatidylglycerol phosphate + phosphatidylglycerol sulfate showed ideal osmometer behavior in 0.005–0.2 M KCl or NaCl. The highly negatively charged total polar lipids of H. cutirubrum thus can form stable lipid bilayers only at low ionic concentrations (0.005–0.2 M), much lower than the salt concentration (4 M) of the growth medium, and the presence of glycolipid sulfate is essential. Stability of the membrane in 4 M salt appears to require direct participation of the protein components.     

Electrostatic and hydrophobic interactions play a major role in the stability and refolding of halophilic proteins

In general, halophilic proteins are stable only in the presence of salts at high concentrations. Not only is high salt concentration important for structural stability of halophilic proteins, but also refolding of a denatured halophilic protein requires high salt concentration. This review summarizes the importance of electrostatic charge shielding and hydrophobic interactions in the stability and refolding of halophilic proteins.     

A two-alpha-helix extra domain mediates the halophilic character of a plant-type ferredoxin from halophilic archaea

The [2Fe-2S] ferredoxin (HsFdx) of the halophilic archaeon Halobacterium salinarum exhibits a high degree of sequence conservation with plant-type ferredoxins except for an insertion of 30 amino acids near its N-terminus which is extremely rich in acidic amino acids. Unfolding studies reveal that HsFdx has an unfolding temperature of approximately 85 degrees C in 4.3 M NaCl, but of only 50 degrees C in low salinity, revealing its halophilic character. The three-dimensional structure of HsFdx was determined by NMR spectroscopy, resulting in a backbone rmsd of 0.6 A for the diamagnetic regions of the protein. Whereas the overall structure of HsFdx is very similar to that of the plant-type ferredoxins, two additional alpha-helices are found in the acidic extra domain. (15)N NMR relaxation studies indicate that HsFdx is rigid, and the flexibility of residues is similar throughout the molecule. Monitoring protein denaturation by NMR did not reveal differences between the core fold and the acidic domain, suggesting a cooperative unfolding of both parts of the molecule. A mutant of the HsFdx in which the acidic domain is replaced with a short loop of the nonhalophilic Anabaena ferredoxin shows a considerably changed expression pattern. The halophilic wild-type protein is readily expressed in large amounts in H. salinarum, but not in Escherichia coli, whereas the mutant ferredoxin could only be overexpressed in E. coli. The salt concentration was also found to play a critical role for the efficiency of cluster reconstitution: the cluster of HsFdx could be reconstituted only in a solution containing molar concentrations of NaCl, while the reconstitution of the cluster in the mutant protein proceeds efficiently in low salt. These findings suggest that the acidic domain mediates the halophilic character which is reflected in its thermostability, the exclusive expression in H. salinarum, and the ability to efficiently reconstitute the iron-sulfur cluster only at high salt concentrations.     

The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases

The extremely halophilic Archae require near-saturating concentrations of salt in the external environment and in their cytoplasm, potassium being the predominant intracellular cation. The proteins of these organisms have evolved to function in concentrations of salt that inactivate or precipitate homologous proteins from non-halophilic species. It has been proposed that haloadaptation is primarily due to clustering of acidic residues on the surface of the protein, and that these clusters bind networks of hydrated ions. The dihydrofolate reductases from Escherichia coli (ecDHFR) and two DHFR isozymes from Haloferax volcanii (hvDHFR1 and hvDHFR2) have been used as a model system to compare the effect of salts on a mesophilic and halophilic enzyme. The KCl-dependence of the activity and substrate affinity was investigated. ecDHFR is largely inactivated above 1M KCl, with no major effect on substrate affinity. hvDHFR1 and hvDHFR2 unfold at KCl concentrations below approximately 0.5M. Above approximately 1M, the KCl dependence of the hvDHFR activities can be attributed to the effect of salt on substrate affinity. The abilities of NaCl, KCl, and CsCl to enhance the stability to urea denaturation were determined, and similar efficacies of stabilization were observed for all three DHFR variants. The DeltaG degrees (H(2)O) values increased linearly with increasing KCl and CsCl concentrations. The increase of DeltaG degrees (H(2)O) as a function of the smallest cation, NaCl, is slightly curved, suggesting a minor stabilization from cation binding or screening of electrostatic repulsion. At their respective physiological ionic strengths, the DHFR variants exhibit similar stabilities. Salts stabilize ecDHFR and the hvDHFRs by a common mechanism, not a halophile-specific mechanism, such as the binding of hydrated salt networks. The primary mode of salt stabilization of the mesophilic and halophilic DHFRs appears to be through preferential hydration and the Hofmeister effect of salt on the activity and entropy of the aqueous solvent. In support of this conclusion, all three DHFRs are similarly stabilized by the non-ionic cosolute, sucrose.     

Functional implications related to the gene structure of the elongation factor EF-Tu from Halobacterium marismortui.

The primary structure of the gene for the elongation factor EF-Tu from the halophilic archaebacterium Halobacterium marismortui (hEF-Tu) is described. It is the first gene of a halophilic elongation factor EF-Tu to be sequenced. When the sequence of hEF-Tu is compared to that of homologous proteins from other organisms, the highest identity (61%) is found with EF-Tu from Methanococcus vannielii, a non-halophilic archaebacterium. In the search for halophilic characteristics therefore the most appropriate comparison is with the M. vannielii sequence. The excess of acidic amino acid residues in the hEF-Tu sequence (already observed in the composition of other halophilic proteins) results to a large extent from changes of Lys, Asn or Gln to Asp or Glu. A structural analysis algorithm applied to the halophilic sequence places these acidic residues on the surface of the protein. The corresponding residues in the crystal structure of the first domain of EF-Tu from E. coli (the only EF-Tu structure available) are grouped in patches on the protein surface, in each of which several residues that may be far apart in the sequence come quite close to each other in the tertiary structure.     

Activation of halophilic nucleoside diphosphate kinase by a non-ionic osmolyte,trimethylamine N-oxide

The folding and activity of halophilic enzymes are believed to require the presence of salts at high concentrations. When the inactivated nucleoside diphosphate kinase (NDK) from extremely halophilic archaea was incubated with low salt media, no activity was regained over the course of 8 days. When it was incubated with 2 M NaCl or 3 M KCl, however, it gradually regained activity. To our surprise, trimethylamine N-oxide (TMAO) also was able to induce activation at 4.0 M. The enzyme activity and secondary structure of refolded NDK in 4 M TMAO were comparable with those of the native NDK or the refolded NDK in 3.8 M NaCl. TMAO is not an electrolyte, meaning that the presence of concentrated salts is not an absolute requirement, and that charge shielding or ion binding is not a sole factor for the folding and activation of NDK. Although both NaCl and TMAO are effective in refolding NDK, the mechanism of their actions appears to be different: the effect of protein concentration and pH on refolding is qualitatively different between these two, and at pH 8.0 NDK could be refolded in the presence of 4 M TMAO only when low concentrations of NaCl are included.