Halophilic Malate Dehydrogenase Research Articles

Influence of an anion-binding site in the stabilization of halophilic malate dehydrogenase from Haloarcula marismortui

Halophilic proteins have evolved to be soluble, stable and active in high salt concentration. Crystallographic studies have shown that surface enrichment by acidic amino acids is a common structural feature of halophilic proteins. In addition, ion-binding sites have also been observed in most of the cases. The role of chloride-binding sites in halophilic adaptation was addressed in a site-directed mutagenesis study of tetrameric malate dehydrogenase from Haloarcula marismortui. The mutation of K 205, which is involved in an inter-subunit chloride-binding site, drastically modified the enzyme stability in the presence of KCl, but not in the presence of KF. The oligomeric state of the [K205A] mutant changes with the nature of the anion. At high salt concentration, the [K205A] mutant is a dimer when the anion is a chloride ion, whereas it is a tetramer when the fluoride ion is used. The results highlight the role of anion-binding sites in protein adaptation to high salt conditions.

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 A) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.

Adaptation to extreme environments: Macromolecular dynamics in complex systems

What we previously thought of as insurmountable physical and chemical barriers to life, we now see as yet another niche harbouring 'extremophiles'. Extremophiles and their macromolecules had to develop molecular mechanisms of adaptation to extreme physico-chemical conditions. Using neutron spectroscopy, we have demonstrated that molecular dynamics represents one of these molecular mechanisms of adaptation. To which extent do hyper-saline conditions and extreme temperatures influence molecular dynamics? First, molecular dynamics were analysed for halophilic malate dehydrogenase from Haloarcula marismortui (Hm MalDH) under different molar solvent salt concentration conditions influencing its stability. Secondly, mean macromolecular motions were measured in-vivo in psychrophile (Aquaspirillum arcticum), mesophile (Escherichia coli and Proteus mirabilis), thermophile (Thermus thermophilus), and hyperthermophile (Aquifex pyrofilus) bacteria. The mean constant force of Hm MalDH increases progessively with increasing stability. The results show that the molecular adaptation of Hm MalDH to hyper-saline conditions is achieved through an increasing resilience of its structure dominated by enthalpic mechanisms. The study of bacteria has provided tools to quantify the macromolecular adaptation to extreme temperatures in the naturally crowded environment of the cell. The macromolecular resilience of bacteria increases with adaptation to high temperatures.

The 2.9 Å Resolution Crystal Structure of Malate Dehydrogenase from Archaeoglobus fulgidus: Mechanisms of Oligomerisation and Thermal Stabilisation

The crystal structure of malate dehydrogenase from the hyperthermophilic archaeon Archeoglobus fulgidus, in complex with its cofactor NAD, was solved at 2.9 Å resolution. The crystal structure shows a compact homodimer with one coenzyme bound per subunit. The substrate binding site is occupied by a sulphate ion. In order to gain insight into adaptation mechanisms, which allow the protein to be stable and active at high temperatures, the 3D structure was compared to those of several thermostable and hyperthermostable homologues, and to halophilic malate dehydrogenase. The hyperthermostable A. fulgidus MalDH protein displays a reduction of the solvent-exposed surface, an optimised compact hydrophobic core, a high number of hydrogen bonds, and includes a large number of ion pairs at the protein surface. These features occur concomitantly with a reduced number of residues in the protein subunit, due to several deletions in loop regions. The loops are further stiffened by ion pair links with secondary structure elements. A. fulgidus malate dehydrogenase is the only dimeric protein known to date that belongs to the [LDH-like] MalDH family. All the other known members of this family are homo-tetramers. The crystal structures revealed that the association of the dimers to form tetramers is prevented by several deletions, taking place at the level of two loops that are known to be essential for the tetramerisation process within the LDH and [LDH-like] MalDH enzymes.

Solvent interactions of halophilic malate dehydrogenase.

Malate dehydrogenase from the extreme halophilic Haloarcula marismortui (Hm MalDH) is an acidic protein that is unstable below molar salt concentrations. The solvated folded protein was studied by small-angle neutron scattering in solvents containing salt: NaCl, NaCH(3)CO(2), KF, NH(4)Cl, NH(4)CH(3)CO(2), (NH(4))(2)SO(4), MgCl(2), and MgSO(4). It was found that the global solvent interactions depend mainly on the nature of the cation. Complementary mass density measurements in MgCl(2), NaCl, NaCH(3)CO(2), and (NH(4))(2)SO(4) allowed determining the partial molal volumes of the protein, which were found to increase slightly with the salt, and the preferential salt binding parameters for each solvent condition. These are strongly dependent on the cation type and salt concentration. Hm MalDH can be modeled as an invariant particle binding 4100 water molecules in MgCl(2) and 2000 +/- 200 in NaCl, NaCH(3)CO(2), or (NH(4))(2)SO(4). The number of salt molecules associated to the particle decreases from about 85 to 0 in the order MgCl(2) > NaCl = NaCH(3)CO(2) > (NH(4))(2)SO(4). Alternatively, we considered exchangeable sites for water and salt with the effects of solvent nonideality. It does not change the description of the solvent interactions. Solvent anions act on Hm MalDH stability through a limited number of strong binding sites, as those seen at the interfaces of Hm MalDH by crystallography. Cations would act through some strong and numerous weak binding sites defined on the folded protein, in possible addition to nonspecific hydration effects.

Non-Ideality by Sedimentation Velocity of Halophilic Malate Dehydrogenase in Complex Solvents

We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D° (1 + k D c), and the reciprocal sedimentation coefficient s = s°/(1 + k s c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k s and s° and also for D° and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k D and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.

Understanding the crystallisation of an acidic protein by dilution in the ternary NaCl–2-methyl-2,4-pentanediol–H 2O system

Halophilic malate dehydrogenase is a negatively charged protein that crystallises well in a dilution process following a complex interplay with the three components of NaCl–MPD–H 2O solvents (MPD: 2-methyl-2,4-pentanediol). The process was characterised by measuring the folding state of the protein, its concentration, its apparent solubility and second virial coefficients in various NaCl–MPD–H 2O ratios representative of the phase diagram of the system. The protein crystallises by vapour diffusion between a drop containing protein in aqueous NaCl and MPD and a bath containing a given percentage of MPD in water. The starting drop is bi-phasic, in which the protein is concentrated in the salt-rich phase. This phase evolves towards a solution less concentrated in both NaCl and protein but more concentrated in MPD, before the system is driven to a single-phase region, where crystals are obtained. The protein stability is preserved during its crystallisation. We showed from second virial coefficient measurements that crystal formation is correlated with a slow evolution from repulsive to attractive protein–protein interactions. The end of the vapour diffusion process between the drop and the bath corresponds to a decrease of the attractive interaction, which we suggest favours crystal growth. The application of these results to crystallisation of other charged proteins and nucleic acids is discussed.

Characterization of a Novel Complex from Halophilic Archaebacteria, Which Displays Chaperone-like Activities in Vitro

We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.

The dependence of a halophilic malate dehydrogenase on ωo and surfactant concentration in reverse micelles

The halophilic malate dehydrogenase (hMDH) activity of Halobacterium salinarum was studied as a function of micelle size ( ω o), in cethyltrimethylammonium bromide (CTAB)/cyclohexane reverse micelles, with 1-butanol as cosurfactant. The velocity dependence of the ω o profile depends on the buffer used, the surfactant concentration, and the salt concentration. In phosphate buffer, the activity increases with increasing water content, while in Tris/HCl buffer a bell-shaped profile is generally observed. Despite a slight change in the ω o-activity profile, the enzymatic activity was higher at low salt concentration even when we employed a different buffer. The ω o value for the maximal activity (optimum ω o) varies directly with the enzyme concentration. The hMDH activity in reverse micelles depends on the surfactant concentration and the dependence of the activity of this enzyme on the surfactant concentration, at constant ω o, is different for each ω o value.

Relative role of anions and cations in the stabilization of halophilic malate dehydrogenase.

Halophilic malate dehydrogenase unfolds at low salt, and increasing the salt concentration stabilizes, first, the folded form and then, in some cases, destabilizes it. From inactivation and fluorescence measurements performed on the protein after its incubation in the presence of various salts in a large range of concentrations, the apparent effects of anions and cations were found to superimpose. A large range of ions was examined, including conditions that are in general not of physiological relevance, to explore the physical chemistry driving adaptation to extreme environments. The order of efficiency of cations and anions to maintain the folded form is, for the low-salt transition, Ca(2+) approximately Mg(2+) > Li(+) approximately NH(4)(+) approximately Na(+) > K(+) > Rb(+) > Cs(+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-), and for the high-salt transition, NH(4)(+) approximately Na(+) approximately K(+) approximately Cs(+) > Li(+) > Mg(2+) > Ca(2+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-) > Br(-) > I(-). If a cation or anion is very stabilizing, the effect of the salt ion of opposite charge is limited. Anions of high charge density are always the most efficient to stabilize the folded form, in accordance with the order found in the Hofmeister series, while cations of high charge density are the most efficient only at the lower salt concentrations and tend to denature the protein at higher salt concentrations. The stabilizing efficiency of cations and anions can be related in a minor way to their effect on the surface tension of the solution, but the interaction of ions with sites only present in the folded protein has also to be taken into account. Unfolding at high salt concentrations corresponds to interactions of anions of low charge density and cations of high charge density with the peptide bond, as found for nonhalophilic proteins.

Protein–solvent and weak protein–protein interactions in halophilic malate dehydrogenase

With the aim to correlate the solvation, stability and solubility properties of halophilic malate dehydrogenase, we characterized its weak interparticle interactions by small-angle neutron scattering in various solvents. The protein concentration dependence of the apparent radius of gyration and forward scattered intensity extrapolated from Guinier plots, and thus the second virial coefficient, A 2, were determined for each solvent condition. In NaCl 1M+2-methylpentane-2,4-diol 30%, a solvent that promotes protein crystallization, A 2 is negative, −0.4×10 −4 ml mol g −2 and indicating attractive interactions; in ammonium sulfate 3M, in which the protein precipitates at high concentrations, A 2∼0. In 2–5M NaCl, 1–3.5M NaOAc, 1–4.5M KF or 1–2M (NH 4) 2SO 4, in which the protein is very soluble, A 2 is positive with a value of the order of 0.4×10 −4 ml mol g −2 which decreases with increasing salt concentration. In MgCl 2 however, A 2 increases with increasing salt concentration from 0.2 to 1.3M.

Stabilisation of Halophilic Malate Dehydrogenase from Haloarcula Marismortui by Divalent Cations

Halophilic malate dehydrogenase is stable in a limited concentration range of MgCl2 or CaCl2. Thermal deactivation of the protein at low concentrations of these divalent salts is very different from that occurring at high concentrations. In low salt, stability always increases as the temperature is lowered. In high salt, stability shows bell-shaped behaviour as a function of temperature: increasing to a maximum at 4 degrees C, and subsequently decreasing as the temperature is lowered. This is in contrast to other salts, for which the deactivation behaviour depends on the salt type but not on its concentration. Cofactor addition or replacement of H2O by D2O modify only the deactivation at low MgCl2 or CaCl2 concentrations. A pH transition between pH 7 and pH 8, however, modified enzyme deactivation at both low and high MgCl2 or CaCl2 concentrations. The pH effect on stability was also observed in other salts. By comparing the effect of CaCl2, MgCl2, and NaCl, a strong correlation was found between the minimum salt concentration required for the stabilisation of halophilic malate dehydrogenase and the hydration of the cation.

Halophilic Protein Stabilization by the Mild Solubilizing Agents Nondetergent Sulfobetaines

In this work, experiments performed on pig heart and halophilic malate dehydrogenase as well as halophilic elongation factor Tu demonstrate a protein stabilization property from the recently described mild solubilizing agents nondetergent sulfobetaines. A practical application is given by the separation of halophilic bacteria elongation factor Tu and halophilic malate dehydrogenase by high-performance ion-exchange chromatography achieved at reduced salt levels without significant loss of activity.

Structural Features That Stabilize Halophilic Malate Dehydrogenase from an Archaebacterium

The high-resolution structure of halophilic malate dehydrogenase (hMDH) from the archaebacterium Haloarcula marismortui was determined by x-ray crystallography. Comparison of the three-dimensional structures of hMDH and its nonhalophilic congeners reveals structural features that may promote the stability of hMDH at high salt concentrations. These features include an excess of acidic over basic residues distributed on the enzyme surface and more salt bridges present in hMDH compared with its nonhalophilic counterparts. Other features that contribute to the stabilization of thermophilic lactate dehydrogenase and thermophilic MDH-the incorporation of alanine into alpha helices and the introduction of negatively charged amino acids near their amino termini, both of which stabilize the alpha helix as a result of interaction with the positive part of the alpha-helix dipole-also were observed in hMDH.

Stability against Denaturation Mechanisms in Halophilic Malate Dehydrogenase "Adapt" to Solvent Conditions

Malate dehydrogenase from Haloarcula marismortui (hMDH) is active, soluble and mildly unstable in an unusually wide range of salt conditions and temperatures, making it a particularly interesting model for the study of solvent effects on protein stability. Its denaturation (loss of activity due to concomitant dissociation and unfolding) kinetics was studied as a function of temperature and concentration of NaCl, potassium phosphate or ammonium sulphate in H 2O or 2H 2O. A transition-state-theory analysis was applied to the data. In all cases, stability (resistance to denaturation) increased with increasing salt concentration, and when 2 H 2O replaced H 2O. Each salt condition was associated with a particular energy regime that dominated stability. In NaCl/H 2O, a positive enthalpy term, Δ H ≠O, always dominated the activation free energy of denaturation, Δ G ≠O. In potassium phosphate/H 2O and ammonium sulphate/H 2O, on the other hand, stability was dominated by a negative activation entropy, Δ S ≠O, and Δ H ≠O changed sign between 10°C and 20°C, consistent with a strong hydrophobic effect contribution, in these salting-out solvents. Decreasing stability at low temperatures, favouring cold denaturation, was observed. Replacing H 2O by 2 H 2O strengthened the hydrophobic effect in all conditions. As a consequence, conditions were found in which HMDH was not halophilic; below 10°C, it was stable in ≈0?1 M NaCl/ 2H 2O. The solution structure and preferential solvent interactions of hMDH in H 2O or 2H 2O solvents containing NaCl were studied by densimetry and neutron scattering. Despite the different stability of the protein in H 2O or 2H 2O, an experimentally identical invariant solution particle was formed in both solvents. It had a total volume of 1.165 cm 3 g -1 and bound about 0.4 g of H 2O (0.44 g of 2H 2O) and about 0.08 g NaCl g protein. The impact of these results on a stabilisation model for hMDH, involving ion binding, is discussed.

Protein and nucleic acid hydration and cosolvent interactions: establishment of reliable baseline values at high cosolvent concentrations

Hydration and cosolvent interactions of biological macromolecules can be derived, subject to excluded volume corrections, from studies of density increments at constant chemical potentials of diffusible solutes through a semipermeable membrane. In addition to precision density determinations of solutions dialyzed to equilibrium, the analytical ultracentrifuge, static and dynamic light and small angle X-ray and neutron scattering, and combined pairwise use of, for instance, ultracentrifugation and neutron scattering, considerably strengthen the experimental analysis and its interpretation. We have examined hydration of bovine serum albumin (BSA) in the native and denatured states, and binding of the denaturant guanidinium chloride (GdmCl) to the latter form; hydration of DNA and interaction with NaCl and CsCl; revised values of the halophilic malate dehydrogenase (hMDH) tetramer hydration and ‘binding’ of salts; probing of nucleosome core particle hydration as distinct from and additionally to the evaluation of volume exclusion (holes), by use of variously sized sugar related probes. Conclusions presented are compared to results from precision calorimetry and from X-ray crystallography structures, whenever applicable, and comparisons made with alternative interpretations and experimental approaches.

Subunit structure of halophilic malate dehydrogenase from Haloarcula marismortui

1. 1. The subunit structure of halophilic malate dehydrogenase from Haloarcula marismortui was studied by the method of crosslinking with bifunctional reagent. 2. 2. Exposure of the enzyme to glutardialdehyde followed by sodium dodecyl sulphate gel electrophoresis resulted in the appearance of four bands with mobilities corresponding to monomeric polypeptide chains and crosslinked polypeptide chain dimers, trimers and tetramers. 3. 3. Our findings are not consistent with the currently accepted dimeric structure of the enzyme. They provide a strong indication that halophilic malate dehydrogenase is composed of four identical subunits.

Biophysical study of halophilic malate dehydrogenase in solution: revised subunit structure and solvent interactions of native and recombinant enzyme

In previous work, malate dehydrogenase from Haloarcula marismortui(hMDH), a halophilic enzyme that is only stable in high concentrations of certain salts, was characterized by different biophysical methods as a dimer of molar mass 87 000 g mol–1 with solvent interactions in multimolar NaCl solutions that are significantly larger than for non-halophilic proteins. A model was proposed in which hMDH is stabilized by different mechanisms in different salt solvents (cf. Eisenberg et al., Adv. Protein Chem., 1992, 43, 1). Recently, the gene coding for hMDH was isolated and sequenced and the recombinant protein expressed in E. coli and renatured (Cendrin et al., Biochemistry, 1993, submitted). A subunit molar mass of 32 638 g mol–1 was calculated from the sequence and confirmed by mass spectrometry. The present study was undertaken in order to resolve the discrepancy between this value and the previously proposed dimer solution structure. The absorption coefficient of the protein was redetermined by amino acid analysis. New densimetry measurements in a large range of salt concentrations, ultracentrifugation and light scattering experiments were performed on the native and recombinant enzymes, and previous ultracentrifugation, X-ray and neutron scattering data were re-analysed. Control experiments on bovine serum albumin (as a model for non-halophilic proteins) were also repeated under similar conditions. All the data are now in agreement with a tetrametric solution structure for hMDH, with solvent interactions (e.g. in multimolar NaCl or KCl solutions: ca. 0.4 g water and 0.1 g salt per g protein) that show water binding comparable to non-halophilic proteins yet an order of magnitude more salt binding than for non-halophilic proteins. The stabilization model for hMDH remains valid. The complementarity and accuracy of the different methods for solution structure analysis are discussed critically in the light of this study.

Biochemical, Structural, and Molecular Genetic Aspects of Halophilism

The study of halobacteria relates to a better understanding of evolutionary relationships. Halophilic enzymes are very unstable in low salt concentrations. Because some of the important fractionation methods in protein chemistry, such as electrophoresis or ion-exchange chromatography, cannot be applied at high salt concentrations, the available fractionation methods are limited. The existing purification procedures fall into two groups: the nonhalophilic approach and the halophilic approach. This chapter reviews the developments in the molecular characterization of halobacterial proteins, starting with the methodology of their purification. The biochemical and biophysical structural analyses of some enzymatic systems for which extensive knowledge is accumulated is described.. Macromolecular structures from halophilic bacteria are discussed to improve the understanding of the molecular mechanisms of adaptation to high salt concentration environments by considering genome organization, genetic tools, isolation of genes, or transcript organization, and structure. The structural aspects of halophilism can be determined by ribosomal subunits, surface layers, purple membrane, or halophilic malate dehydrogenase.

Solution studies of elongation factor Tu from the extreme halophile Halobacterium marismortui

The activity, stability and structure in solution of polypeptide elongation factor hEF-Tu from Halobacterium marismortui have been investigated. The protein is stable in aqueous solutions only at high concentrations of NaCl, KCl or ammonium sulphate, whereas it is more active in exchanging GDP at lower salt concentrations. It is more active and stable at lower pH values than is non-halophilic EF-Tu. The structure in solution of the protein was determined by complementary density, ultracentrifugation, dynamic light-scattering and neutron-scattering measurements. The protein has large hydration interactions, similar to those of other halophilic proteins: 0·4 (±0·1) g of water and 0.20 (±0·05) g of KCl associated with 1 g of protein, with a water/KCl mass ratio always remaining close to 2. The kinetics of inactivation at low salt concentrations showed a stabilizing effect of NaCl when compared to KCl. At low salt concentration, inactivation, protein unfolding and aggregation were strongly correlated. The results suggest that the stabilization model proposed for halophilic malate dehydrogenase by Zaccai et al., involving extensive protein interactions with hydrated salt ions, is also valid for hEF-Tu.