Formation of substrate and transition-state analogue complexes in crystals of phosphoglucomutase after removing the crystallization salt.

Crystals of phosphoglucomutase, grown in 2.1 M ammonium sulfate, "desalted", and suspended in a 30% polyoxyethylene-8000/1 M glycine solution as described in the accompanying paper [Ray, W. J., Jr., Puvathingal, J. M., Bolin, J. T., Minor, W., Liu, Y., & Muchmore, S. W. (1991) Biochemistry 30 (preceding paper in this issue)], were treated with glucose phosphates to form an equilibrium mixture of the catalytically active substrate/product complexes. However, this treatment extensively fractured the crystals, even when very dilute solutions of glucose phosphates were used. But formation of the desired complexes was achieved, without fracturing, by introducing the glucose phosphates at high salt concentration, where they do not bind significantly to the enzyme, and maintaining their presence during subsequent sulfate-removal steps, in order to obtain essentially uniform binding throughout the crystal at all times. Although this procedure produced unfractured crystals of the catalytically active complexes, an adjustment in water activity was required to prevent the crystals from slowly liquefying in the presence of the added glucose phosphates. After this adjustment, the quality of diffraction-grade crystals subjected to this treatment was not significantly altered. An even larger adjustment in water activity was required to stabilize crystals that had been largely converted into a mixture of vanadate-based transition-state analogue complexes [cf. Ray, W. J., Jr., & Puvathingal, J. M. (1990) Biochemistry 29, 2790-2801] by means of an analogous procedure. The rationale for, and the implications of, this adjustment of water activity are discussed. The phenomenon of lattice-based binding cooperativity also is discussed together with a possible role for such cooperativity in the fracturing of protein crystals during formation of ligand complexes and possible ways to circumvent such fracturing based on the annealing of crystals at fractional saturation. An assay for quantifying the extent of formation of the vanadate-based transition-state analogue complexes in crystals of phosphoglucomutase is described. A solution to problems associated with producing and maintaining a steady-state in treated crystals is discussed within the context of maximizing the fraction of the crystalline enzyme present as a complex with one such inhibitor, glucose alpha-1-phosphate-6-vanadate. One of these problems, achieving a substantial reduction in sulfate concentration, could not be successfully addressed by employing the desalting procedure used to produce the substrate/product complexes, because of reduced diffusional rates in the final solution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of polyethylene glycol-400 at low concentrations on long-term growth of muscle phosphoglucomutase crystals from concentrated salt solutions.

Although rabbit muscle phosphoglucomutase occasionally deposits tetragonal crystals from solutions of ammonium sulfate at about 47% of saturation, low concentrations of polyethylene glycol-400 (PEG), 1 to 4.5% w/v, must be included to sustain crystal growth. A comparison of long-term growth rates for macroscopic crystals in the presence and absence of added PEG suggests that at high salt concentration this cosolute exerts its primary effect on disordered protein aggregates, either in the external medium or at the surface of the crystal, and thereby allows the growth of much larger crystals. Since the observed effects may arise from a PEG-induced increase in the "solubility" of the aggregate that exceeds the induced increase in solubility of the crystalline phase under these conditions, the physical basis for a cosolute-induced increase in solubility in the presence of a precipitant is considered. The applicability of such a rationale to the present system is supported by an assessment of the relative effects of polyethylene glycol and beta-octylglucoside on amorphous, salt-induced precipitates of phosphoglucomutase. PEG also produces what appears to be a differential effect on nucleation efficiency and crystal growth rate. Thus, seed crystals cannot be enlarged at a significant rate at high salt concentration without producing showers of extraneous nucleation centers when the concentration of added PEG is 3% or less. But PEG concentrations of 4.5% essentially eliminate the showering problem, ostensibly by increasing the supersaturation required for nucleation to a greater extent than that required for crystal growth. The same type of effect is observed during de novo growth. Again a solubility-based mechanism is posed. Hysteretic effects related to properties of amorphous aggregates of the protein also are described.     

Solutes Involved in Osmotic Adjustment to Increasing Salinity in Suspension Cells of Alternanthera philoxeroides Griseb

Cell recovery from osmotic stress was studied in suspension cell cultures from Alternanthera philoxeroides [Mart.] Griseb. Changes in different classes of cellular solutes were measured after cells were transferred from 0 to 200 mM NaCl (high salt) to obtain an integrated picture of the solute pools involved in osmotic adjustment. By 2 h, cellular [Na⁺] and [Cl⁻] had increased several-fold, potentially accounting for the osmotic adjustment that produced a rapid recovery of cell turgor. There was a four-fold increase in the concentration of quaternary ammonium compounds (QAC) by 12 h and a slower increase for several days afterward. Betaine aldehyde dehydrogenase (BADH) is required for synthesis of glycine betaine, a QAC produced by a range of organisms in response to osmotic stress. Western-blot analysis for BADH suggested that glycine betaine was a significant component of the QAC solutes. The amount of BADH was generally similar at different sampling times for control and high salt cells, unlike previous reports of stimulation by osmotic stress in intact plants of some species. Between 3 and 7 days after cell transfer to high salt, other organic solutes increased in concentration and [Na⁺] and [Cl⁻] decreased. In A. philoxeroides, high [Na⁺] and [Cl⁻] produce rapid osmotic adjustment but organic solutes apparently replace these potentially harmful inorganic ions after the recovery of turgor.     

Intracellular ion and organic solute concentrations of the extremely halophilic bacterium <Emphasis Type="Italic">Salinibacter ruber</Emphasis>

Salinibacter ruber is a red obligatory aerobic chemoorganotrophic extremely halophilic Bacterium, related to the order Cytophagales. It was isolated from saltern crystallizer ponds, and requires at least 150 g l(-1) salt for growth. The cells have an extremely high potassium content, the ratio K(+)/protein being in the same range as in halophilic Archaea of the order Halobacteriales. X-ray microanalysis in the electron microscope of cells grown in medium of 250 g l(-1) salt confirmed the high intracellular K(+)concentrations, and showed intracellular chloride to be about as high as the cation concentrations within the cells. A search for intracellular organic osmotic solutes, using (13)C-NMR and HPLC techniques, showed glutamate, glycine betaine, and N-alpha-acetyllysine to be present in low concentrations only, contributing very little to the overall osmotic balance. The results presented suggest that the extremely halophilic Bacterium Salinibacteruses a similar mode of haloadaptation to that of the Archaea of the order Halobacteriales, and does not accumulate organic osmotic solutes such as are used by all other known halophilic and halotolerant aerobic Bacteria.     

Osmotic second virial cross‐coefficient measurements for binary combination of lysozyme,ovalbumin, and α‐amylase in salt solutions

Interactions measurement is a valuable tool to predict equilibrium phase separation of a desired protein in the presence of unwanted macromolecules. In this study, cross‐interactions were measured as the osmotic second virial cross‐coefficients (B₂₃) for the three binary protein systems involving lysozyme, ovalbumin, and α‐amylase in salt solutions (sodium chloride and ammonium sulfate). They were correlated with solubility for the binary protein mixtures. The cross‐interaction behavior at different salt concentrations was interpreted by either electrostatic or hydrophobic interaction forces. At low salt concentrations, the protein surface charge dominates cross‐interaction behavior as a function of pH. With added ovalbumin, the lysozyme solubility decreased linearly at low salt concentration in sodium chloride and increased at high salt concentration in ammonium sulfate. The B₂₃ value was found to be proportional to the slope of the lysozyme solubility against ovalbumin concentration and the correlation was explained by preferential interaction theory. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1203–1211, 2013     

Correlation of second virial coefficient with solubility for proteins in salt solutions

In this work, osmotic second virial coefficients (B(22)) were determined and correlated with the measured solubilities for the proteins, α-amylase, ovalbumin, and lysozyme. The B(22) values and solubilities were determined in similar solution conditions using two salts, sodium chloride and ammonium sulfate in an acidic pH range. An overall decrease in the solubility of the proteins (salting out) was observed at high concentrations of ammonium sulfate and sodium chloride solutions. However, for α-amylase, salting-in behavior was also observed in low concentration sodium chloride solutions. In ammonium sulfate solutions, the B(22) are small and close to zero below 2.4 M. As the ammonium sulfate concentrations were further increased, B(22) values decreased for all systems studied. The effect of sodium chloride on B(22) varies with concentration, solution pH, and the type of protein studied. Theoretical models show a reasonable fit to the experimental derived data of B(22) and solubility. B(22) is also directly proportional to the logarithm of the solubility values for individual proteins in salt solutions, so the log-linear empirical models developed in this work can also be used to rapidly predict solubility and B(22) values for given protein-salt systems.     

Betaine aldehyde dehydrogenase in sorghum.

The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa.     

Patterns of protein protein interactions in salt solutions and implications for protein crystallization

The second osmotic virial coefficients of seven proteins-ovalbumin, ribonuclease A, bovine serum albumin, alpha-lactalbumin, myoglobin, cytochrome c, and catalase-were measured in salt solutions. Comparison of the interaction trends in terms of the dimensionless second virial coefficient b(2) shows that, at low salt concentrations, protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on the salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate, proteins show a sharp drop in b(2) with increasing salt concentration beyond a particular threshold. The experimental phase behavior of the proteins corroborates these observations in that precipitation always follows the drop in b(2). When the proteins crystallize, they do so at slightly lower salt concentrations than seen for precipitation. The b(2) measurements were extended to other salts for ovalbumin and catalase. The trends follow the Hofmeister series, and the effect of the salt can be interpreted as a water-mediated effect between the protein and salt molecules. The b(2) trends quantify protein-protein interactions and provide some understanding of the corresponding phase behavior. The results explain both why ammonium sulfate is among the best crystallization agents, as well as some of the difficulties that can be encountered in protein crystallization.     

The recovery of transfer ribonucleic acid by binding to DEAE-cellulose induced by ammonium sulfate

tRNA samples may be adsorbed quantitatively to DEAE-cellulose from 2m ammonium sulfate solution, pH 4.5. Washing with 0.3 m NaCl removes the ammonium sulfate and the tRNA is recovered in ethanol-precipitable form by elution with 1.1 m NaCl. The procedure allows convenient and efficient recovery of tRNAs from larger volumes of aqueous salt solutions. A moderate concentration of sodium chloride does not interfere with adsorption.     

Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.     

Stress response by solute accumulation in archaea

The accumulation of organic solutes is a prerequisite for osmotic adjustment of all organisms. Archaea synthesize unusual solutes such as beta-amino acids, Nepsilon-acetyl-beta-lysine, mannosylglycerate and di-myo-inositol phosphate but, as in other cells, uptake of solutes such as glycine betaine is preferred over de novo synthesis. Study of the molecular basis of osmoadaptation and its regulation in archaea is still in its infancy, but genomics and functional genome analyses combined with classical biochemistry shed light on the processes that confer osmoadaptation in archaea. Most interestingly, some solutes are not only produced in response to salt but also to temperature stress.     

BRAIN MITOCHONDRIAL HEXOKINASE AND ADENYLATE KINASE: EFFECT OF OSMOTIC CONDITIONS ON ENZYME ACTIVITIES

The activities of mitochondrial hexokinase and adenylate kinase have been measured in various osmotic conditions. Sucrose, potassium chloride and ammonium acetate were used as solutes. The total hexokinase activity of mitochondrial suspensions increased steadily with decreasing osmolarity of the sucrose or salt solutions. The hexokinase activity of mitochondrial suspensions in water was 93 per cent of that measured in the presence of Triton X-100.The increase in hexokinase activity was irreversible even after very short exposure (90 s) to hypo-osmotic conditions. Total adenylate kinase activity was not affected by osmotic conditions. Adenylate kinase activity increased hyperbolically in supernatants prepared from mitochondrial suspensions with decreasing osmolarity of the sucrose or salt solutions. Besides monitoring adenylate kinase leakage as a measure of outer mitochondrial membrane disruption, mitochondrial swelling was followed by measurement of the turbidity of mitochondrial suspensions at 520 nm. The data has been interpreted in terms of binding of some hexokinase to the inner mitochondrial membrane.     

The catalytic activity of muscle phosphoglucomutase in the crystalline phase

A suspension of microcrystals of phosphoglucomutase in 60% ammonium sulfate exhibits a maximal catalytic activity in substrate-velocity studies that is about 0.2 of that obtained with the soluble enzyme under the same conditions. The apparent Michaelis constants for the reaction in the crystal phase are altered to an even smaller extent, relative to that in solution, although the parameters for the monophosphate and bisphosphate are increased more than 3 and more than 5 orders of magnitude, respectively, by the sulfate present. The compatibility of larger crystals with a reaction that constitutes part of the catalytic process also is demonstrated.     

Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes

Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.     

Compatible-Solute-Supported Periplasmic Expression of Functional Recombinant Proteins under Stress Conditions

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.     

Hydrophobic forces between protein molecules in aqueous solutions of concentrated electrolyte

Protein-protein interactions have been measured for a mutant (D101F) lysozyme and for native lysozyme in concentrated solutions of ammonium sulfate at pH 7 and sodium chloride at pH 4.5. In the mutant lysozyme, a surface aspartate residue has been replaced with a hydrophobic phenylalanine residue. The protein-protein interactions of D101F lysozyme are more attractive than those of native lysozyme for all conditions studied. The salt-induced attraction is correlated with a solvation potential of mean force given by the work required to desolvate the part of the protein surfaces that is buried by the protein-protein interaction. This work is proportional to the aqueous surface-tension increment of the salt and the fractional non-polar surface coverage of the protein. Experimental measurements of osmotic second virial coefficients validate a proposed potential of mean force that ascribes the salt-induced attraction between protein molecules to an enhancement of the hydrophobic attraction. This model provides a first approximation for predicting the protein-protein potential of mean force in concentrated aqueous electrolyte solutions; this potential is useful for determining solution conditions favorable for protein crystallization.     

Identification of an osmotically induced periplasmic glycine betaine-binding protein from Rhizobium meliloti.

The effect of salt stress on glycine betaine-binding activity has been investigated in periplasmic fractions released from Rhizobium meliloti 102F34 by cold osmotic shock. Binding activity was monitored by three techniques: equilibrium dialysis, filter procedure, and detection of 14C ligand-protein binding by direct non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. The three methods demonstrated the existence of a strong glycine betaine-binding activity, but only in periplasmic fractions from cells grown at high osmolarity. The non-denaturing PAGE of such periplasmic shock fluids mixed with [methyl-14C]glycine betaine showed only one radioactive band, indicating the involvement of one glycine betaine-binding protein. To determine the possible implication of this binding protein in glycine betaine uptake, transport activity was measured with cells submitted to cold osmotic shock. No significant decrease of transport activity was noticed. This lack of effect could be explained by the small quantity of periplasmic proteins released as judged by the low activity of phosphodiesterase, a periplasmic marker enzyme, observed in the shock fluid. The specificity of binding was analysed with different potential competitors: other betaines such as gamma-butyrobetaine, proline betaine, pipecolate betaine, trigonelline and homarine, or amino acids like glycine and proline, did not bind to the glycine betaine-binding protein, whereas glycine betaine aldehyde and choline were weak competitors. Optimum pH for binding was around 7.0, but approx. 90% of the glycine betaine-binding activity remained at pH 6.0 or 8.0. The calculated binding affinity (KD) was 2.5 microM. Both glycine betaine-binding activity and affinity were not significantly modified whether or not the binding assays were done at high osmolarity. A 32 kDa osmotically inducible periplasmic protein, identified by SDS-PAGE, apparently corresponds to the glycine betaine-binding protein.     

Phase equilibria in the lysozyme-ammonium sulfate-water system

Ternary phase diagrams were measured for lysozyme in ammonium sulfate solutions at pH values of 4 and 8. Lysozyme, ammonium sulfate, and water mass fractions were assayed independently by UV spectroscopy, barium chloride titration, and lyophilization respectively, with mass balances satisfied to within 1%. Protein crystals, flocs, and gels were obtained in different regions of the phase diagrams, and in some cases growth of crystals from the gel phase or from the supernatant after floc removal was observed. These observations, as well as a discontinuity in protein solubility between amorphous floc precipitate and crystal phases, indicate that the crystal phase is the true equilibrium state. The ammonium sulfate was generally found to partition unequally between the supernatant and the dense phase, in disagreement with an assumption often made in protein phase equilibrium studies. The results demonstrate the potential richness of protein phase diagrams as well as the uncertainties resulting from slow equilibration.     

Amine Oxidases of Microorganisms

A procedure for obtaining crystalline preparations of the amine oxidase of Aspergillus niger has been developed. The method involved fractionations with ammonium sulfate and separation on successive columns of DEAE-cellulose, DEAE-sephadex and hydroxyl-apatite. Crystals were formed when solid ammonium sulfate was added to solutions of the purified enzyme (of about 300- to 350-times the specific activity of the crude mycelium extract). The crystals appeared as fine rods, with a faint pink color. The crystals had a specific activity around 10,000 which was about 20 times as active as that of crystalline preparations of the animal monoamine oxidase.