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)     

Spectroscopic studies on histone-DNA interactions. II. Three transitions in nucleosomes resolved by salt-titration

The multiple-step transitions in DNA-histone interactions in chicken erythrocyte nucleosomes with increasing ionic strength are resolved by salt-titration spectroscopy. Both the circular dichroism of the DNA and the fluorescence of the histones in nucleosomes change during the titration process with concentrations of NaCl from 0.1 M to 2.5 M. By differentiating the titration curves, three distinct peaks corresponding to three structural transitions are observed. The two peaks near 0.95 M and 1.45 M-NaCl are common to the circular dichroism and fluorescence curves. The circular dichroism curve has another peak near 0.55 M-NaCl. Because the derivative of the fluorescence titration curve for the DNA-(H3, H4) complex has only one peak near 1.45 M-NaCl, that peak is attributed to the dissociation of the histone dimer (H3, H4). The peak near 0.95 M-NaCl corresponds to the dissociation of the dimer (H2A, H2B) from the DNA-(H3, H4) complex, as shown by binding experiments of (H2A, H2B) to the DNA-(H3, H4) complex at the salt concentration near this peak. The peak near 0.55 M-NaCl reflects some inner-core structural change. As the change of the circular dichroism signal is reversible, salt-titration spectroscopy is applicable to equilibrium studies of the physical chemical properties of DNA-histone interactions. By the assumption of a non-co-operative model, the binding constant for the chicken erythrocyte (H2A, H2B) dimer to the DNA-(H3, H4) complex is calculated as 2.8 X 10(6) M-1 at 1.0 M-NaCl (20 degrees C, pH 7.6). The DNA sequence dependence of the stability of the DNA-(H3, H4) interaction is observed in the salt-titration profiles of reconstituted material. Decreasing stability of the interaction of (H3, H4) is observed following the order: poly[(dG)-(dC)] much greater than chicken erythrocyte DNA greater than poly[(dA)-(dT)]. It is concluded that histones (H3, H4) have a different DNA sequence dependence from histones (H2A, H2B).     

Solution structure of halophilic malate dehydrogenase from small-angle neutron and X-ray scattering and ultracentrifugation

Data from small-angle X-ray and neutron scattering and ultracentrifugation experiments on solutions of malate dehydrogenase from Halobacterium maris mortui are analysed together to yield a model for the enzyme particle formed by the protein and its interactions with water and salt in the solvent. The halophilic enzyme is stable only in high concentrations of salt and the model has structural features that are absent from non-halophilic malate dehydrogenase. The complementarity of the information derived from the three experimental methods is discussed extensively and quantitatively. It derives from the fact that mass density (ultracentrifugation), electron density (X-rays) and neutron scattering density are independent of each other. Each method gives a different "view" of the same particle, and an analysis of the combined data provided thermodynamic and structural parameters with, apart from the chemical composition of the solutions, only one other assumption: a constant partial specific volume for water equal to 1.00 cm3 g-1. Both the insights gained by this novel approach and its limitations are carefully pointed out. In solvents between 1 M and 5 M-NaCl, the enzyme forms a particle of invariant volume, consisting of a protein dimer (87,000 g mol-1) with which are associated 0.87 g of water and 0.35 g of salt per gram of protein. The partial specific volume of the protein calculated from the combined experimental data is 0.753(+/- 0.030) cm3 g-1, in good agreement with the value calculated from the amino acid composition. The particle has a radius of gyration of 32 A and an equivalent Stokes radius of 43 A. By combining the data from the X-ray and neutron scattering studies, the radii of gyration of the protein moiety alone and of the associated water and salt distribution were calculated. They are 28 A and about 40 A, respectively. The large-angle scattering curves show that the shapes of the particle and of the protein moiety alone are similar. At very low resolution they can be approximated by an ellipsoid of axial ratio 1:1:0.6 (or 1:1:1.5). At higher resolution, it becomes apparent that the particle has a significantly larger interface with solvent than an homogeneous ellipsoid or globular protein. The model has a globular protein core similar to non-halophilic malate dehydrogenase, with about 20% of the protein extending loosely out of the core, forming the large interface with solvent. The main interactions with water and salt take place on this outer part.     

The role of zinc in the stabilization of the dimeric form of bacterial alpha-amylase.

Bacterial alpha-amylase was shown by equilibrium and velocity-sedimentation studies to be a monomer-dimer equilibrium system in 0.10M-NaCl/0.015M-calcium acetate/0.010M-EDTA, pH7.0; an association constant of 2.4 X 10(3)M-1 is obtained. Studies of the binding of Zn2+ to alpha-amylase in 0.10M-NaCl/0.005M-calcium acetate, pH7.0, yielded binding curves that exhibit dependence on the concentration of alpha-amylase (Zn2+-free) used in the equilibrium-dialysis experiments. Results are described very satisfactorily by a reaction scheme in which Zn2+ binds exclusively to the dimer of the above monomer--dimer system with an association constant of 1.0 X 10(6)M-1. The present results refute the earlier scheme for dimer stabilization by Zn2+ in which the metal ion formed a cross-link between two non polymerizing monomer units.     

Testing transport models and transport data by means of kinetic rejection criteria.

The proenzyme form of C1r catalytic domains was generated by limited proteolysis of native C1r with thermolysin in the presence of 4-nitrophenyl-4'-guanidinobenzoate. The final preparation, isolated by high-pressure gel permeation in the presence of 2 M-NaCl, was 70-75% proenzyme and consisted of a dimeric association of two gamma B domains, each resulting from cleavage of peptide bonds at positions 285 and 286 of C1r. Like native C1r, the isolated domains autoactivated upon incubation at 37 degrees C. Activation was inhibited by 4-nitrophenyl-4'-guanidinobenzoate but was nearly insensitive to di-isopropyl phosphorofluoridate; likewise, compared to pH 7.4, the rate of activation was decreased at pH 5.0, but was not modified at pH 10.0. In contrast, activation of the (gamma B)2 domains was totally insensitive to Ca2+. Activation of the catalytic domains, which was correlated with an irreversible increase of intrinsic fluorescence, comparable with that previously observed with native C1r [Villiers, Arlaud & Colomb (1983) Biochem. J. 215, 369-375], was reversibly inhibited at high ionic strength (2 M-NaCl), presumably through stabilization of a non-activatable conformational state. Detailed comparison of the properties of native C1r and its catalytic domains indicates that the latter contain all the structural elements that are necessary for intramolecular activation, but probably lack a regulatory mechanism associated with the N-terminal alpha beta region of C1r.     

Ovarian adenosine 3'':5''-cyclic monophosphate-dependent protein kinase(s). Regulation by choriogonadotropin and lutropin in rat ovarian cells.

Choriogonadotropin and lutropin have been found to activate cyclic AMP-dependent protein kinase in ovarian cells isolated by collagenase dispersion from immature rats. The stimulatory effect of gonadotropins was dependent on both hormone concentration and incubation time. Choriogonadotropin at 1 mug/ml fully stimulated the protein kinase activity within 5 min of incubation, and this effect was specific for choriogonadotropin and lutropin-like activity. In addition, protein kinase activity has been characterized with respect to salt sensitivity, cyclic AMP binding, and its responsiveness to gonadotropins and other peptide hormones. Ovarian protein kinase was susceptible to high salt concentrations. The addition of 0.3-1.0 M-NaCl in incubation medium increased the activity ratio with a concomitant decrease in cycle AMP-dependence. The salt effect on protein kinase was observed both from hormone-treated and untreated cells. The hormone-stimulated and unstimulated protein kinase activity was completely stable in the absence of NaCl. No change in the activity ratio was observed when cellular extracts were assayed for protein kinase activity either immediately or after 2 h in the absence of added salt. Gel filtration in the absence of NaCl of cellular extracts prepared from choriogonadotropin-treated and untreated cells showned only a single peak of protein kinase activity that was sensitive to exogenously added cyclic AMP. By contrast, when 0.5 M-NaCl was included in the column buffer, the chromatography of untreated extract showed two peaks of protein kinase activity. The first peak was sensitive to added cyclic AMP, whereas the second peak was insensitive to it. Under identical experimental conditions, protein kinase from gonadotropin-treated cells showed, on gel filtration, only one peak of activity that was totally insensitive to added cyclic AMP. DEAE-cellulose column chromatography of a 20000 g supernatant fraction resulted in a peak of kinase activity that eluted in approx. 0.15 M-NaCl, similar to the similar to the elution of type II protein kinases as described by Corbin et al. (1975) (J. Biol. Chem. 250, 218-225). Choriogonadotropin stimulation produced a decrease in the capacity of protein kinase to bind exogenous cyclic [3H]AMP, with a concomitant increase in the kinase activity ratio. These results are consistent with the notion that cyclic AMP, GENERATED IN SITU Under hormonal stimulation, binds tot he regulatory subunit of protein kinase with subsequent dissociation of the active catalytic subunit from the holoenzyme.     

Crystallization of the DNA-binding Escherichia coli protein FIS

The specific DNA-binding protein FIS (factor for inversion stimulation), which stimulates site-specific DNA inversion by interaction with an enhancer sequence, was purified from an Escherichia coli strain overproducing the protein. FIS was crystallized at room temperature by microdialysis against 1.2 to 1.5 M-sodium/potassium phosphate containing 10 mM-Tris.HCl, 0.5 to 1 M-NaCl and 1 mM-NaN3 at pH 8.0 to 8.2. The crystals are stout prisms and suitable for X-ray diffraction study beyond 2.5 A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1). The unit cell has dimensions a = 47.57(4) A, b = 51.13(4) A, c = 79.83(6) A and contains one FIS dimer in the asymmetric unit.     

Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution.

The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface.     

The Bohr effect of hemoglobin intermediates and the role of salt bridges in the tertiary/quaternary transitions

Understanding mechanisms in cooperative proteins requires the analysis of the intermediate ligation states. The release of hydrogen ions at the intermediate states of native and chemically modified hemoglobin, known as the Bohr effect, is an indicator of the protein tertiary/quaternary transitions, useful for testing models of cooperativity. The Bohr effects due to ligation of one subunit of a dimer and two subunits across the dimer interface are not additive. The reductions of the Bohr effect due to the chemical modification of a Bohr group of one and two alpha or beta subunits are additive. The Bohr effects of monoliganded chemically modified hemoglobins indicate the additivity of the effects of ligation and chemical modification with the possible exception of ligation and chemical modification of the alpha subunits. These observations suggest that ligation of a subunit brings about a tertiary structure change of hemoglobin in the T quaternary structure, which breaks some salt bridges, releases hydrogen ions, and is signaled across the dimer interface in such a way that ligation of a second subunit in the adjacent dimer promotes the switch from the T to the R quaternary structure. The rupture of the salt bridges per se does not drive the transition.     

Isolation of a 240-kilodalton actin-binding protein from Dictyostelium discoideum

A high molecular weight actin-binding protein with subunit mass of 240 kilodaltons has been purified from vegetative amoebae of Dictyostelium discoideum. Briefly, a cell extract was prepared by homogenizing vegetative amoebae in 5 mM EGTA, 5 mM 1,4-piperazineethanesulfonic acid, 1 mM dithiothreitol, 0.02% NaN3, pH 7.0, followed by ultracentrifugation at 114,000 X g for 1 h. The 240-kDa protein in this extract was separated from actin by chromatography on ATP-saturated DEAE-cellulose and further purified by chromatography on hydroxylapatite and Sephacryl S-300. The 240-kDa protein increases the low shear viscosity of F-actin. Covalent cross-linking with dimethyl suberimidate demonstrates that the 240-kDa protein can form dimers in high salt (500 mM NaCl). Hydrodynamic studies in high salt demonstrate the presence of an asymmetric dimer (Stokes' radius = 8.6 nm, sedimentation coefficient = 12 S, native molecular weight = 434,000, and frictional ratio = 1.7). Rotary shadowing demonstrates that the monomer is a flexible rod of approximately 70 nm in length that can associate end to end to form a dimer of approximately 140 nm in length. The 240-kDa protein cross-reacts with antibodies to chicken gizzard filamin. The properties of the 240-kDa protein suggest that it is a member of the filamin class of actin-associated proteins.     

Small-angle x-ray scattering studies of metastable intermediates of beta-lactoglobulin isolated after heat-induced aggregation

Small-angle x-ray scattering was used for studying intermediate species, isolated after heat-induced aggregation of the A variant of bovine beta-lactoglobulin. The intermediates were separated in two fractions, the heated metastable dimer and heated metastable oligomers larger than the dimer. The pair distance distribution functions for the two intermediate fractions as well as for the native protein have been obtained by indirect Fourier transformation. In addition, the scattering intensity data for samples of the native protein at different concentrations were fitted using a combination of monomer and dimer form factors, which provides an estimate of the amount of monomer in solutions. By subtracting the contribution from the monomer, the scattering intensity from the dimer of the native protein can be determined and compared with the results for the metastable dimer. An ellipsoidal model was used to fit the data for the metastable dimer, and for comparison the same analysis was performed on the dimer of the native protein. The results show that the metastable dimer is more elongated than the dimer of the native protein and it occupies a volume 1.4-fold larger, in agreement with a more loose, partially unfolded conformation. The same ellipsoidal model was used to analyze the data for the fraction of larger metastable oligomers. In this case, an even more elongated ellipsoid was obtained, suggesting a linear association of monomers in the oligomers.     

Salt-dependent monomer-dimer equilibrium of bovine beta-lactoglobulin at pH 3

Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.     

Dimerization of native and proteolytically modified neurophysins as monitored by proton magnetic resonance spectroscopy: proximity of tyrosine-49 to the subunit interface

Neurophysin is a self-associating protein in which peptide-hormone binding and dimerization are thermodynamically linked. The structural basis of the linkage is unknown. We have studied the dimerization of bovine neurophysin I and two proteolytically modified derivatives by proton nuclear magnetic resonance spectroscopy in order to identify residues at the intersubunit contact regions and to evaluate the origin of the reported loss of dimerization associated with tryptic excision of residues 1-8. The concentration dependence at neural pH of the spectra of native neurophysin and des-90-92-neurophysin demonstrated a finite set of dimerization-sensitive resonances that included the ring protons of Tyr-49. Using these to monitor dimerization, we confirmed predictions of a large increase in the dimerization constant associated with carboxyl protonation. By the same criteria, dimerization of the des-1-8 protein, in disagreement with earlier reports, was found to be undiminished relative to that of the native protein. However, spectral changes in the Tyr-49 ring ortho proton region associated with dimerization of the des-1-8 protein differed significantly from those in the native protein and indicated an altered conformation of the des-1-8 dimer apparently restricted to the vicinity of Tyr-49. The results are shown to place Tyr-49 adjacent to both the intersubunit contact region and the 1-8 sequence in the native protein, loss of stabilizing interactions with 1-8 leading to altered interactions of Tyr-49 with the subunit interface. Because Tyr-49 is also close to the peptide-binding site, this arrangement spatially links the peptide-binding and dimerization sites of neurophysin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of nuclear and DNA-binding proteins in proliferating and quiescent mammalian cells.

The dependence of cell proliferation on nuclear protein phosphorylation was studied with exponential-phase and stationary-phase cultures of Chinese-hamster ovary cells. Nuclear proteins were fractionated, according to their DNA-binding affinities, by using sequential extractions of isolated nuclei with increasing concentrations of NaCl. When viable whole cells were labelled with H332PO4, phosphorylation of nuclear proteins was found to be lower in quiescent cells than in proliferating cells. Phosphorylation of nuclear proteins soluble in 0.30M-NaCl (less than 50% of these proteins bind to DNA) was greater than for those proteins soluble in higher salt concentrations (80-100% of these proteins bind to DNA). Cyclic AMP enhanced the phosphorylation of nuclear proteins soluble in 0.3 m-NaCl by 40-50%, and this stimulation was independent of cell growth. Cyclic AMP also increased the phosphorylation of nuclear proteins soluble in 0.6M-NaCl and 2.0M-NaCl by 40-50% in exponential-phase cultures, but not in stationary-phase cultures. Several examples of specific phosphorylation in response to cyclic AMP were observed, including a 35000-mol.wt. protein in the 0.30 M-NaCl-soluble fraction and several proteins larger than 100000 molecular weight within this fraction. A major peptide of molecular weight approx. 31000 extracted with 0.6M-NaCl was also phosphorylated. Its phosphorylation was independent of cyclic AMP in exponential-phase cultures, and it was not phosphorylated in plateau-phase cells. These changes in cell-growth-dependent phosphorylation occurred in the absence of any apparent qualitative changes in the nuclear protein molecular-weight distributions. These data demonstrate that (1) phosphorylation of nuclear proteins is dependent on the culture's proliferative status, (2) both cyclic AMP-dependent and cyclic AMP-independent specific phosphorylation occurs, and (3) the cyclic AMP-dependent growth-independent phosphorylation that occurs does not appear to be a modification of DNA-binding proteins, whereas the cyclic AMP-dependent growth-dependent phosphorylation does involve modification of DNA binding proteins.     

Separation and purification of the alkaline phosphatase and a phosphodiesterase from Halobacterium cutirubrum.

1. Halobacterium cutirubrum alkaline phosphatase is associated in crude extracts with a phosphodiesterase. 2. The enzymes were stabilized in buffers containing both (NH4)2SO4 and 10 mM-Mn2+. 3. Adsorption chromatography on Sepharose 6B/agarose-gel columns in the presence of 1.4M-(NH4)2SO4 gave a phosphatase-free phosphodiesterase and the alkaline phosphatase associated with some phosphodiesterase activity. 4. Further chromatography of the separated enzymes gave a good recovery of greater than 600-fold purified phosphodiesterase and greater than 3000-fold purified alkaline phosphatase. 5. The requirements of these enzymes and their relationship to each other was examined. 6. A detailed study showed that the alkaline phosphatase was adsorbed at least partially to agarose and dextran columns at all (NH4)2SO4 concentrations from 0.25 to 2M. 7. In contrast, no adsorption of the enzyme or protein standards was evident in 2.5M-KCl/l M-NaCl or 0.25 M-KCl/0.1 M-NaCl, in agreement with previous studies by Louis, Peterkin & Fitt [(1971) Biochem. J. 121, 635-641], thus confirming the validity of gel filtration in 2.5 M-KCl/1 M-NaCl as a method for determining the approximate molecular weights of extremehalophile proteins.     

An active covalently linked dimer of human interferon-gamma. Subunit orientation in the native protein.

We have constructed and expressed a covalently linked head to tail dimer of human interferon-gamma (IFN-gamma) in which two monomers are joined head to tail via a rigid peptide hinge using genetic engineering techniques. The hinge was derived from the human immunoglobin IgA1 sequence (Hallewell, R.A., Laria, I., Tabrizi, A., Carlin, G., Getzoff, E.D., Tainer, J.A., Cousens, L.S., and Mullenbach, G.T. (1989) J. Biol. Chem. 264, 5260-5268). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the polypeptide produced by this construction migrates as a 30,000 polypeptide species. The protein elutes as a single species by molecular sieve chromatography under native conditions. The covalently linked dimer exhibits one-half the antiviral activity of native dimeric IFN-gamma; receptor binding assays show the covalently linked dimer binds to the IFN-gamma receptor with one-half the avidity of native IFN-gamma. This difference is not due to conformational differences between the two molecules, as the aromatic region of the NMR spectrum of the purified covalently linked dimer is identical with that of the wild type protein. From these data, we suggest that human IFN-gamma associates in a head to tail dimer in its active configuration. Regions of IFN-gamma are contiguous with the amino and carboxyl termini and are obscured by the hinge peptide in the covalently linked dimer. Our studies demonstrate that these regions may be important for receptor-ligand interaction.     

Characterization of two triacylglycerol lipase activities in pig post-heparin plasma.

Two triacylglycerol lipase activities were characterized after partial purification from pig post-heparin plasma. These two lipase activities were eluted sequentially with a NaCl gradient from columns containing Sepharose with covalently linked heparin. The first lipase activity, which was eluted at 0.75M-NaCl, was not inhibited at 28 degrees C in the presence of 1M-NaCl and was not further activated by plasma apolipoproteins. The absence of this lipase activity from post-heparin plasma from hepatectomized pigs indicates that the liver plays a role in the synthesis of this enzyme. A second lipase activity, which was eluted at 1.2M-NaCl, was inhibited when assayed in the presence of 1.0M-NaCl and was activated 14-fold by an apolipoprotein isolated from human very-low-density lipoprotein. The characteristics are identical with those of lipoprotein lipase purified from pig adipose tissue.     

Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions.

Stefin A folds as a monomer under strongly native conditions. We have observed that under partially denaturing conditions in the temperature range from 74 to 93 degrees C it folds into a dimer, while it is monomeric above the melting temperature of 95 degrees C. Below 74 degrees C the dimer is trapped and it does not dissociate. The dimer is a folded and structured protein as judged by CD and NMR, nevertheless it is no more functional as an inhibitor of cysteine proteases. The monomer-dimer transition proceeds at a slow rate and the activation energy of dimerization at 99 kcal/mol is comparable to the unfolding enthalpy. A large and negative dimerization enthalpy of -111(+/- 8) kcal/mol was calculated from the temperature dependence of the dissociation constant. An irreversible pretransition at 10-15 deg. below the global unfolding temperature has been observed previously by DSC and can now be assigned to the monomer-dimer transition. Backbone resonances of all the dimer residues were assigned using 15N isotopically enriched protein. The dimer is symmetric and the chemical shift differences between the monomer and dimer are localized around the tripartite hydrophobic wedge, which otherwise interacts with cysteine proteases. Hydrogen exchange protection factors of the residues affected by dimer formation are higher in the dimer than in the monomer. The monomer to dimer transition is accompanied by a rapid exchange of all of the amide protons which are protected in the dimer, indicating that the transition state is unfolded to a large extent. Our results demonstrate that the native monomeric state of stefin A is actually metastable but is favored by the kinetics of folding. The substantial energy barrier which separates the monomer from the more stable dimer traps each state under native conditions.     

Dissociation and reconstitution of bovine seminal RNAase: Construction of a hyperactive hybrid dimer

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we describe the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.     

Effect of single point mutations in a form of systemic amyloidosis

Amyloid deposits of light-chain proteins are associated with the most common form of systemic amyloidosis. We have studied the effects of single point mutations on amyloid formation of these proteins using explicit solvent model molecular dynamics simulations. For this purpose, we compare the stability of the wild-type immunoglobulin light-chain protein REI in its native and amyloid forms with that of four mutants: R61N, G68D, D82I, and A84T. We argue that the experimentally observed differences in the propensity for amyloid formation result from two effects. First, the mutant dimers have a lower stability than the wild-type dimer due to increase exposure of certain hydrophobic residues. The second effect is a shift in equilibrium between monomers with amyloid-like structure and such with native structures. Hence, when developing drugs against light-chain associated systemic amyloidosis, one should look for components that either stabilize the dimer by binding to the dimer interface or reduce for the monomers the probability of the amyloid form.