Secondary structure conversions of Mycobacterium tuberculosis ribonucleotide reductase protein R2 under varying pH and temperature conditions

The structural properties of Mycobacterium tuberculosis (Mtb) ribonucleotide reductase R2 protein were studied under varying pH and temperature conditions by circular dichroism (CD) spectroscopy as well as dynamic light scattering (DLS). Under physiological conditions this protein has a high alpha-helical content, similar to the corresponding protein from other species, e.g. mouse. Decreasing the pH induced significant structure conversions. When pH was below 6.5 an aggregated structure was observed and reached a maximum at pH 4. The aggregated state of this protein was verified by DLS and was found to be rich in beta-structure. This amyloid-like structure transformed into a molten globule state with high temperature stability (between 25 and 80 degrees C) at pH below 3. The corresponding mouse protein R2 under similar conditions showed no evidence of an aggregated state around pH 4.     

The major allergen from birch tree pollen, Bet v 1, binds and permeabilizes membranes

The 159 residue Bet v 1 is the major allergen from birch tree pollen. Its natural function is unknown although it is capable of binding several types of physiologically relevant ligands in a centrally placed cavity in the protein structure. Here we use circular dichroism and fluorescence spectroscopy to show that Bet v 1 binds to DOPC and DOPG phospholipid vesicles in a pH-dependent manner. Binding is facilitated by low pH, negatively charged phospholipids, and high vesicle curvature, indicating that electrostatic interactions and vesicle surface defects are important parameters for binding. Binding is accompanied by major structural rearrangements, involving an increase in alpha-helical structure and a decrease in beta-structure. A bilayer structure per se is not a prerequisite for these rearrangements, since they also occur in the presence of the micelle-forming lysophospholipids lysoMPC and lysoMPG. Two major bound states (A and B) with distinct secondary structure compositions were identified, which predominate in the pH ranges approximately 9.5-6.5 and approximately 5-2.5, respectively. Despite the high content of secondary structure, the A- and B-states are partially unfolded as they unfold noncooperatively in CD thermal scans, in contrast to the native state. In addition, the B-state (but not the A-state) shows intermediate proteolysis-resistance and is able to induce complete leakage of calcein from the vesicles, indicating that this state is partially inserted into and significantly perturbs the bilayer structure. We conclude that Bet v 1 is a membrane binding protein, highlighting a possible biological function of this protein.     

Purification and characterization of human transcortin.

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 X 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.     

Crystal structures of [Met5] and [(4-bromo)Phe4,Met5]enkephalins: formation of a dimeric antiparallel beta-structure

The crystal structure of [(4-bromo)Phe4,Met5]enkephalin (Tyr-Gly-Gly-(4-bromo)-Phe-Met) shows two independent molecular conformations. The molecules are arranged in parallel in a head-to-tail fashion and form an antiparallel beta-sheet structure involving intermolecular hydrogen bonds. This dimeric beta-structure is also observed in the [Met5]enkephalin crystal, in spite of their different crystal packing environments, which shows the energetic stability of this molecular conformation. The three-dimensional similarity between the dimeric beta-structure and the beta-turn form is discussed in the relation to the opioid delta and mu receptors.     

Stability and transformation of the glucocorticoid receptor under acidic conditions

The [3H]triamcinolone acetonide ([3H]TA)-binding ability of the rat liver glucocorticoid receptor (GR) was investigated under acidic conditions, ranging from pH 2 to 7.3. Both in the presence and absence of 10 mM molybdate, the [3H]TA-binding ability decreased below pH 6.5 and was almost completely lost below pH 5, pH 5.9 +/- 0.1 giving 50% [3H]TA-binding. The binding ability was recovered when the pH of the cytosol was reversed to 7.3 or the precipitate obtained on acidification was dissolved in a buffer of pH 7.3. Moreover, in the absence of molybdate, the [3H]TA-GR complexes formed at pH 7.3 remained unchanged until pH 5. Then they decreased, pH 3.9 +/- 0.1 giving 50% binding, and completely disappeared at pH 3. [3H]TA-binding activity recovered from the precipitate also decreased in a similar pH region (a 50% decrease in binding being observed at pH 4.2 +/- 0.04). These results suggest that rat liver GR is rather resistant under acidic conditions and that it exists in a peculiar state below pH 5.9 to approximately 4 as to its ligand binding property: unoccupied GR has no [3H]TA-binding ability but [3H]TA-GR complexes once formed at neutral pH do not dissociate. [3H]TA-GR complexes recovered from the precipitate at pH 5 had a Stokes radius of 7.5 nm, little DNA-cellulose-binding ability and sedimented at 8.6S on glycerol gradient centrifugation, indicating that the receptor existed in a nontransformed state. In addition, both occupied and unoccupied GR were transformed at about pH 4, their being 50% transformation. This transformation was accompanied by irreversible denaturation of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acridine dimers: influence of the intercalating ring and of the linking-chain nature on the equilibrium and kinetic DNA-binding parameters

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA-binding properties of DNA bifunctional intercalators, fluorescent 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine analogues with chains of variable rigidity were synthesized. 1H-NMR studies show that the conformation of 9-aminoacridine dimers is almost independent of the nature of the linking chain. A strong self-stacking of the aromatic rings of the 2-methoxy-6-chloro-9-aminoacridine is observed for dimers with flexible chains but not for those with rigid chains. All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly[d(A-T)].poly[d(A-T)] are in the same range (2-4 x 10(7) M-1 s-1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant. The effect of chain rigidity and pH on the calf thymus DNA binding of 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine dimers is quite different. In the series of 9-aminoacridine the pKa of the dimers remains high and therefore no difference of DNA-binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA-binding affinity. In the 2-methoxy-6-chloro-9-aminoacridine series, the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA-binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A-T)].poly[d(A-T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.     

Effect of transcortin on the corticosterone-transforming activity of cytosol from the rat liver

The study of transcortin role in 3H-corticosterone metabolism has shown that transcortin of blood plasma from rats bearing Walker carcinosarcoma preserves the hormone conversion to dihydrocompounds 4 time less intensively than transcortin taken from healthy rats. Inactivated transcortin exerts no effect on the rate of formation of 5 beta-metabolites. Under the influence of homogeneous transcortin samples, a decrease in the content of 5 beta-reduced corticosterone metabolites is revealed to occur depending on transcortin concentration in the system. It is shown that in incubation systems where hormone is in the bound state the metabolism preserving capacity of transcortin depends on the temperature degree. The transcortin activity on corticosterone metabolism is supposed to be closely related to the intensity of its complexing with transcortin.     

The influence of transcortin on adrenocorticotropin-stimulated corticosterone production in monolayer cultured rat adrenal cells

To verify the influence of the protein binding status of steroids adjacent to adrenal cells on steroidogenesis, the effect of transcortin, a specific binding protein of cortisol or corticosterone, on adrenocorticotropin (ACTH)-stimulated corticosterone production in monolayer cultured rat adrenal cells was studied. The transcortin in concentration of 5 x 10(-7) M was loaded with 0, 2.5, 5 and 10 pg/ml ACTH-(1-24), and the cells were incubated for 2 and 4 hours. Since molar concentrations of corticosterone produced in the medium were below the transcortin concentration at all levels of stimulation, protein-unbound corticosterone in the medium may have been largely reduced by the addition of transcortin. However, the total corticosterone production was not influenced by the transcortin added to the medium. It was speculated that protein-unbound steroid within the concentration range modulated by transcortin in the area surrounding the adrenal cells may not affect adrenal steroidogenesis.     

Human liver nuclear transcortin its postulated role in glucocorticoid regulation of genetic activity

Highly purified human transcortin was injected into rabbits, and the antibody subsequently obtained was employed for the demonstration of transcortin-like molecules within various subcellular fractions of the human liver cell. Results obtained via quantitative double diffusion ouchterlony procedures indicate that proteins extracted from the nucleus or from chromatin form continuous precipitin lines of identity with those of transcortin. Fluorescein-tagged anti-transcortin permitted the visual localization of this molecule within isolated nuclei. Cortisol binding studies of all the subcellular fractions, particularly that extracted from the chromatin, suggest that such proteins do indeed bind cortisol specifically, as well as responding to exogeneous additions to the buffer (sulfhydryl reagents) as does purified transcortin. Purified transcortin when dialyzed exhaustively loses its cortisol binding ability, although the latter can be restored after its incubation with chromatin at 4°C. The restoration of such activity is dependent upon a dialyzable, heat-resistant chromatin component which itself lacks cortisol binding activity and which increases the sedimentation characteristics of dialyzed transcortin. The effect of transcortin on the in vitro synthesis of RNA in an Escherichia coli RNA polymerase human liver chromatin system is also presented. All of the above results are interpreted to indicate that transcortin is involved directly in the regulation of that genetic activity observed subsequent to the administration of cortisol.     

Comparative study of primates' transcortin: immunoreactivity and steroid-binding activity

To investigate the phylogenic aspect of transcortin (corticosteroid-binding globulin, CBG), the immunoreactivity of transcortin with anti-human transcortin antiserum was studied in primates. The anti-human transcortin antibody was recognized by plasma proteins obtained from Catarrhini, taxonomically the most evolved monkey group. The immunoreactivity was not observed in plasma obtained from Platyrrhini and Prosimiae, classified as less evolved monkey groups than Catarrhini. Though comparison of immunoreactivity among different classes of Catarrhini was difficult because of non-parallelism of their displacement curves, displacement of 125I-labelled human transcortin from the antiserum by 1:10 and 1:100 diluted plasma was highest in human followed by Pongidae, Cercopithecoidea. The immunoreactivity of thyroxine-binding globulin (TBG) with anti-human TBG antiserum was also examined. The anti-human TBG antibody was only recognized in plasma from Pan (anthropoid ape) among Pongidae, highly evolved monkeys among Catarrhini. The existence of immunoreactive transcortin and TBG to respective human protein antibody in the highly evolved ape agreed well with the cladogenetic division of primate species delineated by Goodman and Moore (1971). Cortisol-binding activity of transcortin was detected in all monkeys except three, tafted capuchin monkey, night monkey and cotton-headed tamarin, which belong to Platyrrhini. The absence of cortisol-binding activity in these animals might be attributed to high levels of endogenous cortisol and low cortisol-binding capacity of transcortin. It is speculated that the structure of the immunoreactive site in transcortin could be modified by evolution without affecting the biologically important site, the site for cortisol binding.     

Interaction of elongation factor 1alpha from Zea mays (ZmEF-1alpha) with F-actin and interplay with the maize actin severing protein, ZmADF3.

EF-1alpha is an abundant eukaryotic protein whose principle function appears to be to bind aminoacyl-tRNA to the ribosome. However, it is also known that EF-1alpha from other sources binds both microtubules and microfilaments. We report the expression of Zea mays EF-1alpha (ZmEF-1alpha) in bacteria and that this protein has similar actin-binding properties as other EF-1alpha members. ZmEF-1alpha bundles actin filaments at low pH (6.5) and inhibits the addition of monomer at both filament ends, possibly as a consequence. ZmEF-1alpha binds actin filaments at all pH values tested (pH 6.0-8.0), indicating that one actin binding site is not pH sensitive. One of the actin-binding sites was determined to reside within domain I (1-223) of ZmEF-1alpha, but this domain did not affect the kinetics of polymerisation. We show that the bundling activity of ZmEF-1alpha is modulated by ZmADF3 a (a Zea mays ADF/cofilin), an actin filament severing protein, in vitro. Bundling of actin filaments caused by ZmEF-1alpha was enhanced in the presence of ZmADF3. The pH-dependent activities of both proteins in vitro suggests that they may work together to respond to temporal and spatial intracellular pH changes to regulate the pattern of the growth of plant cells.     

A specific effect of calcium ion on the polymerization-depolymerization of tobacco mosaic virus protein.

Tobacco mosaic virus protein stored in the cold at low ionic strength between pH 5 and 5.5 is highly polymerized. When such protein is brought to room temperature and mixed with acetate buffer and additional electrolyte to give a final pH of 6.5 and ionic strength of 0.1, the protein is still in the polymerized state. When the temperature is dropped to about 5 °C, the protein depolymerizes rapidly, in the normal manner, if the added electrolyte is barium chloride, magnesium chloride, or potassium chloride. However, if it is 0.01 m calcium chloride, the depolymerization is slow, requiring about 12 h to reach completion. When the temperature of this depolymerized solution is raised, the protein polymerizes rapidly; when the temperature is dropped, the protein depolymerizes rapidly, just as in solutions free of calcium.Ion-binding studies show that calcium is bound to the protein during the initial step when it is brought to pH 6.5 and room temperature in the presence of calcium. The calcium is released during the slow depolymerization when the temperature is dropped and is not bound again during polymerization at pH 6.5, brought about by an increase in temperature. This means that polymerized protein at pH 5.5 has a structure capable of binding calcium ions, probably a helical structure like that of the protein in the virus. When pH is raised to pH 6.5 at room temperature, this structure remains long enough for calcium to be bound when present. These calcium ions stabilize the polymer, resulting in slow depolymerization when the temperature is lowered. When the temperature is raised at pH 6.5, a different, looser polymer structure is obtained, one not capable of binding calcium.     

Physical studies on the conformation of ribosomal protein S4 from Escherichia coli.

Proton magnetic resonance, circular dichroism and infrared spectroscopy were used to investigate the secondary and tertiary structure of the 16-S RNA binding protein S4 from Escherichia coli ribosomes. The proton magnetic resonance spectra of protein S4 in ribosomal reconstitution and low-salt buffers were identical and showed little dipolar broadening of the peaks, suggesting that the protein had an open extended structure. A ring-current-shifted apolar methyl resonance in the high-field region of the spectrum, together with a perturbation of the tyrosine ring proton resonance in the low-field region, indicated the existence of a specific tertiary fold in the polypeptide chain. This structure disappeared on lowering the pH below 5 or on heating above 30 degrees C, both processes being reversible. Circular dichroism measurements on protein S4 showed an alpha-helix content of 32% in reconstitution buffer compared with 26% in low-salt buffer. Heating the protein solution in reconstitution buffer above 35 degrees C reversibly disrupted this extra helix. Infrared studies on both solid films and solutions of protein S4 indicated the presence of little or no beta-structure. These results correlate well with the known RNA binding properties of protein S4.     

Effect of pH on Muscarinic Acetylcholine Receptors from Rat Brainstem

The effect of hydrogen ion concentration on ligand binding to muscarinic acetylcholine receptors was studied in membranes isolated from rat brainstem. The binding of [3H]methylscopolamine was constant between pH 7 and 10. The affinity, but not the number, of [3H]methylscopolamine binding sites decreased below pH 7; at pH 4 little binding was detected. When brainstem membranes were incubated at various pH levels from 3 to 11 for 1 h and then returned to pH 8, [3H]methylscopolamine binding affinity was restored to control levels. Carbamylcholine binding affinity was also depressed in media of low pH. However, this decrease was permanent after a 1-h incubation at pH 4 (i.e. carbamylcholine affinity was not restored on raising the pH to 8). The capacity of a guanine nucleotide to affect carbamylcholine was also abolished by a 1-h incubation at pH 4, and was not restored by raising the pH. The guanine nucleotide-dependent regulatory protein may be irreversibly inactivated or dissociated from the receptor at low pH. The receptor's binding subunit, on the other hand, appears to be much less sensitive to hydrogen ion concentration.     

Characterization of a common intermediate of pea lectin in the folding pathway induced by TFE and HFIP

When pea lectin was exposed to a low pH range, it was found that the secondary structure of the lectin resisted conformational changes to a large extent up to pH 2.4 and below this pH, a sharp transition was observed which could be due to the presence of 27 acidic amino acid residues present in the protein. The effects of 1,1,1,3,3,3 hexafluoro-isopropanol (HFIP) and 2,2,2-Trifluoroethanol (TFE) on the conformation of pea lectin at pH 2.4 were studied using circular dichroism and fluorescence spectroscopy. Analysis varying the TFE concentration showed that up to 80% TFE (v/v) protein retained the residual beta-structure accompanied by a loss in tertiary structure. A similar conformation is presumed to exist at 4% HFIP (v/v), with an increase in HFIP concentration structural rearrangements occurred and a transition from beta-structure to alpha-helical structure started from 12% HFIP which completed at 30% HFIP. Our studies show the occurrence of a common intermediate in the folding pathway of pea lectin induced by two different fluoroalcohols, which differ in their mode of action to stabilize the secondary structure of a given protein. While TFE was not found to induce any alpha-helical structure, HFIP caused the transition of pea lectin, which is predominantly a beta-sheet protein, to a structure rich in alpha-helical contacts. Thus, our results also point out the possibility of a non-hierarchical model of protein folding in lectins.     

The heat denaturation of bacterial ribonuclease studied by infrared spectroscopy]

The thermal denaturation of bacterial ribonuclease in the interval of pH 2.5-7.0 has been investigated by means of infra-red spectroscopy method. The protein melting for pH 2.5 begins at the temperature 25 degrees C and is accompanied by secondary protein structure reconstruction, partially destroying native beta-structure and leading to new denatured conformation appearance of different types of beta-turns. Spectral changes for pH 3.5 and 7.0 are significantly less in the same frequency areas. At the temperature more than 50 degrees C protein aggregation takes place with inter-molecule-beta-form formation.     

Hormone-protein interactions and functional activity of the adrenal cortex during adaptation to pressure chamber hypoxia

Blood plasma and adrenals of rats subjected to the exposure of deep pressure-chamber hypoxia are studied. The method of high-performance liquid chromatography is used to determine the content of corticosterone and deoxycorticosterone as well as the binding ability of transcortin relative to hydrocortisone and corticosterone before and after ectomy of endogenic hormones. It is shown that hyperactivation of adrenals occurs already the first 20 min of the experiment, which is preserved during the whole experiment lowering, to some extent, by 150, 240 min. The binding ability of transcortin considerably lowers in the acute phase of stress and after the prolonged hypoxic exposure due to a 1.5-1.9-fold decrease in the number of the binding sites in the protein. The equilibrium redistribution is revealed in the plasma between free corticosterone and corticosterone bound to transcortin at the different stages of the hypoxic exposure.     

Circular dichroic study of the conformational stability of sulfhydryl-blocked bovine serum albumin

1. The blockage of the single sulfhydryl-group of bovine serum albumin does not alter the secondary structure, although the alpha-helical structure is destabilized since lower concentrations of guanidine and of urea unfold the protein. 2. What happens to the previously helical structure depends upon the reagent used to block the sulfhydryl-group. Bovine serum albumin derivatized with 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetate preferentially acquire the beta-structure in high concentrations of guanidine and urea, whereas iodoacetamide-derivatized bovine serum albumin acquires primarily the random coil structure. 3. Part of the helical structure is also lost in 5-6 mM sodium dodecyl sulfate; thionitrobenzoate-bovine serum albumin shows an increase in the random coil, whereas the two alkylated proteins display the increase both in beta-structure and random coil. 4. Carboxymethylation or carboxamidomethylation of fully reduced bovine serum albumin results in a drastic change in the secondary structure of the protein with a substantial decrease in alpha-helix and a corresponding increase in both beta-structure and random coil. These extensively alkylated proteins also display differences in denaturation profiles in solutions of guanidine and urea.     

pH-Dependent Conformational Changes in Proteins and Their Effect on Experimental pKas: The Case of Nitrophorin 4

The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis, substrate binding and protein structure. Due to interactions with the protein environment, their pK_as can be shifted from their solution values and, if a protein has two stable conformations, it is possible for a residue to have different “microscopic”, conformation-dependent pK_a values. In those cases, interpretation of experimental measurements of the pK_a is complicated by the coupling between pH, protonation state and protein conformation. We explored these issues using Nitrophorin 4 (NP4), a protein that releases NO in a pH sensitive manner. At pH 5.5 NP4 is in a closed conformation where NO is tightly bound, while at pH 7.5 Asp30 becomes deprotonated, causing the conformation to change to an open state from which NO can easily escape. Using constant pH molecular dynamics we found two distinct microscopic Asp30 pK_as: 8.5 in the closed structure and 4.3 in the open structure. Using a four-state model, we then related the obtained microscopic values to the experimentally observed “apparent” pK_a, obtaining a value of 6.5, in excellent agreement with experimental data. This value must be interpreted as the pH at which the closed to open population transition takes place. More generally, our results show that it is possible to relate microscopic structure dependent pKa values to experimentally observed ensemble dependent apparent pK_as and that the insight gained in the relatively simple case of NP4 can be useful in several more complex cases involving a pH dependent transition, of great biochemical interest.     

Reversible dissociation of cortisol-transcortin complex by sodium para-chloromercuribenzoate

Mercurials are considered as sulphydryl group specific reagents and one of them, sodium para-chloromercuribenzoate (PCMB), is currently used for SH titration. It has been shown that cellular steroid receptors are reversibly inactivated by mercurials even when the binding site is occupied by the steroid (Coty, W.A. (1980) J. Biol. Chem. 255, 8035-8037). This is a striking difference with alkylating SH reagents such as iodoacetic acid or N-ethylmaleimide, since these reagents inactivate only steroid-free receptors. In order to explain this discrepancy, we tested, in the present study, the specificity of PCMB on a blood plasma steroid binding protein: human transcortin. This protein presents the advantage, over cellular receptors, of being well characterized and to be available in a pure state. The transcortin-cortisol complex was also reversibly inactivated by PCMB when the reaction was carried out at a high excess of reagent over protein; such conditions are those previously used with steroid receptors. The reversibility was obtained not only with a reducing agent (dithiothreitol) but also with EDTA, which suggests a poor stability of the protein mercurial bond and therefore a nonspecific action. The decrease of activity was the result of a loss of binding sites and Scatchard plot analysis did not reveal any detectable decrease of the affinity constant for cortisol. Transcortin possesses two SH groups per molecule, one of these being buried in native conformation. After blockage of the accessible SH group by aminoethylation, transcortin kept the same activity, but when this aminoethylated transcortin was incubated with PCMB a loss of activity was obtained, although the residual buried SH group was again titrable with Ellman's reagent. Therefore, we can conclude that the action of PCMB on proteins must be interpreted with precaution, since it can induce an inactivation that is SH-independent.