Differences between the pressure- and temperature-induced denaturation and aggregation of beta-lactoglobulin A, B, and AB monitored by FT-IR spectroscopy and small-angle X-ray scattering.

We examined the temperature- and pressure-induced unfolding and aggregation of beta-lactoglobulin (beta-Lg) and its genetic variants A and B up to temperatures of 90 degrees C in the pressure range from 1 bar to 10 kbar. To achieve information simultaneously on the secondary, tertiary, and quaternary structures, we have applied Synchrotron small-angle X-ray diffraction and Fourier transform infrared spectroscopy. Upon heating a beta-Lg solution at pH 7.0, the radius of gyration Rg first decreases, indicating a partial dissociation of the dimer into the monomers, the secondary structures remaining essentially unchanged. Above 50 degrees C, the infrared spectroscopy data reveal a decrease in intramolecular beta-sheet and alpha-helical structures, whereas the contribution of disordered structures increases. Within the temperature range from 50 to 60 degrees C, the appearance of the pair distance distribution function is not altered significantly, whereas the amount of defined secondary structures declines approximately by 10%. Above 60 degrees C the aggregation process of 1% beta-Lg solutions is clearly detectable by the increase in Rg and intermolecular beta-sheet content. The irreversible aggregation is due to intermolecular S-H/S-S interchange reactions and hydrophobic interactions. Upon pressurization at room temperature, the equilibrium between monomers and dimers is also shifted and dissociation of dimers is induced. At pressures of approximately 1300 bar, the amount of beta-sheet and alpha-helical structures decreases and the content of disordered structures increases, indicating the beginning unfolding of the protein which enables aggregation. Contrary to the thermal denaturation process, intermolecular beta-sheet formation is of less importance in pressure-induced protein aggregation and gelation. The spatial extent of the resulting protein clusters is time- and concentration-dependent. The aggregation of a 1% (w/w) solution of A, B, and the mixture AB results in the formation of at least octameric units as can be deduced from the radius of gyration of about 36 A. No differences in the pressure stability of the different genetic variants of beta-Lg are detectable in our FT-IR and SAXS experiments. Even application of higher pressures (up to 10 kbar) does not result in complete unfolding of all beta-Lg variants.     

Enhanced thermodynamic stability of beta-lactoglobulin at low pH. A possible mechanism.

The thermodynamic stability of beta-lactoglobulin (beta-Lg) was studied at acidic and near-neutral pH values using equilibrium thermal-unfolding measurements. Transition temperature increased with a decrease in pH from 7.5 to 6.5 and 3.0 to 1.5, suggesting an increase in the net protein stability. Determination of the change in free energy of unfolding and extrapolation into the nontransition region revealed that beta-Lg increases its stability by increasing the magnitude of the change in free energy of unfolding at the temperature of maximum stability, as well as by increasing the temperature of maximum stability. The relative difference in the change in free energy of unfolding at 70 degrees C (with a reference pH of 7.5) was positive and its magnitude increased with a decrease in pH from 7.0 to 1.5 van't Hoff plots of thermal unfolding of beta-Lg at all pH values studied were non-linear and the measured changes in the enthalpy and entropy of unfolding for beta-Lg were high and positive. The relative magnitude of change of both enthalpy and entropy at 70 degrees C (compared with pH 7.5) increased with a decrease in pH up to 1.5. A possible mechanism for the increased stability of beta-Lg at low pH is discussed.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Pressure denaturation of the yeast prion protein Ure2

Denaturation of the Saccharomyces cerevisiae prion protein Ure2 was investigated using hydrostatic pressure. Pressures of up to 600 MPa caused only limited perturbation of the structure of the 40-kDa dimeric protein. However, nondenaturing concentrations of GdmCl in combination with high pressure resulted in complete unfolding of Ure2 as judged by intrinsic fluorescence. The free energy of unfolding measured by pressure denaturation or by GdmCl denaturation is the same, indicating that pressure does not induce dimer dissociation or population of intermediates in 2 M GdmCl. Pressure-induced changes in 5 M GdmCl suggest residual structure in the denatured state. Cold denaturation under pressure at 200 MPa showed that unfolding begins below -5 degrees C and Ure2 is more susceptible to cold denaturation at low ionic strength. Results obtained using two related protein constructs, which lack all or part of the N-terminal prion domain, were very similar.     

Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids

We report steady-state and time-resolved fluorescence studies with the single tryptophan protein, Staphylococcus aureus A, and several of its site-directed mutants. A couple of these mutants, nuclease-conA and nuclease-conA-S28G (which are hybrid proteins containing a six amino acid beta-turn substitute from concanavalin A), are found to have a much lower thermodynamic stability than the wild type. The thermal transition temperatures for nuclease-conA and S28G are 32.8 and 30.5 degrees C, which are about 20 degrees C lower than the Tm for wild-type nuclease A. These mutant proteins also are denatured by a much lower concentration of the denaturants urea and guanidine hydrochloride. We also show that an unfolding transition in the structure of the nuclease-conA hybrids can be induced by relatively low hydrostatic pressure (approximately 700 bar). The free energy for unfolding of nuclease-conA (and nuclease-conA-S28G) is found to be only 1.4 kcal/mol (and 1.2 kcal/mol) by thermal, urea, guanidine hydrochloride, and pressure unfolding. Time-resolved fluorescence intensity and anisotropy measurements with nuclease-conA-S28G show the temperature-, urea-, and pressure-perturbed states each to have a reduced average intensity decay time and to depolarize with a rotational correlation time of approximately 1.0 ns (as compared to a rotational correlation time of 11 ns for the native form of nuclease-conA-S28G at 20 degrees C).     

Role of the disulphide bridge in folding, stability and function of porcine odorant binding protein: spectroscopic equilibrium studies on C63A/C155A double mutant

Porcine odorant binding protein (pOBP) contains a single disulphide bridge linking residues Cys63 and Cys155. In order to get information on the role played by this crosslink in determining the structural and functional properties of the protein, we substituted these two Cys residues with two Ala residues by site directed mutagenesis and investigated the changes in folding, stability and functional features, as detected by fluorescence and circular dichroism measurements. In particular, we studied both chemical and thermal unfolding/refolding processes under equilibrium conditions, the first induced by guanidinium hydrochloride and the second by raising the temperature from 15 to 90 degrees C. Chemical unfolding curves, as obtained from intrinsic fluorescence and far-UV circular dichroism data, can be fitted by a simple two-state cooperative sigmoidal function; however, their partial overlap (C(1/2)=0.57+/-0.05 from fluorescence and 0.66+/-0.03 from CD) suggests the formation of an intermediate, which lacks tertiary structural features. Thermal unfolding was found to be reversible if the protein was heated up to 65 degrees C, but irreversible above that temperature because of aggregation. The thermodynamic unfolding parameters of this double mutant protein, when compared to those of the wild type protein, clearly point out the important role played by the disulphide bridge on the stability and function of this protein family and probably of many other lipocalins.     

Inhibitor and Substrate Binding Induced Stability of HIV-1 Protease against Sequential Dissociation and Unfolding Revealed by High Pressure Spectroscopy and Kinetics

High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of −32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.     

Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance

The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy. The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical. The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two-state transition. The circular dichroism at 222 nm decreases in two phases as urea is added. Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate. Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content. The second transition is the dissociation and unfolding of the partially unfolded dimer. At high concentrations of protein (500 microM) about 73% of the repressor exists as the intermediate in 4 M urea. The apparent dissociation constant is about 10(-4) M; the subunits are probably strongly stabilised by the subunit interaction. The native repressor is stable up to at least 70 degrees C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature approximately 60 degrees C, delta H approximately 230 kJ/mol).     

Structural basis for thermal stability of human low-density lipoprotein

The stability of human low-density lipoprotein (LDL), the major cholesterol carrier in plasma, was analyzed by heating samples of different concentrations at a rate from 11 to 90 K/h. Correlation of the calorimetric, circular dichroism, fluorescence, turbidity, and electron microscopic data shows that thermal disruption of LDL involves irreversible changes in the particle morphology and protein conformation but no global protein unfolding. Heating to 85 degrees C induces LDL conversion into smaller and larger particles and apparent partial dissociation, but not unfolding, of its sole protein, apoB. Further heating leads to partial unfolding of the beta-sheets in apoB and to fusion of the protein-depleted LDL into large aggregated lipid droplets, resulting in a previously unidentified high-temperature calorimetric peak. These lipid droplets resemble in size and morphology the extracellular lipid deposits formed in the arterial wall in early atherosclerosis. The strong concentration dependence of LDL fusion revealed by near-UV/visible CD, turbidity, and calorimetry indicates high reaction order, and the heating rate dependence suggests high activation energy that arises from transient disruption of lipid and/or protein packing interactions in the course of particle fusion and apparent apoB dissociation. Consequently, thermal stability of LDL is modulated by kinetic barriers. Similar barriers may confer structural integrity to LDL subclasses in vivo.     

Reversible unfolding of bovine odorant binding protein induced by guanidinium hydrochloride at neutral pH

An analysis of the unfolding and refolding curves at equilibrium of dimeric bovine odorant binding protein (bOBP) has been performed. Unfolding induced by guanidinium chloride (GdnHCl) is completely reversible as far as structure and ligand binding capacity are concerned. The transition curves, as obtained by fluorescence and ellipticity measurements, are very similar and have the same protein concentration-independent midpoint (C1/2 approximately 2.6 M). This result implies a sequential, rather than a concerted, unfolding mechanism, with the involvement of an intermediate. However, since it has not been detected, this intermediate must be present in small amounts or have the same optical properties of either native or denatured protein. The thermodynamic best fit parameters, obtained according to a simple two-state model, are: deltaG degrees un,w = 5.0 +/- 0.6 kcal mol(-1), m = 1.9 +/- 0.2 kcal mol(-1) M(-1) and C1/2 = 2.6 +/- 0.1 M. The presence of the ligand dihydromyrcenol has a stabilising effect against unfolding by GdnHCl, with an extrapolated deltaG degrees un,w of 22.2 +/- 0.9 kcal mol(-1), a cooperative index of 3.2 +/- 0.3 and a midpoint of 4.6 +/- 0.4 M. The refolding curves, recorded after 24 h from dilution of denaturant are not yet at equilibrium: they show an apparently lower midpoint (C1/2 = 2.2 M), but tend to overlap the unfolding curve after several days. In contrast to chromatographic unfolding data, which fail to reveal the presence of folded intermediates, chromatographic refolding data as a function of time clearly show a rapid formation of folded monomers, followed by a slower step leading to folded dimers. Therefore, according to this result, we believe that the preferential unfolding/refolding mechanism is one in which dimer dissociation occurs before unfolding rather than the reverse.     

Unfolding transitions of fibronectin and its domains. Stabilization and structural alteration of the N-terminal domain by heparin.

Changes in the conformational state of human plasma fibronectin and several of its fragments were studied by fluorescence emission, intrinsic fluorescence polarization and c.d. spectroscopy under conditions of guanidinium chloride-and temperature-induced unfolding. Fragments were chosen to represent all three types of internal structural homology in the protein. Low concentration (less than 2 M) of guanidinium chloride induced a gradual transition in the intact protein that was not characteristic of any of the isolated domains, suggesting the presence of interdomain interactions within the protein. Intermediate concentrations of guanidinium chloride (2-3 M) and moderately elevated temperatures (55-60 degrees C) induced a highly co-operative structural transition in intact fibronectin that was attributable to the central 110 kDa cell-binding domain. High temperatures (greater than 60 degrees C) produced a gradual unfolding in the intact protein attributable to the 29 kDa N-terminal heparin-binding and 40 kDa collagen-binding domains. Binding of heparin to intact fibronectin and to its N-terminal fragment stabilized the proteins against thermal unfolding. This was reflected in increased delta H for the unfolding transitions of the heparin-bound N-terminal fragment, as well as decreased accessibility to solvent perturbants of internal chromophores in this fragment when bound to heparin. These results help to account for the biological efficacy of the interaction between the fibronectin N-terminal domain and heparin, despite its relatively low affinity.     

Characterization of the pressure-induced intermediate and unfolded state of red-shifted green fluorescent protein--a static and kinetic FTIR,UV/VIS and fluorescence spectroscopy study

The green fluorescence proteins (GFP) are widely used as reporters in molecular and cell biology. For their use it in high-pressure microbiology and biotechnology studies, their structural properties, thermodynamic parameters and stability diagrams have to be known. We investigated the pressure stability of the red-shifted green fluorescent protein (rsGFP) using Fourier-transform infrared spectroscopy, fluorescence and UV/Vis spectroscopy. We found that rsGFP does not unfold up to approximately 9kbar at room temperature. Its unique three-dimensional structure is held responsible for the high-pressure stability. At higher temperatures, its secondary structure collapses below 9kbar (e.g. the denaturation pressure at 58 degrees C is 7.8kbar). The analysis of the IR data shows that the pressure-denatured state contains more disordered structures at the expense of a decrease of intramolecular beta-sheets. As indicated by the large volume change of DeltaV degrees (u) approximately -250(+/-50)mlmol(-1) at 58 degrees C, this highly cooperative transition can be interpreted as a collapse of the beta-can structure of rsGFP. For comparison, the temperature-induced unfolding of rsGFP has also been studied. At high temperature (T(m)=78 degrees C), the unfolding resulted in the formation of an aggregated state. Contrary to the pressure-induced unfolding, the temperature-induced unfolding and aggregation of GFP is irreversible. From the FT-IR data, a tentative p,T-stability diagram for the secondary structure collapse of GFP has been obtained. Furthermore, changes in fluorescence and absorptivity were found which are not correlated to the secondary structural changes. The fluorescence and UV/Vis data indicate smaller conformational changes in the chromophore region at much lower pressures ( approximately 4kbar) which are probably accompanied by the penetration of water into the beta-can structure. In order to investigate also the kinetics of this initial step, pressure-jump relaxation experiments were carried out. The partial activation volumes observed indicate that the conformational changes in the chromophore region when passing the transition state are indeed rather small, thus leading to a comparably small volume change of -20 ml mol(-1) only. The use of the chromophore absorption and fluorescence band of rsGFP in using GFP as reporter for gene expression and other microbiological studies under high pressure conditions is thus limited to pressures of about 4kbar, which still exceeds the pressure range relevant for studies in vivo in micro-organisms, including piezophilic bacteria from deep-sea environments.     

Unfolding of a leucine zipper is not a simple two-state transition

Temperature-induced unfolding of the leucine zipper, an alpha-helical, double-stranded, coiled-coil, was studied by circular dichroism spectroscopy, spectrofluorimetry and heat capacity scanning calorimetry. It is shown that this process does not represent a simple two-state transition, as previously believed, but consists of several stages. The first transition starts at the very beginning of heating from 0 degrees C and proceeds with significant heat absorption and decrease of ellipticity. This transition does not depend on the concentration of protein and is sensitive to modification of the N terminus; it is therefore associated with unfolding or fraying of this part of the leucine zipper. The second transition takes place at a considerably higher temperature; it is more pronounced than the first one and does not depend on the concentration of protein, i.e. it is unimolecular. This transition is sensitive to modification of both termini of the leucine zipper and affects the optical properties of a tryptophan residue placed in the central part of the zipper. It therefore involves the whole dimer but does not result in its dissociation, presumably being associated with some repacking of the coiled-coil. This second transition is followed at higher temperatures by the concentration-dependent cooperative unfolding/dissociation of the two strands. The enthalpy and entropy of the temperature-induced structural changes of the leucine zipper that take place before its cooperative unfolding/dissociation comprises almost 40% of the total enthalpy and entropy of unfolding of the completely folded coiled-coil, the state in which it appears to be below 0 degrees C. Comparison of the total enthalpy of leucine zipper unfolding with that of a single-stranded alpha-helix shows that their temperature-dependence correlates with the extent of intramolecular non-polar contacts and allows an assessment of the enthalpy of hydrogen bonding in alpha-helices, which appears to be about 3.3kJmol(-1) at 20 degrees C.     

Structural stability and unfolding properties of thermostable bacterial alpha-amylases: a comparative study of homologous enzymes

In a comparative investigation on two thermostable alpha-amylases [Bacillus amyloliquefaciens (BAA), T(m) = 86 degrees C and Bacillus licheniformis (BLA), T(m) = 101 degrees C], we studied thermal and guanidine hydrochloride (GndHCl)-induced unfolding using fluorescence and CD spectroscopy, as well as dynamic light scattering. Depletion of calcium from specific ion-binding sites in the protein structures reduces the melting temperature tremendously for both alpha-amylases. The reduction is nearly the same for both enzymes, namely, in the order of 50 degrees C. Thus, the difference in thermostability between BLA and BAA (DeltaT(m) approximately 15 degrees C) is related to intrinsic properties of the respective protein structures themselves and is not related to the strength of ion binding. The thermal unfolding of both proteins is characterized by a full disappearance of secondary structure elements and by a concurrent expansion of the 3D structure. GndHCl-induced unfolding also yields a fully vanishing secondary structure but with more expanded 3D structures. Both alpha-amylases remain much more compact upon thermal unfolding as compared to the fully unfolded state induced by chemical denaturants. Such rather compact thermal unfolded structures lower the conformational entropy change during the unfolding transition, which principally can contribute to an increased thermal stability. Structural flexibilities of both enzymes, as measured with tryptophan fluorescence quenching, are almost identical for both enzymes in the native states, as well as in the unfolded states. Furthermore, we do not observe any difference in the temperature dependence of the structural flexibilities between BLA and BAA. These results indicate that conformational dynamics on the time scale of our studies seem not to be related to thermal stability or to thermal adaptation.     

Thermodynamics of binding interactions between bovine beta-lactoglobulin A and the antihypertensive peptide beta-Lg f142-148

The binding capacity of bovine beta-lactoglobulin variant A (beta-Lg A) for six peptides derived from beta-Lg was evaluated using an ultrafiltration method under the following conditions: pH 6.8, 40 degrees C, and a beta-Lg A/peptide molar ratio of 1:5. Only peptides beta-Lg f102-105, f142-148, and f69-83 bound in significant amounts to beta-Lg A corresponding to 1.5, 1.1, and 0.7 mol of peptide per mole of beta-Lg A, respectively. The interaction between beta-Lg A and the antihypertensive peptide beta-Lg f142-148 was investigated further by isothermal titration calorimetry. The binding isotherms at pH 6.8 and 25 degrees C confirmed that beta-Lg f142-148 bound to beta-Lg A and that the interaction followed a sequential three-site binding model with constants of association of 2 x 10(3), 1 x 10(3), and 0.4 x 10(3) M(-1) for the first, second, and third binding sites, respectively. The enthalpy of binding was exothermic for the first and second binding sites and endothermic for the third binding site. Binding of the peptide to all three sites was spontaneous as shown by the negative free energy values. These results show for the first time that beta-Lg A can bind bioactive peptides. This potential could be exploited to transport bioactive peptides and protect them in the gastrointestinal tract following their oral administration as nutraceuticals.     

Pressure-induced unfolding of lysozyme in aqueous guanidinium chloride solution.

The pressure-induced unfolding of lysozyme was investigated in an aqueous guanidinium chloride solution by means of ultraviolet spectroscopy. Assuming a two-state transition model, volume changes were calculated from the slope of free energy vs. pressure plots over a temperature range of 10 to 60 degrees C. Between 25 and 60 degrees C, almost constant volume changes were observed in the transition region, which was reflected in almost identical slopes of the free energy change vs. pressure plots. On the other hand, the different slopes were observed in the pressure dependence of free energy change at temperatures lower than 25 degrees C. These data were interpreted as suggesting that a two-state model is not appropriate at low temperature, but instead one or more intermediates are present under these conditions. The volume changes for unfolding became less negative at temperatures higher than 25 degrees C.     

A comparison of the urea-induced unfolding of apoflavodoxin and flavodoxin from Desulfovibrio vulgaris.

The kinetics and thermodynamics of the urea-induced unfolding of flavodoxin and apoflavodoxin from Desulfovibrio vulgaris were investigated by measuring changes in flavin and protein fluorescence. The reaction of urea with flavodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s(-1) in 3 m urea in 25 mm sodium phosphate at 25 degrees C), and it results in the dissociation of FMN. The rate of unfolding of apoflavodoxin depends on the urea concentration, while the reaction with the holoprotein is independent of urea. The rates decrease in high salt with the greater effect occurring with apoprotein. The fluorescence changes fit two-state models for unfolding, but they do not exclude the possibility of intermediates. Calculation suggests that 21% and 30% of the amino-acid side chains become exposed to solvent during unfolding of flavodoxin and apoflavodoxin, respectively. The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength. This effect is larger with phosphate than with chloride, and with apoflavodoxin than with flavodoxin. In low salt the conformational stability of the holoprotein is greater than that of apoflavodoxin, but in high salt the relative stabilities are reversed. It is calculated that two ions are released during unfolding of the apoprotein. It is concluded that the urea-dependent unfolding of flavodoxin from D. vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that flavodoxin dissociates. Small changes in flavin fluorescence occur at low concentrations of urea and these may reflect binding of urea to the holoprotein.     

Conformational studies of Actinobacillus actinomycetemcomitans leukotoxin: partial denaturation enhances toxicity

A 114 kDa, water-soluble, cytotoxin secreted by the Gram-negative bacterium Actinobacillus actinomycetemcomitans (Aa) is similar in sequence to Escherichia coli alpha-hemolysin, but is non-hemolytic, killing leukocytes of select species, including humans. In this work, we investigated aspects of the water-soluble conformation of Aa toxin which relate to its biological, pore-forming activity. The toxin has five native tryptophans and fluorescence spectra were monitored in aqueous solutions in the presence of varying denaturants. Significant changes in the fluorescence spectra, without significant wavelength shifts, were induced by small additions of denaturants and changes in the temperature or pH. The fluorescence changes suggested that small perturbations in the aqueous environment resulted in structural changes in the toxin related not to a large unfolding but to more subtle conformational changes. Analytical ultracentrifugation showed the toxin to be a globular monomer in dilute aqueous solution. Circular dichroism spectroscopy showed about 25% alpha-helical structure which is largely maintained up to a temperature (65 degrees C) known to deactivate toxin activity. Changes in the cytotoxic properties of the toxin were monitored with flow cytometric analysis following preincubation of the toxin under mild conditions similar to those used in the fluorescence studies. These experiments showed that the pretreated toxin exhibited enhanced cell-killing potency on toxin-sensitive cells. The correlation of cytotoxicity with the changes in Trp fluorescence is consistent with the idea that partial unfolding of Aa toxin is an early, obligate step in toxin-induced cell kill.     

Thermodynamic effects of active-site ligands on the reversible, partial unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric studies.

Dodecameric glutamine synthetase (GS) from Escherichia coli undergoes reversible, thermally induced partial unfolding without subunit dissociation. A single endotherm for Mn.GS (+/- active-site ligands) in the presence of 1 mM free Mn2+ and 100 mM KCl at pH 7 is observed by differential scanning calorimetry (DSC). Previous deconvolutions of DSC data for Mn.GS showed only two two-state transitions (with similar tm values; 51.6 +/- 2 degrees C), and indicated that cooperative interactions link partial unfolding reactions of all subunits within the Mn.enzyme dodecamer [Ginsburg, A., & Zolkiewski, M. (1991) Biochemistry 30, 9421]. A net uptake of 8.0 equiv of H+ by Mn.GS occurs during partial unfolding, as determined in the present DSC experiments conducted with four buffers having different heats of protonation at 50 degrees C. These data gave a value of 176 +/- 12 kcal (mol of dodecamer)-1 for delta Hcal corrected for buffer protonation. L-Glutamine and L-Met-(SR)-sulfoximine stabilize the Mn.GS dodecamer through the free energies of ligand binding, and these were shown to be partially and totally released, respectively, from the 12 active sites at high temperature. Ligand effects on Tm values from DSC were similar to those from spectral measurements of Trp and Tyr exposures in two subunit domains. Effects of varying [ADP] on DSC profiles of Mn.GS were complex; Tm is increased by low [ADP] and decreased by > 100 microM free ADP. This is due to the exposure of an additional low-affinity ADP binding site per GS subunit at high temperature with log K1' = 4.3 and log K2' = 3.6 at 60 degrees C relative to log K' = 5.5 for ADP binding at 30 degrees C, as determined by isothermal calorimetric and fluorescence titrations. Moreover, delta Hcal at > 27% saturation with ADP (corrected for ADP binding/dissociation) is approximately 80-100 kcal/mol more than in the absence of ligands. Changes in domain interactions could result from ADP bridging subunit contacts in the dodecamer. Each of the active-site ligands investigated here produces different effects on DSC profiles without uncoupling the extremely cooperative, partial unfolding reactions in the Mn.GS dodecamer.     

The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure.

The heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75 degrees C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety. In contrast, the mature form, which lacks the propeptide composed of 91 amino acid residues, showed more complex behaviour: differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition. These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarboxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding. The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding.