Dissociation/association properties of a dodecameric cyclomaltodextrinase. Effects of pH and salt concentration on the oligomeric state

As an effort to elucidate the quaternary structure of cyclomaltodextrinase I-5 (CDase I-5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I-5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain-swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I-5 was present as a dimer in the pH range of 5.0-6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I-5 dissociated into dimers at a rate of 8.58 x 10(-2) h(-1) at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild-type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH-dependent process, the dissociation of wild-type CDase I-5 proceeded very fast at pH 7.0 in the presence of 0.2-1.0 M of KCl. Stopped-flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s(-1) and 7.99 s(-1) in the presence of 0.2 M and 1.0 M of KCl, respectively.     

Reassessment of protein stability, DNA binding, and protection of Mycobacterium smegmatis Dps

The structure and function of Mycobacterium smegmatis Dps (DNA-binding proteins from starved cells) and of the protein studied by Gupta and Chatterji, in which the C terminus that is used for binding DNA contains a histidine tag, have been characterized in parallel. The native dodecamer dissociated reversibly into dimers above pH 7.5 and below pH 6.0, with apparent pK(a) values of approximately 7.65 and 4.75; at pH approximately 4.0, dimers formed monomers. Based on structural analysis, the two dissociation steps have been attributed to breakage of the salt bridges between Glu(157) and Arg(99) located at the 3-fold symmetry axes and to protonation of Asp(66) hydrogen-bonded to Lys(36) across the dimer interface, respectively. The C-terminal tag did not affect subunit dissociation, but altered DNA binding dramatically. At neutral pH, protonation of the histidine tag promoted DNA condensation, whereas in the native C terminus, compensation of negative and positive charges led to DNA binding without condensation. This different mode of interaction with DNA has important functional consequences as indicated by the failure of the native protein to protect DNA from DNase-mediated cleavage and by the efficiency of the tagged protein in doing so as a result of DNA sequestration in the condensates. Chemical protection of DNA from oxidative damage is realized by Dps proteins in a multistep iron oxidation/uptake/mineralization process. Dimers have a decreased protection efficiency due to disruption of the dodecamer internal cavity, where iron is deposited and mineralized after oxidation at the ferroxidase center.     

Dissociation and reassociation of prolyl 4-hydroxylase subunits after cross-linking of monomers

1. Incubation of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate : oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with H2O2 leads to a decrease of 50% in the specific activity of enzyme tetramers, followed by dissociation into inactive dimers in which the monomers are covalently cross-linked by S-S bridge formation. 2. Incubation of the enzyme with K3Fe(CN)6 leads to a comparable decrease in activity of enzyme tetramers. Addition of urea leads to dissociation into inactive dimers with similarly cross-linked monomers. 3. Removal of the dissociating agent leads to reassociation of cross-linked dimers to tetramers and to about 50% reactivation. The enzyme is further reactivated by preincubation with dithiothreitol. 4. Dissociation of the enzyme with dithiothreitol, urea or LiCl, or at low pH (4.15) produces inactive monomers, which could not be reassociated.     

Subunit interactions in horse spleen apoferritin. Dissociation by extremes of pH

1. The dissociation of horse spleen apoferritin as a function of pH was analysed by sedimentation-velocity techniques. The oligomer is stable in the range pH2.8-10.6. Between pH2.8 and 1.6 and 10.6 and 13.0 both oligomer and subunits can be detected. At pH values between 1.6 and 1.0 the subunit is the only species observed, although below pH1.0 aggregation of the subunits to a particle sedimenting much faster than the oligomer occurs. 2. When apoferritin is first dissociated into subunits at low pH values and then dialysed into buffers of pH1.5-5.0, the subunit reassociates to oligomer in the pH range 3.1-4.3. 3. U.v.-difference spectroscopy was used to study conformational changes occurring during the dissociation process. The difference spectrum in acid can be accounted for by the transfer of four to five tyrosine residues/subunit from the interior of the protein into the solvent. This process is reversed on reassociation, but shows the same hysteresis as found by sedimentation techniques. The difference spectrum in alkali is more complex, but is consistent with the deprotonation of tyrosine residues, which appear to have rather high pK values. 4. In addition to the involvement of tyrosine residues in the conformational change at low pH values, spectral evidence is presented that one tryptophan residue/subunit also changes its environment before dissociation and subsequent to reassociation. 5. Analysis of the dissociation and reassociation of apoferritin at low pH values suggests that this is a co-operative process involving protonation and deprotonation of at least two carboxyl functions of rather low intrinsic pK. The dissociation at alkaline pH values does not appear to be co-operative. 6. Of the five tyrosine residues/subunit only one can be nitrated with tetranitromethane. Guanidination of lysine residues results in the modification of seven out of a total of nine residues/subunit. Nine out of the ten arginine residues/subunit react with cyclohexanedione.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

X-ray structure of recombinant horse L-chain apoferritin at 2.0 Å resolution: implications for stability and function

The X-ray structure of recombinant horse L-chain (rL) apoferritin, solved at 2.0?Å resolution with a final R factor of 17.9%, gives evidence that the residue at position 93 in the sequence is a proline and not a leucine, as found in earlier sequencing studies. The structure is isomorphous with other apoferritin structures, and we thus draw particular attention to those structural features which can be related to the stability and function of the protein. Analysis of hydrogen bonding and salt bridge interactions shows that dimers and tetramers are the most stable molecular entities within the protein shell: a result confirming earlier biophysical experiments. The stability of horse rL apoferritin to both dissociation into subunits at acidic pH values and to complete unfolding in guanidine chloride solutions is compared with that of other apoferritins. This emphasizes the role played by the salt bridge in the stability of this protein family. The horse rL apoferritin is significantly more resistant to denaturation than horse spleen ferritin, which in turn is more resistant than any human rH apoferritins, even those for which a salt bridge is restored. Finally, this structure determination not only establishes that a preformed pocket exists in L-chain apoferritin, at a site known to be able to bind porphyrin, but also underlines the particular function of a cluster of glutamic acids (E53, E56, E57 and E60) located at the entrance of this porphyrin-binding pocket.     

The dodecameric ferritin from Listeria innocua contains a novel intersubunit iron-binding site

Ferritin is characterized by a highly conserved architecture that comprises 24 subunits assembled into a spherical cage with 432 symmetry. The only known exception is the dodecameric ferritin from Listeria innocua. The structure of Listeria ferritin has been determined to a resolution of 2.35 A by molecular replacement, using as a search model the structure of Dps from Escherichia coli. The Listeria 12-mer is endowed with 23 symmetry and displays the functionally relevant structural features of the ferritin 24-mer, namely the negatively charged channels along the three-fold symmetry axes that serve for iron entry into the cavity and a negatively charged internal cavity for iron deposition. The electron density map shows 12 iron ions on the inner surface of the hollow core, at the interface between monomers related by two-fold axes. Analysis of the nature and stereochemistry of the iron-binding ligands reveals strong similarities with known ferroxidase sites. The L. innocua ferritin site, however, is the first described so far that has ligands belonging to two different subunits and is not contained within a four-helix bundle.     

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.     

Molecular heterogeneity of the hemocyanin isolated from the king crab Paralithodes camtschaticae.

Native Paralithodes camtschaticae hemocyanin is found as a mixture of dodecamers (24S; 80%) and hexamers (16S; 20%). Removal of Ca2+ ions by dialysis against EDTA-containing buffer solution at neutral pH induces complete dissociation of the 24S form into the 16S form. Under these conditions, a further increase in pH to 9.2 produces complete dissociation of the hexamers into monomers (5S). In both cases, the dissociation process is reversible. The dodecamer (24S) is composed of two different hexamers which can be discriminated only by ion-exchange chromatography in the presence of Ca2+ ions. At alkaline pH and in the presence of EDTA, two major monomeric fractions can be separated by ion-exchange chromatography: ParcI (60%) and ParcII (40%). The reassociation properties of the two fractions were studied separately to define their ability to form hexamers and dodecamers. The oxygen-binding properties of the different aggregation states were investigated. Native hemocyanin binds O2 co-operatively (nH = 3) and with low affinity (p50 approximately 103 Torr). The two monomeric fractions, ParcI and ParcII, are not co-operative and the affinity is twice that of the native protein (p50 approximately 65 and 52 Torr). Oxygen-binding measurements of native hemocyanin carried out at different pH values indicate a strong positive Bohr effect within the pH range 6.5-8.0 and an increase in oxygen affinity at pH below 6.5.     

Effect of pH on the structure and aggregation of human glycodelin A. A comparison with beta-lactoglobulin A

The effect of pH on the structure of glycodelin A (GdA) and of beta-lactoglobulin A (beta-LgA) has been investigated by means of circular dichroism, steady state fluorescence, synchrotron radiation small angle X-ray scattering (SR-SAXS) and gel permeation chromatography. The comparison between GdA and beta-LgA shows that, at pH 7.0, both proteins are dimers with an extended content of beta-sheet conformation, but pH 2.0 and 9.0 yield a different secondary, tertiary and quaternary structural organisation. Whilst beta-LgA is a monomer, that conserves beta-sheet conformation at pH 2.0 and 9.0, GdA has a stable dimeric structure at alkaline pH, but at pH 2.0 increases its alpha-helix content and it aggregates soon. SR beam has been used to perform SAXS comparative measurements of the two proteins. SR-SAXS data provide the radius of gyration and the radii of the cross-section and of the thickness. GdA aggregation at acid pH has been characterised by calculating the distance distribution function (P(r)). Isoelectric focusing and chromatofocusing data show a different charge distribution on the surfaces of the two proteins, supporting the hypothesis that the presence of oligosaccharides deeply influences the conformational state and the aggregation process of GdA at different pH values. In particular, the presence of sialic acid residues, within the oligosaccharide moiety of the GdA, might be responsible for the differences observed between the two proteins.     

Yeast pyruvate decarboxylase tetramers can dissociate into dimers along two interfaces. Hybrids of low-activity D28A (or D28N) and E477Q variants, with substitution of adjacent active center acidic groups from different subunits, display restored activity

The tetrameric enzyme yeast pyruvate decarboxylase (YPDC) has been known to dissociate into dimers at elevated pH values. However, the interface along which the dissociation occurs, as well as the fundamental kinetic properties of the resulting dimers, remains unknown. The active sites of YPDC are comprised of amino acid residues from two subunits, a property which we utilize to address the issue as to which dimer interface is cleaved under different conditions of dissociation. Hydroxide-induced dissociation of the active site D28A (or D28N) and E477Q variants, each at least 100 times less reactive than wild-type YPDC, followed by reassociation of D28A (or D28N) and E477Q variants led to a remarkable 35-50-fold increase in activity. This result is possible only if the hydroxide-induced dissociation results in a cleavage along the interface between two subunits so that residues D28 and E477 are now separated. Upon reassociation, one of the two active sites of the hybrid dimer will have both residues substituted, whereas the second one will be of the wild-type phenotype. In contrast to the hydroxide-induced dimers, the urea-induced dissociation recently proposed results in dissociation along dimer-dimer interfaces, without separating the active sites, and therefore, on reassociation, these dimers do not regain activity. The significance of the results is discussed in light of a recently proposed alternating sites mechanism for YPDC. A preparative ion-exchange method is reported for the separation and purification of hybrid enzymes.     

Detergent-solubilized bovine cytochrome c oxidase: dimerization depends on the amphiphilic environment

The extent to which bovine cytochrome c oxidase (COX) dimerizes in nondenaturing detergent environments was assessed by sedimentation velocity and equilibrium. In contrast to generally accepted opinion, the COX dimer is difficult to maintain and is the major oligomeric form only when COX is solubilized with a low concentration of dodecylmaltoside, i.e., approximately 1 mg/mg protein. The dimer form is intrinsically unstable and dissociates into monomers with increased detergent concentration, i.e., >5 mg/mg protein. The structure of the solubilizing detergent, however, greatly alters detergent effectiveness by inducing either monomerization or aggregation. Triton X-100 is most effective at solubilizing COX, but it destabilizes COX dimers, even at low concentration. Undecylmaltoside, decylmaltoside, and octaethyleneglycolmonododecyl ether (C(12)E(8)) are less effective at solubilizing COX. Each prevents COX aggregation at high detergent concentration, but also destabilizes the COX dimer. Other detergents, e.g., Tween 20, sodium cholate, sodium deoxycholate, CHAPS, or CHAPSO, are completely ineffective COX solubilizers and do not prevent aggregation even at 10-40 mg/mL. The transition from dimers to monomers depends on many factors other than detergent structure and concentration, e.g., protein concentration, phospholipid content and pH. We conclude that the intrinsic dimeric structure of COX can be maintained only after solubilization with low concentrations of dodecylmaltoside at near neutral pH, and even then precautions must be taken to prevent its dissociation into monomers.     

Chicken liver bile acid-binding protein is in a compact partly folded state at acidic pH. Its relevance to the interaction with lipid membranes

Chicken liver bile acid-binding protein (formerly known as chicken liver basic fatty acid-binding protein) binds to anionic lipid membranes acquiring a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B., and Montich, G. (2003) Biochim. Biophys. Acta 1611, 98-106]. To understand the mechanisms of its interactions with membranes, we have investigated the presence of partly folded states in solution. Using fluorescence spectroscopy of the single Trp residue, circular dichroism in the far- and near-UV, Fourier transform infrared spectroscopy, and size-exclusion chromatography, we found that L-BABP was partly unfolded at pH 2.5 and low ionic strength, retaining some of its secondary structure. Addition of 0.1 M NaCl at pH 2.5 or decreasing the pH to 1.5 produced a more compact partly folded state, with a partial increase of secondary structure and none of tertiary structure. Fluorescence emission spectra of this state indicate that the Trp residue is within an environment of low polarity, similar to the native state. This environment is not produced by the insertion of the Trp into soluble aggregates as revealed by size-exclusion chromatography, fluorescence anisotropy, and infrared spectroscopy. The presence of partly folded states under acidic conditions in solution suggests the possibility that membrane binding of L-BABP occurs via this state.     

The mutations Lys 114 --> Gln and Asp 126 --> Asn disrupt an intersubunit salt bridge and convert Listeria innocua Dps into its natural mutant Listeria monocytogenes Dps. Effects on protein stability at Low pH

The stability of the dodecameric Listeria monocytogenes Dps has been compared with that of the Listeria innocua protein. The two proteins differ only in two amino acid residues that form an intersubunit salt-bridge in L. innocua Dps. This salt-bridge is replaced by a hydrogen bonding network in L. monocytogenes Dps as revealed by the X-ray crystal structure. The resistance to low pH and high temperature was assayed for both Dps proteins under equilibrium conditions and kinetically. Despite the identical equilibrium behavior, significant differences in the kinetic stability and activation energy of the unfolding process are apparent at pH 1.5. The higher stability of L. monocytogenes Dps has been accounted for in terms of the persistence of the hydrogen bonding network at this low pH value. In contrast, the salt-bridge between Lys 114 and Asp 126 characteristic of L. innocua Dps is most likely abolished due to protonation of Asp 126.     

Urea-induced dissociation and unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric and spectral studies.

Urea-induced dissociation and unfolding of manganese.glutamine synthetase (Mn.GS) have been studied at 37 degrees C (pH 7) by spectroscopic and calorimetric methods. In 0 to approximately 2 M urea, Mn.GS retains its dodecameric structure and full catalytic activity. Mn.GS is dissociated into subunits in 6 M urea, as evidenced by a 12-fold decrease in 90 degrees light scattering and a monomer molecular weight of 51,800 in sedimentation equilibrium studies. The light scattering decrease in 4 M urea parallels the time course of Trp exposure but occurs more rapidly than changes in secondary structure and Tyr exposure. Early and late kinetic steps appear to involve predominantly disruption of intra-ring and inter-ring subunit contacts, respectively, in the layered hexagonal structure of Mn.GS. The enthalpies for transferring Mn.GS into urea solutions have been measured by titration calorimetry. After correcting for the enthalpy of binding urea to the protein, the enthalpy of dissociation and unfolding of Mn.GS is 14 +/- 4 cal/g. A net proton uptake of approximately 50 H+/dodecamer accompanies unfolding reactions. The calorimetric data are consistent with urea binding to multiple, independent sites in Mn.GS and the number of binding sites increasing approximately 9-fold during the protein unfolding.     

Evidence of hemoglobin dissociation

Bovine carbonmonoxy hemoglobin investigated with light scattering studies is found to dissociate from its native tetramer structure into dimers and monomers. The values of the hydrated tetramer radius, R_T = 32.1 Å, and the fractional dissociation vs pH, have been obtained at different ionic strengths from the autocorrelation function of the scattered light. The results suggest that a relevant contribution to Hb dissociation is due to electrostatic effects and, by means of a model derived by Tanford, it has been possible to predict the behavior of dissociation. Among the findings of this approach, we recall the estimates of the electrostatic energy contributions to Hb dissociation, up to ? GRT, and the predicted charge of tetrameric Hb vs pH, which agrees very well with the experimental data. © 1994 John Wiley & Sons, Inc.     

The H(+)-induced dissociation of human plasma alpha 2-macroglobulin. An investigation using small-angle neutron scattering and test of trypsin binding activity.

The dissociation of the tetrameric alpha 2-macroglobulin molecule into two half-molecular fragments, which occurs at pH less than 4.5, has been investigated using the small-angle neutron scattering method, and test of trypsin binding activity. Best fit with the relative forward scattering of neutrons is obtained for a model where the dissociation of the protein is driven by the uptake of H+ on altogether four acid-base groups, one per monomeric subunit of alpha 2-macroglobulin. These groups are not (or only slightly) accessible in the native tetramer, but become exposed to the solvent after dissociation of the protein. The H(+)-binding constant obtained for these groups, after dissociation of the protein, log K1 in the range 4.2-4.5, suggests that they are most probably carboxylate groups. From the about 10% increase in the radius of gyration, which occurs when lowering the pH from 4.5 to 2.0, we can conclude that the dissociation is associated with a change in structure of the protein. Tests of trypsin binding show that there is also an irreversible loss in trypsin binding activity, which is directly related to the fraction of dissociated protein. Thus, at pH less than 4.5, there is a transition of alpha 2-macroglobulin which results simultaneously in dissociation, disorganisation of the conformation of the subunits and loss in activity.     

Structural study of Carcinus maenas hemocyanin by native ESI‐MS: Interaction with L‐lactate and divalent cations

The interaction of L ‐lactate and divalent cations with Carcinus maenas hemocyanin has been probed by electrospray ionization mass spectrometry under conditions preserving noncovalent interactions (native ESI‐MS). C. maenas native hemocyanin in the hemolymph occurs mainly as dodecamers and to a lesser extent as hexamers. A progressive acidification with formic acid after alkaline dissociation resulted in the preferential recruitment of the two lightest subunits into light dodecamers, a molecular complex absent from native hemolymph, in addition to regular dodecamers and hexamers. Addition of L ‐lactic acid also induced the recruitment of these subunits, even at alkaline pH. A dodecamer‐specific subunit is needed to enable aggregation over the hexameric state. Experiments with EDTA suggested the existence of different binding sites and association constants for divalent cations within hexameric structures and at the interface between two hexamers. L ‐lactic acid specific interaction with the lightest subunits was not inhibited by removal of the divalent cations. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Effects of pH on structural and functional properties of porin from the outer membrane of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>. I. Functionally important conformational transitions of yersinin

The changes in the structural and functional properties of yersinin, a porin from the outer membrane of Yersinia pseudotuberculosis, were studied in the pH range 8.0–2.0 using SDs-PAGE, scanning microcalorimetry, optical spectroscopy and bilayer lipid membrane technique. It was found that in the pH range under study the changes in the spatial structure of yersinin were biphasic. In the first steps of pH titration (pH 8.0–4.5), porin underwent a series of conformational transitions, which did not affect the trimeric structure of its molecule. In the second step (pH 4.0–2.0), structural rearrangements led to dissociation of the protein trimers into monomers. It is noteworthy that complete unfolding of the polypeptide chain of the protein was not observed even at low values of pH. Thus, at pH 2.0 the conformational intermediate of the protein retained up to 50% of its regular secondary structure. Studies of current fluctuations in the bilayer lipid membrane revealed that in weakly acidic media the conductivity of yersinin pores was decreased by one order of magnitude. The most drastic changes in the conductivity of the model membrane were observed at pH 5.8, whereas a further decrease of pH to 5.0 resulted in the closure of porin channels. It was concluded that the observed changes in the pore-forming properties of yersinin in a narrow range of pH represent an early step in the adaptation of bacteria to the changing conditions of the environment and entail control over the biosynthesis of nonspecific porins. The pH-dependent changes in the structure and pore-forming properties of yersinin provide additional evidence in favor of conformational and functional plasticity of porins.     

Apolipoprotein A-I(Milano) unfolds via an intermediate state as studied by differential scanning calorimetry and circular dichroism.

In this study the thermal and denaturant induced denaturation behaviors of apolipoprotein A-I(Milano) (apo A-IM) have been studied by differential scanning calorimetry and circular dichroism spectroscopy, as well as solution properties by analytical ultracentrifugation. Thermal denaturation is dependent on pH, sodium phosphate concentration and NaCl concentration. The protein is highly self-associated at the protein concentrations used in this study. Denaturation of apo A-IM at pH 7.4 and 8.0 occurs in two steps. The midpoint between the transition is at 37 degrees C. The first step at 31 degrees C involves melting of tertiary structure and rearrangement of protein association complexes, i.e. a transition into an intermediate molten globular-like state. Subsequent melting of this intermediate state into an unfolded state occurs at 52 degrees C. At pH 2.8 the protein lacks all tertiary structure and denaturation occurs over a large temperature interval, indicating the induction of a molten globular-like state at low pH.