Early stage aggregation of human serum albumin in the presence of metal ions

The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of Cu(II) and Zn(II) was investigated by using optical absorption, fluorescence, AFM and EPR spectroscopy. Turbidity experiments as a function of temperature indicate that the protein aggregation occurs after the melting of the protein. The kinetic of HSA aggregation, investigated between 60 and 70 °C by monitoring the optical density changes at 400 nm on a 180 min time window, shows an exponential growth with a rate that increases with the temperature. Fluorescence of the thioflavin T evidences a significant increase of the intensity at 480 nm at increasing incubation time. These results combined with AFM experiments show that the protein aggregates are elongated oligomers with fibrillar-like features. The absence of a lag-phase suggests that the early stage aggregation of HSA follows a downhill pathway that does not require the formation of an organized nucleus. The presence of Cu(II) and Zn(II) ions does not affect the thermally induced aggregation process and the morphology of HSA aggregates. The result is compatible with the binding of the metal ions to the protein in the native state and with the high conformational stability of HSA.     

Thermal induced conformational changes involved in the aggregation pathways of beta-lactoglobulin

Aggregation of proteins appears to be associated most often with conformational and structural changes that lead to exposure of some apolar residues. Depending on the native structure of the protein in exam, aggregation is a process that involves different mechanisms, whose time of occurrence and interplay can depend upon temperature. To single out information about the multistages of the aggregation pathway, here we investigate the thermally induced conformational and structural changes of the beta-lactoglobulin (BLG). The experimental approach consists in studying steady-state fluorescence spectra of intrinsic chromophores, two tryptophans, and Anylino-Naphthalene-Sulfonate dye (ANS) molecular probe. This technique revealed to be particularly suitable in investigating samples in the low concentration range and at the initial steps of the aggregation process. The Rayleigh scattering of the exciting light follows the growth of the intermolecular interactions at the same time. Complementary information is also obtained by circular dichroism (CD) measurements on samples in the same experimental conditions. The obtained data indicate a well-defined interconversion between quaternary, ternary and secondary structures, together with conformational rearrangements driven by hydrophobic interactions and intermolecular bonds. The results are also discussed in comparison with similar studies on BSA aggregation.     

Complexes of DNA with trivalent metal ions in presence of manganese ions

The interaction of DNA with Fe3+, Al3+, Co(NH3)6(3+) in a solution containing MnCl2 was studied. It was shown that there exists a competition for the binding sites between Mn2+ and Al3+, while the binding of Mn2+ to DNA does not depend on the presence of Fe3+ and Co(NH3)6(3+) in solution. We proposed that Fe3+ and Co(NH3)6(3+) ions prefer to bind to phosphates, and Al3+ ions are capable to bind to the nitrogen bases of DNA.     

Thermal stability of beta-lactoglobulin in the presence of aqueous solution of alcohols and polyols

A systematic study concerning the effect of aqueous solution of alcohols and polyols with four carbon atoms on β-lactoglobulin stability is presented. The protein was chosen due to its functional properties and applications in food and pharmaceutical industries and because its structure and properties in aqueous solution have been widely described. The alcohols having a four carbon chain were selected to examine the effect of the gradual increase in the number of OH groups on protein stability.

Protein thermal stability in water, buffers and dilute aqueous solutions of 1-butanol, 1,2-butanediol, 1,2,4-butanetriol and 1,2,3,4-butanetetrol was evaluated by fluorescence spectroscopy. The results were used to determine the temperature range in which the unfolding process is reversible and the protein denaturation temperature in acetate buffer pH 5.5 and in the aqueous mixed solvents. Thermodynamic results show that alcohol denaturating effect diminishes gradually as the number of OH groups increase.     

Formation of gels in the presence of metal ions

A small library of stereoisomeric pseudopeptides able to make gels in different solvents has been prepared and their attitude to make gels in the presence of several metal ions was evaluated. Four benzyl esters and four carboxylic acids, all containing a moiety of azelaic acid (a long chain dicarboxylic acid) coupled with four different pseudopeptide moieties sharing the same skeleton (a phenyl group one atom apart from the oxazolidin-2-one carboxylic group), were synthesized in solution, by standard coupling reaction. The tendency of these pseudopeptides to form gels was evaluated using the inversion test of 10 mM solutions of pure compounds and of stoichiometric mixtures of pseudopeptides and metal ions. To obtain additional information on the molecular association, the gel samples were left dry in the air to form xerogels that were further analyzed using SEM and XRD. The formation of gel containing Zn(II) or Cu(II) ions gave good results in term of incorporation of the metal ions, while the presence of Cu(I), Al(III) and Mg(II) gave less satisfactory results. This outcome is a first insight in the formation of stable LMWGs formed by stoichiometric mixtures of pseudopeptides and metal ions. Further studies will be carried out to develop similar compounds of pharmacological interest.     

Template-directed synthesis of oligoguanylates in the presence of metal ions

We have studied the condensation reaction of ImpG² on a poly(C) template, in the presence of various metal ions. With Mg²⁺ as co-catalyst we confirmed that Pb²⁺ and Zn²⁺ are effective catalysts. A catalytic effect was also observed for Bi³⁺, Sb³⁺ and Mn²⁺. Bi³⁺ and Sn²⁺, like Pb²⁺, favored the formation of 2′-5′ linkages. With Mn²⁺ a rather complex mixture of oligomers is formed, some of which contain pyrophosphate linkages. None of the metal ions investigated behaved like Zn²⁺ in favoring the formation of the naturally occurring 3′-5′ linkages.     

Bipyridine-modified oligonucleotides: Aggregation in the presence of metal ions

To the 5′-end of the palindromic oligonucleotide sequence d(CGCGAATTCGCG) was appended an artificial 2,2′-bipyridine-based nucleoside, resulting in the formation of regular DNA double helices that contain bidentate ligands as single-nucleotide overhangs. Due to their limited size, these duplexes are too small to be resolved by atomic force microscopy (AFM). Therefore, only a homogeneous surface can be detected after their deposition on mica. In the presence of the octahedrally coordinating transition metal ion iron(II), an entirely different surface topology is observed, however. On mica support, two types of aggregates are formed, namely a monolayer of interconnected DNA double helices and a three-dimensional disc-like structure that with time rearranges into fibers with lengths of several micrometers. On highly ordered pyrolytic graphite (HOPG), two-dimensional structures resembling a labyrinth are observed in the presence of iron(II). These observations can be explained by the formation of artificial three-way junctions between DNA double helices, mediated by octahedral iron bipyridine complexes. Hence, the incorporation of artificial ligand-containing nucleosides into oligonucleotides opens up the way to DNA-based nanostructures that assemble only in the presence of suitable metal ions.     

Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.     

Role of calcium as trigger in thermal beta-lactoglobulin aggregation

Divalent calcium ions have been suggested to be involved in intermolecular protein-Ca2+-protein cross-linking, intramolecular electrostatic shielding, or ion-induced protein conformational changes as a trigger for protein aggregation at elevated temperatures. To address the first two phenomena in the case of beta-lactoglobulin, a combination of chemical protein modification, calcium-binding, and aggregation studies was used, while the structural integrity of the modified proteins was maintained. Although increasing the number of carboxylates on the protein by succinylation results in improved calcium-binding, calcium appears to be less effective in inducing protein aggregation. In fact, the larger the number of carboxylates, the higher the concentration of calcium that is required to trigger the aggregation. Lowering the number of negative charges on the protein surface via methylation of carboxylates reduces calcium-binding properties, but calcium-induced aggregation at low concentration is improved. Monovalent sodium ions cannot take over the specific role of calcium. The relation between net surface charge and number of calcium ions bound required to trigger the aggregation suggests that calcium needs to bind site specific to carboxylates with a threshold affinity. Subsequent site-specific screening of surface charges results in protein aggregation, driven by the partial unfolding of the protein at elevated temperatures, which is then facilitated by the absence of electrostatic repulsion.     

Disulfide-bonded polymerization of plasma fibronectin in the presence of metal ions

Incubation of human plasma fibronectin in the presence of low concentrations of FeCl3 or CuSO4 led to the formation of disulfide-bonded multimers as revealed by analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing or reducing conditions. The polymers induced by FeCl3 did not enter the spacer gel, and those induced by CuSO4 migrated to the top of the running gel, indicating that the former polymers were larger than the latter, which in gel filtration experiments appeared to be larger than Mr 670,000. The polymerization occurred between pH 7 and 9 and more rapidly at 22 or 37 degrees C than at 4 degrees C and was inhibited by metal-chelating reagents. NaCl, heparin, spermine, urea, or guanidine hydrochloride did not appreciably affect the reaction, whereas dithioerythritol enhanced the CuSO4-induced polymerization of fibronectin. When incubated in the presence of FeCl3, the Mr 30,000 NH2-terminal, Mr 40,000 gelatin-binding, and the Mr 120,000-140,000 COOH-terminal fragments of fibronectin formed disulfide-bonded polymers, whereas only the Mr 140,000 fragment was polymerized in the presence of CuSO4. Disulfide-bonded polymers were also formed in the presence of FeCl3 but not CuSO4 when the free sulfhydryl groups of fibronectin were blocked by N-ethylmaleimide. The results suggest that in the presence of CuSO4, disulfide-bonded polymerization of fibronectin may involve predominantly the free sulfhydryl groups, whereas in the presence of FeCl3, also the intramolecular disulfides may exchange to form disulfides between separate fibronectin molecules. Thus, under different conditions, different parts of fibronectin may be susceptible to disulfide-bonded polymerization.     

Dimerization of human growth hormone in the presence of metal ions

Zn(2+) and Co(2+) ions are known to promote human growth hormone reversible dimerization. In these studies, dimerization was also shown to be initiated by nine other metal ions: Cd(2+), Hg(2+), Cu(2+), Ag+, Au(3+), Au+, Pd(2+), Ni(2+), and Pt(4+). In some cases (Hg(2+), Ag(+), Au(3+), and Ni(2+)) formation of higher oligomers also took place. In addition further detailed investigation of dimerization in the presence of Zn(2+) ions was carried out.     

Effect of divalent metal ions on annexin-mediated aggregation of asolectin liposomes

Annexins belong to a family of Ca2+- and phospholipid-binding proteins that can mediate the aggregation of granules and vesicles in the presence of Ca2+. We have studied the effects of different divalent metal ions on annexin-mediated aggregation of liposomes using annexins isolated from rabbit liver and large unilamellar vesicles prepared from soybean asolectin II-S. In the course of these studies, we have found that annexin-mediated aggregation of liposomes can be driven by various earth and transition metal ions other than Ca2+. The ability of metal ions to induce annexin-mediated aggregation decreases in the order: Cd2+ > Ba2+, Sr2+ > Ca2+ > Mn2+ > Ni2+ > Co2+. Annexin-mediated aggregation of vesicles is more selective to metal ions than the binding of annexins to membranes. We speculate that not every type of divalent metal ion can induce conformational change sufficient to promote the interaction of annexins either with two opposing membranes or with opposing protein molecules. Relative concentration ratios of metal ions in the intimate environment may be crucial for the functioning of annexins within specialized tissues and after treatment with toxic metal ions.     

Role of metal ions in aggregation of intrinsically disordered proteins in neurodegenerative diseases

Neurodegenerative diseases constitute a set of pathological conditions originating from the slow, irreversible, and systematic cell loss within the various regions of the brain and/or the spinal cord. Depending on the affected region, the outcomes of the neurodegeneration are very broad and diverse, ranging from the problems with movements to dementia. Some neurodegenerative diseases are associated with protein misfolding and aggregation. Many proteins that misfold in human neurodegenerative diseases are intrinsically disordered; i.e., they lack a stable tertiary and/or secondary structure under physiological conditions in vitro. These intrinsically disordered proteins (IDPs) functionally complement ordered proteins, being typically involved in regulation and signaling. There is accumulating evidence that altered metal homeostasis may be related to the progression of neurodegenerative diseases. This review examines the effects of metal ion binding on the aggregation pathways of IDPs found in neurodegenerative diseases.     

Effect of metal ions on de novo aggregation of full-length prion protein

It is well established that the prion protein (PrP) contains metal ion binding sites with specificity for copper. Changes in copper levels have been suggested to influence incubation time in experimental prion disease. Therefore, we studied the effect of heavy metal ions (Cu(2+), Mn(2+), Ni(2+), Co(2+), and Zn(2+)) in vitro in a model system that utilizes changes in the concentration of SDS to induce structural conversion and aggregation of recombinant PrP. To quantify and characterize PrP aggregates, we used fluorescently labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system. We found a specific strong pro-aggregatory effect of Mn(2+) at low micromolar concentrations that could be blocked by nanomolar concentration of Cu(2+). These findings suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo.     

The titration curve of insulin in the presence of various bivalent metal ions

1. Titration curves of insulin in the presence and absence of various metal ions are reported. 2. The difference in base consumption with and without the metal ions is compared with calculated curves. 3. These experiments suggest that in dilute solutions Zn(2+) and Cu(2+) ions are bound to alpha-amino groups.     

Ordered association of tobacco mosaic virus in the presence of divalent metal ions

The formation of ordered aggregates of tobacco mosaic virus (TMV) in the presence of divalent metal ions has been studied in concentrated (1-25 mg/ml) solutions of the virus. The divalent metal cations Cd2+, Zn2+, Pb2+, Cu2+, and Ni2+ have been found to promote TMV precipitation from solution at a critical concentration Ccrit, which for a given metal depends on the pH and the ionic strength of the solution, but is largely independent of the virus concentration. The TMV precipitate behaves as a nematic liquid crystal and on drying at a glass surface produces highly ordered, optically birefringent films. However, precipitation is not observed with alkali-earth metals such as Ca2+ and Mg2+. The experimental data suggest that, apart from two 'internal' metal-binding sites in each TMV subunit, the virus contains metal-binding sites of a lower affinity which promote cross-linking of TMV rods via metal bridges. The latter seem to be responsible for the precipitation of TMV in the presence of divalent cations at neutral pH. We propose that the metal-induced cross-linking may be the predominant mechanism to account for the limited solubility of a variety of proteins in solution containing metal cations with valence 2 and higher.     

Destabilization of non-pathological variants of ataxin-3 by metal ions results in aggregation/fibrillogenesis

Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e. murine (Q6), human non-expanded (Q26) and human moderately expanded (Q36). AT3 variants showed an intrinsic propensity to misfolding/aggregation; on the other hand, Zn(2+) and Al(3+) strongly stimulated the amplitude and kinetics of these conformational conversions. While both metal ions induced a time-dependent aggregation into amyloid-like fibrillar forms, only small oligomers and/or short protofibrillar species were detected for AT3s alone. The rate and extent of the metal-induced aggregation/fibrillogenesis processes increased with the size of the polyglutamine stretch. Mn(2+) and Cu(2+) had no effect on (Q6) or actually prevented (Q26 and Q36) the AT3 structural transitions. The observation that Zn(2+) and Al(3+) promote AT3 fibrillogenesis is consistent with similar results found for other amyloidogenic molecules, such as beta-amyloid and prion proteins. Plausibly, these metal ions are a major common factor/cofactor in the etiopathogenesis of neurodegenerative diseases. Studies of liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of AT3, which were enhanced after supplementing the protein with Zn(2+) and Al(3+). This suggests that cell membranes could be a potential primary target in the ataxin-3 pathogenesis and metals could be a biological factor capable of modulating their interaction with AT3.     

Effects of gamma radiation on beta-lactoglobulin: oligomerization and aggregation

The conformational changes and aggregation process of beta-lactoglobulin (beta-LG) subjected to gamma irradiation are presented. Beta-LG in solutions of different protein concentrations (3 and 10 mg/ml) and in solid state with different water activities (a(w)) (0.22; 0.53; 0.74) was irradiated using a Cobalt-60 radiation source at dose level of 1-50 kGy. Small-angle X-ray scattering (SAXS) was used to study the conformational changes of beta-LG due to the irradiation treatment. The irradiated protein was also examined by high performance size exclusion chromatography (HPSEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing and reducing conditions and fluorescence. SAXS analysis showed that the structural conformation of irradiated beta-LG in solid state at different a(w) and dose level was essentially the same as the nonirradiated beta-LG. The scattering data also showed that the irradiation of beta-LG in solution promoted the formation of oligomers. Interestingly, from the data analysis and model building, it could be shown that the formed oligomers are linear molecules, built by linear combinations of beta-LG dimers (tetramers, hexamers, etc). The formation of oligomers was also evidenced by SDS-PAGE analysis and HPSEC chromatograms, in which products with higher molecular mass than that of the dimeric beta-LG were detected. Formation of intermolecular cross-linking between tyrosyl radicals are proposed to be at least partially responsible for this occurrence. From the results it could be shown that the samples irradiated in solution presented some conformational changes under gamma irradiation, resulting in well ordered oligomers and aggregates formed by cross-linking of beta-LG dimers subunits, while the samples irradiated in the solid state were not modified.