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α-synuclein amyloid fibrils are found in surviving neurons of Parkinson's disease affected patients, but the role they play in the disease development is still under debate. A growing number of evidences points to soluble oligomers as the major cytotoxic species, while insoluble fibrillar aggregates could even play a protection role. In this work, we investigate α-synuclein fibrils dissociation induced at high pressure by means of Small Angle X-ray Scattering and Fourier Transform Infrared Spectroscopy. Fibrils were produced from wild type α-synuclein and two familial mutants, A30P and A53T. Our results enlighten the different reversible nature of α-synuclein fibrils fragmentation at high pressure and suggest water excluded volumes presence in the fibrils core. Wild type and A30P species stabilized at high pressure are highly amyloidogenic and quickly re-associate into fibrils upon decompression, while A53T species shows a partial reversibility of the process likely due to the presence of an intermediate oligomeric state stabilized at high pressure. The amyloid fibrils dissociation process is here suggested to be associated to a negative activation volume, supporting the notion that α-synuclein fibrils are in a high-volume and high-compressibility state and hinting at the presence of a hydration-mediated activated state from which dissociation occurs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Plasma membrane calcium pumps (PMCAs) sustain a primary transport system for the specific removal of cytosolic calcium ions from eukaryotic cells. PMCAs are characterized by the presence of a C-terminal domain referred to as a regulatory domain. This domain is target of several regulatory mechanisms: activation by Ca²+-calmodulin complex and acidic phospholipids, phosphorylation by kinase A and C, proteolysis by calpain and oligomerization. As far as oligomerization is concerned, the C-terminal domain seems to be crucial for this process. We have cloned the C-terminal domain of the human PMCA isoform 1b, and characterized its properties in solution. The expressed protein maintains its tendency to oligomerize in aqueous solutions, but it is dissociated by amphipathic molecules such as diacylglycerol and sodium dodecyl sulphate. The presence of sodium dodecyl sulphate stabilizes the domain as a compact structure in monomeric form retaining the secondary structure elements, as shown by small angle neutron scattering and circular dichroism measurements. The importance of oligomerization for the regulation of PMCA activity and intracellular calcium concentration is discussed., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Hemocyanins from Crustacea usually are found as 1x6 or 2x6-meric assemblies. An exception is the hemocyanin isolated from thalassinidean shrimps where the main component is a 24-meric structure. Our analysis of oxygen binding data of the thalassinidean shrimp Upogebia pusilla based on a three-state MWC-model revealed that despite the 24-meric structure the functional properties can be described very well based on the hexamer as allosteric unit. In contrast to the hemocyanins from other thalassinidean shrimps the oxygen affinity of hemocyanin from U. pusilla is increased upon addition of l-lactate. A particular feature of this hemocyanin seems to be that l-lactate already enhances oxygen affinity under resting conditions which possibly compensates the rather low intrinsic affinity observed in absence of l-lactate. The fast rate of oxygen dissociation might indicate that in this hemocyanin a higher cooperativity is less important than a fast response of saturation level to changes in oxygen concentration., (2010 Elsevier Inc. All rights reserved.)

The effects of temperature and oxygen saturation on the respiration rate of two cold stenothermal chironomids, Diamesa insignipes and Pseudodiamesa branickii were investigated. Fourth instar larvae were collected in winter in a glacio-rhithral stream (1300 m a.s.l., Alps, NE-Italy) and their respiration rate was measured with a Clark's electrode in the range 0-14 degrees C. The respiration rate was significantly higher in D. insignipes than in P. branickii at low temperatures (

The functional differences between the oxygen transport protein Hemocyanin and the enzymes Tyrosinase and Catechol oxidase are believed to be governed, at least in part, by the tertiary structure, which differs in these molecules and controls the accessibility of their copper containing active site for substrate(s). Accordingly, Octopus vulgaris Hemocyanin catalyses the o-diphenol oxidation to o-quinone at a very low rate. The crystallographic structure of one of the functional units (called Odg) of O. dofleini Hemocyanin shows two domains, a mainly alpha-helical domain that directly binds the copper ions of the reaction center and a beta-strand domain that precludes access to the active site to ligands bigger than molecular oxygen. In this work, we have first cleaved the whole protein and then purified different oxygen binding functional units from O. vulgaris Hemocyanin. These functional units were used in activity assays with l-DOPA, the paradigmatic substrate for Catechol oxidase. All functional units show a negligible enzymatic activity. The procedure to generate the functional units induces in only one of them a proteolytic cleavage. Amino terminal sequencing and mass spectroscopy of the fragments allow to place the cleavage site between the alpha and beta domains of the functional unit homologous to Odd, in the O. dofleini sequence. An increase, up to three orders of magnitude, of Tyrosinase-like activity was observed when the cleaved Odd-like was incubated with the substrate in the presence of trifluoroethanol or hexafluoroisopropanol.

Ceruloplasmin is a copper protein found in vertebrate plasma, which belongs to the family of multicopper oxidases. Like transferrin of the blood plasma, lactoferrin, the iron-containing protein of human milk, saliva, tears, seminal plasma and of neutrophilic leukocytes tightly binds two ferric ions. Human lactoferrin and ceruloplasmin have been previously shown to interact both in vivo and in vitro forming a complex. Here we describe a study of the conformation of the human lactoferrin/ceruloplasmin complex in solution using small angle X-ray scattering. Our ab initio structural analysis shows that the complex has a 1:1 stoichiometry and suggests that complex formation occurs without major conformational rearrangements of either protein. Rigid-body modeling of the mutual arrangement of proteins in the complex essentially yields two families of solutions. Final discrimination is possible when integrating in the modeling process extra information translating into structural constraints on the interaction between the two partners.

We have investigated the effect of copper binding on the structural properties of hemocyanin (Hc). To this aim, we have studied the holo- and apo-form of the protein, both in the hexameric and in the monomeric state (CaeSS2 subunit), with experimental approaches that report on the protein aggregation and conformational stability. The results of gel-filtration chromatography and small angle X-ray scattering (SAXS) provide evidence that the hydrodynamic and gyration radius (R(g)) of Hc in the hexameric form only slightly increase upon copper removal, whereas a remarkable enhancement in the R(g) value is observed for the CaeSS2 monomer. CD measurements in the far- and near-UV region indicate that removal of copper only marginally affects the conformation of the hexameric Hc. Instead, copper depletion in the CaeSS2 strongly alters the tertiary structure of the monomer (near-UV CD), even though it is almost inconsequential on the secondary structure content (far-UV CD). These findings are fully consistent with the results of limited proteolysis experiments showing that the hexameric Hc is similarly resistant to proteolysis by trypsin both in the holo- and apo-form. Conversely, the apo-form of CaeSS2 monomer is much more susceptible to proteolytic attack by trypsin than the holo-form. Based on SAXS measurements, the concentration-dependent oligomerization process for apo-CaeSS2 has been analyzed on the basis of a thermodynamic model involving a concentration-dependent equilibrium between a monomer in a native-like and an hexameric aggregate of monomers.

The hemocyanin of the crab Carcinus aestuarii contains a carbohydrate moiety that represents 1.6% of protein mass. This carbohydrate content is higher than that exhibited by other arthropod hemocyanins so far investigated. By combination of FPLC ion exchange chromatography and reverse-phase HPLC, the native oligomeric protein can be resolved into three major and one minor electrophoretically pure fractions that are found to be homogeneous by N-terminal sequencing and correspond to the subunit polypeptide chains. Sugar analysis on the different subunits reveals that the subunit referred to as Ca2 is glycosylated, with a carbohydrate content of 6.3%. By Ca2 trypsin digestion, separation of glycopeptides, and amino acid sequencing, three consensus sequences for O-glycosylation and one for N-glycosylation were found. MALDI-MS was applied for the determination of the molecular masses of the various glycopeptides and peptides after removal of carbohydrates by neuraminidase and alpha-N-acetylgalactosaminidase., (Copyright 2000 Academic Press.)

When added to human blood serum, the iron-binding protein lactoferrin (LF) purified from breast milk interacts with ceruloplasmin (CP), a copper-containing oxidase. Selective binding of LF to CP is evidenced by the results of polyacrylamide gel electrophoresis, immunodiffusion, gel filtration, and affinity chromatography. The molar stoichiometry of CP:LF in the complex is 1:2. Near-uv circular dichroism spectra of the complex showed that neither of the two proteins undergoes major structural perturbations when interacting with its counterpart. K(d) for the CP/LF complex was estimated from Scatchard plot as 1.8 x 10(-6) M. The CP/LF complex is found in various fluids of the human body. Upon injection into rat of human LF, the latter is soon revealed within the CP/LF complex of the blood plasma, from where the human protein is substantially cleared within 5 h., (Copyright 2000 Academic Press.)

Rapana venosa hemocyanin (Hc) is a giant oxygen-binding protein consisting of different subunits assembled in a hollow cylinder. The polypeptide chain of each subunit is believed to be folded in several oxygen-binding functional units of molecular mass 50 kDa, each containing a binuclear copper active site. Limited proteolysis with alpha-chymotrypsin of native R. venosa hemocyanin allows the separation of three functional proteolytic fragments with molecular masses of approximately 150, 100, and 50 kDa. The functional fragments, purified by combining gel filtration chromatography and ion-exchange FPLC, were analyzed by means of small-angle X-ray scattering (SAXS). The gyration radius of the 50-kDa Rapana Hc fraction (2.4 nm) agrees well with that calculated on the basis of the dimensions determined by X-ray crystallography for one functional unit of Octopus Hc (2.1 nm). Independent shape determination of the 50- and 100-kDa proteolytic fragments yields consistent low-resolution models. Simultaneous fitting of the SAXS data from these fragments provides a higher-resolution model of the 100-kDa species made of two functional units tilted with respect to each other. The model of the 150-kDa proteolytic fragment consistent with the SAXS data displays a linear chain-like aggregation of the 50-kDa functional units. These observations provide valuable information for the reconstruction of the three-dimensional structure of the minimal functional subunit of gastropod hemocyanin in solution. Furthermore, the spatial relationships among the different functional units within the subunit will help in elucidation of the overall quaternary structure of the oligomeric native protein., (Copyright 2000 Academic Press.)

RtH1 and RtH2, the two hemocyanin isoforms of the prosobranch gastropod Rapana thomasiana, have been purified by anion-exchange chromatography and studied by SDS-PAGE and immunoelectrophoresis. Both subunit types are built up of eight functional units (FUs). Under reducing conditions subunit RtH2 splits into two fragments, RtH2-a-f and RtH2-gh, suggesting the presence of a disulfide bridge between FU2-f and FU2-g. By proteolytic cleavage of the subunits into three-, two-, and single-FU fragments, purification of fragments by HPLC, N-terminal sequencing of the peptides, and crossed-line immunoelectrophoresis, FUs-a-h of RtH2 and FU-a, FU-d, FU-e, and FU-f of RtH1 were identified and correlated to the eight-FUs pattern of immunoelectrophoresis. FU-a, FU-e, and FU-f of RtH1 and RtH2 are very closely related immunologically. RtH1 and RtH2 both correspond immunologically to KLH2, one of the two hemocyanin isoforms of the prosobranch gastropod Megathura crenulata., (Copyright 1999 Academic Press.)

Copper ions of different types were gradually eliminated from ceruloplasmin (CP1; ferro-O2-oxidoreductase, EC 1.16.3.1.) by dialyzing the enzyme against KCN. Protein was sampled 2, 4, 6, 22, and 28 h after the dialysis started. Atomic absorption allowed us to estimate the amount of copper atoms per CP molecule. Light absorption in the UV and visible regions along with fluorescence and EPR spectra were also registered. Oxidase and dismutase activities of the enzyme were measured at each step. The combination of the data thus obtained allowed us to trace the sequence of CP depletion of certain copper ions. The same methods were applied in reconstitution studies to detect the return of different types of Cu2+. The experiments were performed on CP samples differing in the amount of copper still bound after CN- treatment. It is shown that the oxidase activity is efficiently brought back to CP if, after the dialysis against cyanide, the catalytic center had preserved its type 3 Cu2+. Dismutase activity of CP did not depend greatly on the presence or absence of type 1 and type 2 copper ions. The results obtained allow a more precise evaluation of the role of different types Cu2+ in the assembly of the complex catalytic center of CP and in the accomplishment by the enzyme of its multiple functions.

The deoxygenated form of hemocyanin, containing a dinuclear Cu(I) active site, emits luminescence in the red with maximum around 1.54 microns-1 (650 nm). The luminescence of deoxyhemocyanin (deoxy-Hc) from arthropod species is detectable at room temperature, the quantum yield being 2.4-2.7 x 10(-3); in contrast, the emission from molluscan proteins can be detected only at liquid nitrogen temperature. The luminescence emission is an inherent property of the bis[Cu(I)-(histidine)3] complex of the deoxygenated form of the protein to which both Cu(I) ions contribute equally to the overall emission. Luminescence is not observed with the oxygenated and the oxidized forms of hemocyanin, in which the metal is in the Cu(II) state, and in the metal-depleted or apo-Hc form. Based on steady-state and time-resolved measurements and references to Cu(I) model compounds, the luminescence emission is attributed to a triplet excited state of a Cu(I)-to-N (histidine) charge transfer transition 3d-pi*. Acrylamide quenching experiments indicate that the metal active site is very shielded from the solvent. This property of deoxy-Hc enables us to directly follow reactions that modify either the copper oxidation number or the metal-to-protein stoichiometry.

The interaction of Carcinus hemocyanin with Cd(II) was studied. The incubation of the apoprotein with the metal yields a derivative containing 1 g-at. of EDTA-stable Cd(II) per 75 kDa. Spectroscopic data ruled out Cd(II) coordination to tryptophan or cysteine residues. The optical activity and fluorescence properties of the protein are affected by Cd(II) binding and indicate a rearrangement of tryptophan residues. The poor Cd(II) binding to the oxy-form and the resistance of Cd(II)-hemocyanin to EDTA treatment and to the regeneration by Cu(I) strongly indicate that Cd(II) binding to apohemocyanin occurs at the copper-free active site. During the metal-binding process, a marked increase of light scattering is observed. This effect, however, is reversible provided that the incubation medium contains SCN- and glycine as exogenous ligands of the metal in the bulk solution.

The reconstitution of Neurospora apotyrosinase was studied in the presence of Cu(I) or Cu(II) ions. The kinetics and the mechanism of reactivation were found to differ markedly for the two metal ions. Thus the reconstitution with Cu(I) was found to be very fast and complete following an all or none process; in contrast the reaction with Cu(II) proved to be rather slow and incomplete with the two Cu(II) ions binding with different rate constants.

Some structural properties of Octopus vulgaris hemocyanin have been investigated by fluorescence spectroscopy. The three-dimensional structure of Octopus hemocyanin is remarkably tight, resulting in a deep burial of almost all the tryptophyl residues of the protein. The hemocyanin conformation has been studied in the two main aggregation states (11 S, 50 S) of the protein, and with respect to the presence or absence of copper in the active site. Upon changing the pH of the solution, Octopus hemocyanin in the 50 S aggregation state can assume at least three different conformations. During the transition between each conformation the fluorescence quantum yield changes, but the environment of tryptophans does not change. Dissociation of the protein from 50 S to 11 S strongly enhances its susceptibility toward denaturating agents such as pH or temperature, and modifies the effects of fluorescence quenchers such as acrylamide. Moreover, these effects are more pronounced when copper is removed from the active site. A comparative analysis of the results shows that the subunit-subunit interactions exerted within the 50 S species are more important in the maintenance of the conformational stability than the copper ions present in the active sites. This behavior can be accounted for by the large amount of Ca(II) ions linked to 50 S hemocyanin.

The reaction between Carcinus maenas hemocyanin and cyanide has been used for probing protein conformation in the presence of perturbants such as various anions, cations (Ca2+, Mg2+), and aliphatic alcohols. The kinetic parameters of the reaction are strongly affected by these agents, suggesting that the induced conformational modifications change the reactivity of the active site toward exogenous ligands. Different patterns are observed according to the perturbant used. As indicated by the mathematical treatment of the kinetic curves the affinity of the active site for CN- and O2 is affected much more than the rate constant of copper removal.

Octopus vulgaris hemocyanin in 11 S aggregation state binds oxygen following a noncooperative oxygen saturation curve with Hill coefficient n = 1. Under the same conditions the equilibrium and kinetics of the reaction with cyanide and other ligands are indicative of an anticooperative behavior displaying different characteristics for the different ligands. The data are consistent with an induced-fit type allosteric model which assumes for the 11 S subunit of O. vulgaris hemocyanin an annular structure made up by five identical domains each containing one binding site whose reactivity is near-neighbor regulated.