Structural role of the copper ions in the dinuclear active site of Carcinus aestuarii hemocyanin

In this work we show, by a combination of biochemical and biophysical approaches, that the copper ions bound in the binuclear active site of Carcinus aestuarii hemocyanin play a stabilizing role on the tertiary structure of the protein. Upon removal of copper, the monomeric hemocyanin, but not the hexameric oligomer, undergoes changes at the level of tertiary structure while the secondary structure is almost unaffected. By Small-Angle X-Ray Scattering, supported by gel chromatography measurements, it can be concluded that the apo-monomer, but not the holo form or the hexameric form, undergoes a slow time-dependent oligomerization process.     

Spectroscopic Properties and Conformational Stability of Concholepas concholepas Hemocyanin

The structure in solution and conformational stability of the hemocyanin from the Chilean gastropod mollusk Concholepas concholepas (CCH) and its structural subunits, CCH-A and CCH-B, were studied using fluorescence spectroscopy and differential scanning calorimetry (DSC). The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Besides tryptophan residues buried in the hydrophobic interior of the protein molecule also exposed fluorophores determine the fluorescence emission of the oxy- and apo-forms of the investigated hemocyanins. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-forms of CCH and its subunits. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophan residues in the respective apo-forms. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the CCH and its subunits exist in different conformations. The thermal denaturation of the hemocyanin is an irreversible process, under kinetic control. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. Arrhenius equation parameter for the two-state irreversible model of the thermal denaturation of oxy-CCH at pH 7.2 was estimated. Both factors, oligomerization and the copper-dioxygen system at the active site, are important for stabilizing the structure of the hemocyanin molecule.     

Structural quantitative information on the active sites of hemocyanins and related model compounds by the XAS approach: the role of multiple-scattering calculations

In this contribution, we will present an overview of the role of the multiple scattering (MS) calculations in the X-ray absorption spectroscopy (XAS) approach in order to extract from experimental data quantitative structural information on the active sites of the hemocyanin derivatives and of the related model compounds considered.     

Toward oxygen binding curves of single respiratory proteins

Oxygen binding curves of single molecules promise to discriminate between different models describing cooperativity because load distributions are accessible. Individual tarantula hemocyanins could be detected by fluorescence correlation spectroscopy using intrinsic tryptophan fluorescence as sensor of bound oxygen. However, imaging of immobilized proteins was not possible due to fast photo-bleaching. It is shown that tetra-methyl-carboxy-rhodamine (TAMRA), commonly used as a fluorescence label in single-molecule spectroscopy, can also be applied to monitor bound oxygen. The dye's fluorescence is quenched due to F?rster energy transfer to the oxygenated active sites of hemocyanin.     

Model building of a molluscan hemocyanin from X-ray solution scattering

Molluscan hemocyanins are proteins of a truly enormous size. Because of this, determination of their quaternary structure at high resolution cannot easily be obtained by standard methods such as X-ray crystallography and NMR. Therefore, different approaches, using several low-resolution techniques are currently necessary to understand hemocyanin structure. In this work a model of the Rapana venosa hemocyanin has been obtained from a template model and small angle X-ray scattering (SAXS) data. The template model was built from the electron density of the closely related Haliotis tuberculata hemocyanin and a computer program was written to fit this model to the SAXS data using the simulated annealing algorithm.     

Comparative 11A structure of two molluscan hemocyanins from 3D cryo-electron microscopy

Hemocyanins are giant extracellular proteins that transport oxygen in the hemolymph of many molluscs. Molluscan hemocyanins are cylindrical decamers or didecamers of a 350-400 kDa subunit that contains seven or eight different covalently linked globular functional units (FUs), arranged in a linear manner. Each FU carries a single copper active site and reversibly binds one dioxygen molecule. As a consequence, the decamer can carry up to 70 or 80 O(2) molecules. Although complete sequence information is now available from several molluscan hemocyanins, many details of the quaternary structure are still unclear, including the topology of the 10 subunits within the decamer. Here we show 3D reconstructions from cryo-electron micrographs of the hemocyanin decamer of Nautilus pompilius (Cephalopoda) and Haliotis tuberculata (Gastropoda) at a resolution of 11A (FSC(1/2-bit) criterion). The wall structure of both hemocyanins is very similar and shows, as in previous reconstructions, three tiers with 20 functional units each that encircle the cylinder wall, and the 10 oblique minor and major wall grooves. However, the six types of wall FUs of the polypeptide subunit, termed a-b-c-d-e-f, are now for the first time individually discernable by their specific orientation, shape, and connections. Also, the internal collar complex of the decamers shows superior resolution which, in this case, reveals striking differences between the two hemocyanins. The five arcs (FU-g pairs) of the central collar (in both hemocyanins) and the five slabs (FU-h pairs) of the peripheral collar (only present in Haliotis hemocyanin), as well as their connections to the wall and to each other are now more clearly defined. The arc is attached to the wall through a feature termed the anchor, a previously undescribed structural element of the hemocyanin wall.     

3D reconstruction of the hemocyanin subunit dimer from the chiton Acanthochiton fascicularis

Procedures are presented for the purification of the subunit dimer from Acanthochiton fasicularis hemocyanin. Electron microscopy of negatively stained specimens revealed a uniform population of macromolecules possessing the characteristic "boat shape". A 3D reconstruction from this EM data generated a approximately 3 nm resolution model that correlates well with earlier data of the purported subunit dimer, extracted from the 3D reconstruction of the didecamer of Haliotis tuberculata hemocyanin type 1.     

Haliotis tuberculata hemocyanin (HtH): analysis of oligomeric stability of HtH1 and HtH2, and comparison with keyhole limpet hemocyanin KLH1 and KLH2

The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31–41; Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43–56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329–339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8 MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100 mM concentrations of calcium and magnesium ions in Tris–HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100 mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100 mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.     

Immobilization and AFM of single 4 x 6-mer tarantula hemocyanin molecules

The high molecular mass respiratory protein of the tarantula Eurypelma californicum, a 4 × 6-mer hemocyanin, was investigated by atomic force microscopy (AFM). Various substrates and methods were evaluated for immobilization of individual hemocyanin molecules on a solid surface. Samples were imaged after physisorption on mica and self-assembled monolayers, and after chemisorption on Au(111) and N-hydroxy-succinimide (NHS) functionalized surfaces. AFM measurements were carried out preferable in solution and contact mode, but also in Tapping mode and on air-dried samples. Adsorption of the protein on mica followed by drying and carrying out the measurements in Tapping mode gave the best results. In the AFM images the four hexamers of the native 4 × 6-mer hemocyanin have been defined. The results were compared with independent available structural data and represent a validation case for this technique applied for the first time on such giant and complex molecules. As observable in images taken by transmission electron microscopy and also proposed from SAXS data, 4 × 6-mers could be found where the half-molecules are tilted against each other. This study is a step in resolving conformational heterogeneities, involved in oxygen binding of hemocyanins, at the single-molecule level by AFM.     

High pressure stability of protein complexes studied by static and dynamic light scattering

The high pressure dissociation of hemocyanin prepared from the lobster Homarus americanus and casein micelles from cow milk were observed by in situ light scattering. The hemocyanin dodecamer dissociated via a hexamer into monomers in a two-step three-species reaction. The influence of ligands and the effector l-lactate on the dissociation behavior was investigated. While no effect by carbon monoxide after exchanging the ligand oxygen was observed, the addition of the effector l-lactate led to a decrease in the pressure stability. Due to a trimer intermediate which was found to be stabilized by l-lactate, the dissociation reaction in the presence of the effector was analyzed by a three-step four-species reaction. In the case of casein micelles, a two-step dissociation mechanism was found. The stabilizing interactions of casein micelles were identified and separated.     

Comparative analysis of structural properties of Portunid Crab Hemocyanin

In order to explore the hemocyanin adaptative potential and evolutive dynamics, we have analyzed the structural properties of this oxygen-carrier protein, in some species of portunid Crabs, (Brachyura, Portunida). We have compared the intra- and interspecific subunits patterns, in native and denaturant conditions, to estimate the phenetic relationships and the different stabilities of the protein.     

Rapana thomasiana hemocyanin (RtH): dissociation and reassociation behavior of two isoforms, RtH1 and RtH2

Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin isoforms, termed RtH1 and RtH2. The two subunit types, purified by ion exchange chromatography, were used for macromolecular reassociation studies. In vitro reassociation was achieved with Tris-saline stabilizing buffer at pH 7.4, containing 100mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation was monitored, and the different oligomeric forms of RtH1 and RtH2 were studied by transmission electron microscopy, using samples negatively stained with 1% (w/v) uranyl acetate or 5% (w/v) ammonium molybdate containing 1% (w/v) trehalose at pH 7.0. The two subunits reassociate to produce characteristic didecamers, oligomeric and polymeric forms depending on the dissociated material and the reassociation conditions (i.e. divalent ion concentration, duration). In contrast to the didecamers of the freshly isolated RtH preparations, RtH1 and RtH2 show after 2 weeks' reassociation a clear tendency to generate multidecameric structures. The behavior of the native RtH1 and RtH2 during reassociation in the presence of 100mM calcium and magnesium chloride corresponds to the reported common oligomerization characteristics of KLH1/HtH1 and KLH2/HtH2, respectively. It is important to note that during the reassociation of the RtH isoforms: (I) no smaller diameter tubular polymers (ca. 25-27nm) were formed from the subunits as well as from the decamers; (II) multidecamers with one or more 'nucleating' didecamers were detected in addition to the multidecamers, composed of didecamers with associated decamers at one or both ends.     

Characterization of keyhole limpet hemocyanin (KLH) glycans sharing a carbohydrate epitope with Schistosoma mansoni glycoconjugates

Keyhole limpet hemocyanin (KLH) is known to share carbohydrate epitopes with Schistosoma mansoni. In order to define the structural basis for the observed serological cross-reactivity, KLH glycans were released either by enzyme treatment or by hydrazinolysis and probed with a rabbit hyperimmune serum directed against S. mansoni egg antigen. Both major, non-reacting oligosaccharide species as well as the minor compounds recognized were isolated by two-dimensional high performance liquid chromatography and in part by lectin affinity chromatography, and characterized by mass spectrometry. The results revealed that KLH carries predominantly high mannose-type glycans as well as short sugar chains. As a characteristic feature, a number of the latter glycans contained a Gal(beta1-6)Man-unit, which has not yet been found in glycoprotein-N-glycans. Oligosaccharides cross-reacting with schistosomal glycans comprised a terminal Fuc(alpha1-3)GalNAc-motif, which appears to represent the main carbohydrate epitope mediating cross-reactivity of KLH with glycoconjugates from S. mansoni.     

Rapana thomasiana hemocyanin (RtH): comparison of the two isoforms, RtH1 and RtH2, at 19A and 16A resolution

Three-dimensional (3D) reconstructions of the two 8.4 MDa Rapana thomasiana hemocyanin isoforms, RtH1 and RtH2, have been obtained by cryoelectron microscopy of molecules embedded in vitreous ice and single particle image processing. The final 3D structures of the RtH1 and RtH2 didecamers at 19 A and 16 A resolution, respectively, are very similar to earlier reconstructions of gastropodan hemocyanins, revealing structural features such as the obliquely oriented subunits, the five- and two-fold symmetrical axes. Three new interactions are defined; two of them connecting the arch and the wall while the third is formed between the collar and the wall. The collar-wall connection and one of the arch-wall connections are positioned between two individual subunit dimers, while the second arch-wall connection is located between two subunits within the subunit dimer. All three interactions establish connections to the first tier of the wall. Furthermore, for each interaction we have allocated two first tier functional units most likely involved in forming the connections.     

Keyhole limpet hemocyanin (KLH): a biomedical review

In this review we present a broad survey of fundamental scientific and medically applied studies on keyhole limpet hemocyanin (KLH). Commencing with the biochemistry of KLH, information on the biosynthesis and biological role of this copper-containing respiratory protein in the marine gastropod Megathura crenulata is provided. The established methods for the purification of the two isoforms of KLH (KLH1 and KLH2) are then covered, followed by detailed accounts of the molecular mass determination, functional unit (FU) structure, carbohydrate content, immunological analysis and recent aspects of the molecular genetics of KLH. The transmission electron microscope (TEM) has contributed significantly to the understanding of KLH structure, primarily from negatively stained images. We give a brief account of TEM studies on the native KLH oligomers, the experimental manipulation of the oligomeric states, together with immunolabelling data and studies on subunit reassociation. The field of cellular immunology has provided much relevant biomedical information on KLH and has led to the expansion of use of KLH in experimental immunology and clinically as an immunotherapeutic agent; this area is presented in some detail. The major clinical use of KLH is specifically for the treatment of bladder carcinoma, with efficacy probably due to a cross-reacting carbohydrate epitope. KLH also has considerable possibilities for the treatment of other carcinomas, in particular the epithelially derived adenocarciomas, when used as a carrier for carcinoma ganglioside and mucin-like epitopes. The widespread use of KLH as a hapten carrier and generalised vaccine component represent other major on-going aspects of KLH research, together with its use for the diagnosis of Schistosomiasis, drug assay and the treatment of drug addiction. Immune competence testing, assessment of stress and the understanding of inflammatory conditions are other areas where KLH is also making a useful contribution to medical research.     

Motion-triggered cine MR imaging of active joint movement.

MRI cine studies of active physiological joint movement can provide additional functional information as a supplement to standard examinations. With the ankle joint as an example, it is shown that it is possible to measure kinematic MRI presentations of active joint movement. A pneumatic pressure transducer, a respiratory monitor, and an active differentiator transformed the skin muscle shifting of periodically performed joint movement to a pseudo-ECG, which finally triggered the MRI scanner as in cardiac cine MR imaging.     

Active spot-scanning test with heavy ions at HIRFL-CSR

An active spot beam delivery system for heavy ion therapy has been developed based on the Cooling Storage Ring at HIRFL-CSR, where the pencil carbon-ion beams were scanned within a target volume transversely by a pair of orthogonal (horizontal and vertical) dipole magnets to paint the slices of the target volume and longitudinally by active energy variation of the synchrotron slice by slice. The unique techniques such as dose shaping via active energy variation and magnetic deflection constitute a promising three-dimensional conformal even intensity-modulated radiotherapy with heavy ions at HIRFL-CSR. In this paper, the verification of active energy variation and the calibration of steerable beam deflection are shown, as the basic functionality components of the active spot-scanning system. Additionally, based on the capability of creating homogeneous irradiation fields with steerable pencil beams, a radiobiological experiment like cell survival measurement has been performed aiming at comparison of the radiobiological effects under active and passive beam deliveries.     

An active vertical suspension for track/vehicle systems

Optimal control theory is used to formulate and solve the problem of design of an active suspension system to control vertical vibration of a track/vehicle system. The active suspension system is taken as a cascade arrangement of a Kalman filter and the optimal controller. A noisy measured data sequence of the track unevenness is used as the input. As a numerical example, an active suspension of a simple carbody of the Indian Railways is presented.     

An urban ecosystem as a superposition of interrelated active media

A space-time model that treats the urban ecosystem as a superposition of active media is expanded to take the heterogeneity of anthropogenic and natural factors into account. The approach is based on representation of urban ecosystems as conjugated active media and is aimed at identifying the threshold values of control parameters. The theoretical basis of the system analysis of the stability of urban ecosystems is provided by synergetic ideas concerning autowave self-organization in active media.