Mg2+ binding triggers rearrangement of the IM30 ring structure, resulting in augmented exposure of hydrophobic surfaces competent for membrane binding

The "inner membrane-associated protein of 30 kDa" (IM30), also known as "vesicle-inducing protein in plastids 1" (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg2+ However, the precise role of Mg2+ in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg2+ binds directly to IM30 with a binding affinity of ∼1 mm …

Complete subunit sequences, structure and evolution of the 6 x 6-mer hemocyanin from the common house centipede, Scutigera coleoptrata.

Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure-function relationship and evolution. S. coleoptrata hemocyanin is a 6…

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.

Keyhole limpet haemocyanin: negative staining in the presence of trehalose

Abstract Samples of unpurified and purified haemocyanin from the giant keyhole limpet Megathura crenulata have been studied by transmission electron microscopy (TEM) using mixtures of trehalose with the negative stains, uranyl acetate and ammonium molybdate. Trehalose is a known protein preservative during air and freeze drying, UV irradiation and high temperatures, and therefore offers the possibility of protecting proteins during the drying of negatively-stained specimens and their subsequent electron microscopical study. Evidence is presented that trehalose possesses satisfactory stability within the electron beam during conventional room temperature, negative-staining studies. The combi…

Comparison of testing acute toxicity on embryo of zebrafish, Brachydanio rerio and RTG-2 cytotoxicity as possible alternatives to the acute fish test

Abstract The present study compares two possible alternative methods to replace the acute fish test. Fertilized eggs of zebrafish, Brachydanio rerio were used to investigate the acute toxicity of chemicals. Different toxicological endpoints such as coagulation of the eggs, development of gastrulation, number of somites, development of organs, circulation, heartbeat, otolithanlage and pigmentation have been examined during the embryonic development of zebrafish within the first 48 h. Differences to the normal development were measured by these endpoints. Chemicals have been examined in a preliminary approach with respect to their cytotoxicity on the RTG-2 cells (Rainbow Trout Gonad). The vit…

3-D reconstruction of hemocyanins and other invertebrate hemolymph proteins by cryo-TEM: an overview.

Recombinant expression of gastropod (Biomphalaria glabrata) haemoglobin

Topology of the 10 subunits within the decamer of KLH, the hemocyanin of the marine gastropod Megathura crenulata.

Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of th…

Hemocyanin subunit organization of the gastropod Rapana thomasiana

Abstract 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 i…

Keyhole Limpet Hemocyanin Type 2 (KLH2): Detection and Immunolocalization of a Labile Functional Unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of …

10-A cryoEM structure and molecular model of the Myriapod (Scutigera) 6x6mer hemocyanin:understanding a giant oxygen transport protein

Oxygen transport in Myriapoda is maintained by a unique 6x6mer hemocyanin, that is, 36 subunits arranged as six hexamers (1x6mers). In the sluggish diplopod Spirostreptus, the 1x6mers seem to operate as almost or fully independent allosteric units (h approximately 1.3; P(50) approximately 5 torr), whereas in the swift centipede Scutigera, they intensively cooperate allosterically (h approximately 10; P(50) approximately 50 torr). Here, we show the chemomechanical basis of this differential behavior as deduced from hybrid 6x6mer structures, obtained by single-particle cryo-electron microscopy of the Scutigera 6x6mer (10.0 A resolution according to the 0.5 criterion) and docking of homology-m…

The Tegument Protein pp65 of Human Cytomegalovirus Acts as an Optional Scaffold Protein That Optimizes Protein Uploading into Viral Particles

ABSTRACT The mechanisms that lead to the tegumentation of herpesviral particles are only poorly defined. The phosphoprotein 65 (pp65) is the most abundant constituent of the virion tegument of human cytomegalovirus (HCMV). It is, however, nonessential for virion formation. This seeming discrepancy has not met with a satisfactory explanation regarding the role of pp65 in HCMV particle morphogenesis. Here, we addressed the question of how the overall tegument composition of the HCMV virion depended on pp65 and how the lack of pp65 influenced the packaging of particular tegument proteins. To investigate this, we analyzed the proteomes of pp65-positive (pp65pos) and pp65-negative (pp65neg) viri…

Rhogocytes (pore cells) as the site of hemocyanin biosynthesis in the marine gastropod Haliotis tuberculata.

Rhogocytes (pore cells) are specific molluscan cell types that are scattered throughout the connective tissues of diverse body parts. We have identified rhogocytes in large numbers in tissue taken from mantle, foot and midgut gland of the abalone Haliotis tuberculata (Vetigastropoda). Within cisternae of the endoplasmic reticulum, particles are visible that resemble, in shape and size, hemocyanin molecules, the respiratory protein of many molluscs. Immunohistochemical experiments using hemocyanin-specific antibodies demonstrated that these cells contain hemocyanin. In situ hybridization with a cDNA probe specific for Haliotis hemocyanin showed that hemocyanin-specific mRNA is present in rho…

A simple method to estimate the isoelectric point of modified Tomato bushy stunt virus (TBSV) particles

We present a simple method to estimate the isoelectric point (pI) of Tomato Bushy Stunt particles. We demonstrate that the combination of agarose gels with different pH buffers can be used to electrophorese the virus particles and their migration patterns can be compared. This method allows us to estimate the pI of the virus particles (wild type, wt, and genetically modified particles) and to monitor the effect of the pI of modified peptide side chains of the viral capsid subunit on the pI of the whole virus particle.

Controlled cleavage of KLH1 and KLH2 by the V8 protease from Staphylococcus aureus reassociation, electrophoretic and transmission electron microscopy study of peptide fragments.

The reassociation behaviour of protease V8-cleaved peptides from KLH1 and KLH2, the two hemocyanin isoforms from the giant keyhole limpet Megathura crenulata, has been studied by transmission electron microscopy of negatively stained specimens and SDS/PAGE. Reassociation of the complete mixture of protease cleavage products and of combinations of peptide fragments purified by HPLC was performed in the presence of 100 mm CaCl2 and 100 mm MgCl2 at pH 7.4, over a period of 1 to 4 weeks. The V8 protease splits KLH1 into peptide fragments containing the functional units abc, def, defg, defgh, g and h. This mixture of peptide fragments reassociated to form helical tubular polymers, with a diamete…

Keyhole limpet hemocyanin (KLH), II: Characteristic reassociation properties of purified KLH1 and KLH2.

Subunits of the two types of keyhole limpet hemocyanin (KLH1 and KLH2), purified by gel filtration chromatography and preparative polyacrylamide gel electrophoresis from Immucothel, have been used for macromolecular reassociation studies. In-vitro reassociation has been achieved with a standardized system using a Tris-saline stabilizing buffer at pH 7.4 containing 100 mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation has been monitored and the varying oligomeric forms of KLH1 and KLH2 produced have been studied by transmission electron microscopy, using specimens negatively stained with 5% ammonium molybdate containing 1% trehalose. Specimens ha…

8 Å cryo-EM structure of the giant hemoglobin from the planorbid snail Biomphalaria glabrata

Until 2006, snail red hemoglobin remained a phylogenetic enigma because it occurs quite isolated in a single gastropod family, the Planorbidae, whereas all other gastropods use blue hemocyanin as a respiratory protein (for recent cryo-EM of hemocyanin, see [1,2]). Moreover, sequence data on this snail hemoglobin were completely lacking. In 2006, our group published the complete cDNA and predicted amino acid sequence of two Biomphalaria glabrata hemoglobin polypeptides, termed BgHb1 and BgHb2 [3]. (Biomphalaria is intermediate host of the human parasite Schistosoma mansoni that causes Bilharziosis.) Resembling pearl-chains, both polypeptide subunits encompass 13 different, cysteine-free glob…

Identification, structure, and properties of hemocyanins from Diplopod myriapoda.

Hemocyanins are copper-containing, respiratory proteins that occur in the hemolymph of many arthropod species. Here we report for the first time the presence of hemocyanins in the diplopod Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two immunologically distinct subunits in the 75-kDa range that are most likely arranged in a 36-mer (6 x 6) native molecule. It has a high oxygen affinity (P(50) = 4.7 torr) but low cooperativity (h = 1.3 +/- 0.2). Spirostreptus hemocyanin is structurally similar to the single known hemocyanin from the myriapod taxon,…

Red blood with blue-blood ancestry: Intriguing structure of a snail hemoglobin

The phylogenetic enigma of snail hemoglobin, its isolated occurrence in a single gastropod family, the Planorbidae, and the lack of sequence data, stimulated the present study. We present here the complete cDNA and predicted amino acid sequence of two hemoglobin polypeptides from the planorbid Biomphalaria glabrata (intermediate host snail for the human parasite Schistosoma mansoni ). Both isoforms contain 13 different, cysteine-free globin domains, plus a small N-terminal nonglobin “plug” domain with three cysteines for subunit dimerization (total M r ≈ 238 kDa). We also identified the native hemoglobin molecule and present here a preliminary 3D reconstruction from electron microscopical …

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; H…

Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel® followed by separation of KLH1 and KLH2

Abstract Studies of keyhole limpet hemocyanin (KLH) normally require purification of functional complexes directly from living animals. An alternative procedure is described wherein a commercial preparation of KLH which is fully dissociated into its subunits (Immucothel®, biosyn Arzneimittel GmbH) is reassociated in the presence of a high concentration of calcium and magnesium. The reassociation products, when observed by electron microscopy, consist of didecamers, multidecamers and flexible tubules of varying length. The two forms of KLH described previously and designated KLH1 and KLH2, are present in the reassocated mixture as homo-oligomers/polymers and can be separated by selective dis…

Subunit organization of the abalone Haliotis tuberculata hemocyanin type 2 (HtH2), and the cDNA sequence encoding its functional units d, e, f, g and h.

We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtH2) of the European abalone Haliotis tuberculata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, PAGE, N-terminal protein sequencing and cDNA sequencing, we have identified eight different 40-60-kDa functional units (FUs) in HtH2, termed HtH2-a to HtH2-h, and determined their linear arrangement within the elongated 400-kDa subunit. From a Haliotis cDNA library, we have isolated and sequenced a cDNA clone which encodes the five C-terminal FUs d, e, f, g and h of HtH2. As shown by multiple sequence alignments, defg of HtH2 correspond structurally to defg from Octo…

Acetylcholine-binding protein in the hemolymph of the planorbid snail Biomphalaria glabrata is a pentagonal dodecahedron (60 subunits).

Nicotinic acetylcholine receptors (nAChR) play important neurophysiological roles and are of considerable medical relevance. They have been studied extensively, greatly facilitated by the gastropod acetylcholine-binding proteins (AChBP) which represent soluble structural and functional homologues of the ligand-binding domain of nAChR. All these proteins are ring-like pentamers. Here we report that AChBP exists in the hemolymph of the planorbid snail Biomphalaria glabrata (vector of the schistosomiasis parasite) as a regular pentagonal dodecahedron, 22 nm in diameter (12 pentamers, 60 active sites). We sequenced and recombinantly expressed two ∼25 kDa polypeptides (BgAChBP1 and BgAChBP2) wit…

Keyhole limpet haemocyanin (KLH): Purification of intact KLH1 through selective dissociation of KLH2

Abstract Keyhole limpet haemocyanin (KLH) from almost all newly captive animals contains a mixture of KLH1 and KLH2. We show that the dissociation of KLH2 can be produced during EM specimen preparation by the negative staining-carbon film (NS-CF) procedure and in solution by ammonium molybdate-PEG solutions at slightly acidic pHs. The KLH2 multidecamers split apart in the pH range 7.5-6.5 and in the pH range 6.5-6.0 the individual decamers break open and start to dissociate. At pH 5.9 the dissociation of KLH2 yields predominantly a mixture of single subunits and what appear to be subunit dimers. Over the pH range 7.0-5.7 the KLH1 didecamer remains stable. Separation of intact KLH1, in the f…

Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and htH.

Keyhole limpet hemocyanin (KLH) is a well-established immune stimulant and hapten carrier, and Haliotis tuberculata hemocyanin (HtH) is a related product. Biologically, KLH and HtH are blue copper proteins which serve as oxygen carriers in the blood of the keyhole limpet Megathura crenulata and the abalone H. tuberculata, respectively, two marine gastropods. Both hemocyanins occur as two distinct isoforms, termed KLH1 KLH2, HtH1, and HtH2. Each of these molecules is based on a very large polypeptide chain, the subunit (molecular mass ca 400 kDa), which is folded into a series of eight globular functional units (molecular mass ca 50 kDa each). Twenty copies of this subunit form a cylindrical…

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light sca…

Blood cells and the biosynthesis of hemocyanin in Sepia embryos.

Immunoelectron Microscopy of Hemocyanin from the Keyhole Limpet (Megathura crenulata): A Parallel Subunit Model

Abstract Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin (KLH) subunit 2 di- and multidecamers with domain-specific monoclonal antibodies. One antibody (KLH2 a macr 1) links the hemocyanin molecules in a side-to-side pattern, whereas the other antibody (KLH2 fg macr 1) links the molecules end-to-end. From existing knowledge of the domain sequence of KLH subunit 2, these data provide support for a parallel arrangement of subunits within each decamer. Ten N-terminal a macr: domains are then present at the noncollar region of each decamer with 10 C-terminal g macr domains at the collar region. The immunonegative staining data …

Primary structure and unusual carbohydrate moiety of functional unit 2-c of keyhole limpet hemocyanin (KLH)

Abstract The complete amino acid sequence of the Megathura crenulata hemocyanin functional unit KLH2-c was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein, and of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin and cyanogen bromide. This is the first complete primary structure of a functional unit c from a gastropod hemocyanin. KLH2-c consists of 420 amino acid residues. Circular dichroism spectra indicated approx. 31% β-sheet and 29% α-helix contents. A multiple sequence alignment with other molluscan hemocyanin functional units revealed average identities between 41 and 49%, but 55% in case of Octopus he…

Keyhole Limpet Hemocyanin (KLH): Slow In Vitro Reassociation of KLH1 and KLH2 from Immucothel®

Abstract Following our in vitro reassociation of keyhole limpet hemocyanin subunits in the presence of high concentrations (100 mM each) of calcium and magnesium chloride (Harris et al., 1997a, Micron 28, 31–41; 1997b, Micron 28, 43–56), we have now extended our investigations by using a buffer system containing a lower concentration of the two divalent cations (10 mM each). Reassociation of mixed KLH subunits present in the commercially available product Immucothel® was performed using a standardized buffer solution containing 50 mM Tris–HCl, 150 mM NaCl, 10 mM CaCl2 and 10 mM MgCl2 (pH 7.4) over a minimum period of one week, at 4°C. This solution was selected as being close to our KLH sta…