Homology modelling of hemocyanins and tyrosinases: pitfalls in automated approaches.
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Animals↗
Biomedical subjects
Heinz Decker
Publications and source records attributed to Heinz Decker.
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 Förster energy transfer to the oxygenated active sites of hemocyanin.
Animals↗
Urate as effector for crustacean hemocyanins.
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Animals↗
Small-angle scattering techniques for analyzing conformational transitions in hemocyanins.
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Algorithms↗
Structure-based calculation of multi-donor multi-acceptor fluorescence resonance energy transfer in the 4x6-mer tarantula hemocyanin.
Hemocyanins are oxygen carriers of arthropods and molluscs. The oxygen is bound between two copper ions, forming a Cu(II)-O(2)(2-)-Cu(II) complex. The oxygenated active sites create two spectroscopic signals indicating the oxygen load of the hemocyanins: first, an absorption band at 340 nm which is due to a ligand-to-metal charge transfer complex, and second, a strong quenching of the intrinsic tryptophan fluorescence, the cause of which has not been definitively identified. We showed for the 4x6-mer hemocyanin of the tarantula Eurypelma californicum that the fluorescence quenching of oxygenated hemocyanin is caused exclusively by fluorescence resonance energy transfer (FRET). The tarantula hemocyanin consists of 24 subunits containing 148 tryptophans acting as donors and 24 active sites as acceptors. The donor-acceptor distances are determined on the basis of a closely related crystal structure of the horseshoe crab Limulus polyphemus hemocyanin subunit II (68-79% homology). Calculation of the expected fluorescence quenching and the measured transfer efficiency coincided extraordinary well, so that the fluorescence quenching of oxygenated tarantula hemocyanin can be completely explained by Förster transfer. This results explain for the first time, on a molecular basis, why fluorescence quantum yield can be used as an intrinsic signal for oxygen load of at least one arthropod hemocyanin, in particular that from the tarantula.
Animals↗
Isolation and characterization of haemoporin, an abundant haemolymph protein from Aplysia californica.
In the present study, we show the isolation and characterization of the protein haemoporin, which constitutes the second most abundant protein fraction in the haemolymph of the marine gastropod Aplysia californica. Although Aplysia is commonly used to investigate the molecular basis of learning, not much is known about the proteins in its haemolymph, which is in contact with the neurons owing to the open circulatory system of molluscs. In the native state, haemoporin is a macromolecular complex forming a cylinder with a central solvent-filled pore. The native complex most probably is a homopentamer made up from 70 kDa subunits with a molecular mass of 360 kDa and a sedimentation coefficient of 11.7 S. Prediction of the secondary structure by CD spectroscopy revealed that haemoporin contains 36% alpha-helices and 19% beta-strands. An absorption band in the 300-400 nm region indicates that haemoporin probably contains a bound substance. Haemoporin also contains a below average amount of tryptophan as evident from absorption and fluorescence spectra. The specific absorption coefficient at 280 nm (a (280 nm, 1 mg/ml)) varies between 0.42 and 0.59 l x g(-1) x cm(-1) depending on the method. The function of the protein is not yet known, but there are structural parallels between haemoporin and a pore protein reported previously in the haemolymph of another marine gastropod Megathura crenulata. The alanine-rich N-terminal sequence (AAVPEAAAEATAEAAPVSEF) is unique among protein sequences and indicates an alpha-helical structure. Whereas one side of the helix is hydrophobic and faces the interior of the protein, the other side contains a glutamic cluster, which may form the channel of the pore in the quaternary structure. Thus both proteins might belong to a new class of haemolymph proteins present in the haemolymph of marine gastropods.
Amino Acid Sequence↗
Small-angle X-ray scattering-based three-dimensional reconstruction of the immunogen KLH1 reveals different oxygen-dependent conformations.
For decades the respiratory protein keyhole limpet hemocyanin (KLH1) from the marine gastropod Megathura crenulata has been used widely as a potent immunostimulant, useful hapten carrier, and valuable agent in the treatment of bladder carcinoma. Although much information on the immunological properties of KLH1 is available, biochemical and structural data are still incomplete. Small-angle x-ray scattering revealed the existence of two conformations, an oxy state being slightly more compact than the deoxy state. Based on small-angle scattering curves, a newly developed Monte Carlo algorithm delivered a surface representation of proteins. The massive changes of the surfaces of reconstructed didecameric KLH1 molecules are explained as a twist of the two non-covalently associated decameric half-molecules. Upon oxygenation, the KLH1 molecule becomes longer and skinnier. This study provides the first real evidence how a molluscan hemocyanin changes conformation during an allosteric transition.
Allosteric Regulation↗
Nested allosteric interactions in extracellular hemoglobin of the leech Macrobdella decora.
Hemoglobin from the leech Macrobdella decora belongs to the class of giant extracellular hexagonal bilayer globin structures found in annelid and vestimentiferan worms. These complexes consist of 144 heme-bearing subunits, exhibit a characteristic quaternary structure (2 x (6 x (3 x 4))), and contain tetramers as basic substructures that express cooperative oxygen binding and thus provide a structural basis for a hierarchy in allosteric interactions. A thorough analysis of the isolated tetramer indicates that it functions as a trimer of cooperatively interacting subunits and a non-cooperative monomer rather than as four interacting subunits. A thermodynamic analysis of the whole molecule favors the application of a nested Monod-Wyman-Changeux model with six cooperatively interacting 12-mer allosteric units. In contrast to the isolated tetramers, all subunits of the tetramers seem to be coupled cooperatively within the oligomerized 144-mer. Thus, besides hemocyanins and GroEL, the hexagonal bilayer hemoglobins represent another class of proteins in which the hierarchical quaternary structure provides the basis for nested interaction in their functional properties.
Animals↗
Effects of ultrahigh dilutions of 3,5-dichlorophenol on the luminescence of the bacterium Vibrio fischeri.
There is a great need for research in the field of homeopathy for laboratory test systems to investigate the actions of ultrahighly diluted biological effectors. With this in mind, we used the luminescent bacterium Vibrio fischeri, which is used throughout the world in testing water quality. Luminescence inhibition is utilized as a test parameter for the toxicity of a sample. We used ultrahigh dilutions (UHD) of 3,5-dichlorophenol as effector and adapted the standard test procedure for water toxicity in a way that let us evaluate very minute effects. Three groups of samples were prepared and then blinded: 45 dilutions of 3,5-dichlorophenol in steps of 10, starting with 4.2 x 10(-2) M, with vigorous shaking between dilution steps; 45 identical dilutions of 3,5-dichlorophenol without vigorous shaking; and 49 control samples of the diluent. The results of, and the discussion based on, a thorough statistical analysis led to the conclusion that an effect based on UHD, which results in an inhibition of luminescence of less than 1.5%, can be confirmed for some of the potency samples. There were both effective and ineffective samples in the three sample groups. The size of the effect was very small (ca. 1.5%), though statistically significant. The number of effective samples was significantly higher among the vigorously shaken samples than among the controls and the unshaken samples (14, 6 and 7 effective samples, respectively).
Chlorophenols↗
Tyrosinases from crustaceans form hexamers.
Tyrosinases, which are widely distributed among animals, plants and fungi, are involved in many biologically essential functions, including pigmentation, sclerotization, primary immune response and host defence. In the present study, we present a structural and physicochemical characterization of two new tyrosinases from the crustaceans Palinurus elephas (European spiny lobster) and Astacus leptodactylus (freshwater crayfish). In vivo, the purified crustacean tyrosinases occur as hexamers composed of one subunit type with a molecular mass of approx. 71 kDa. The tyrosinase hexamers appear to be similar to the haemocyanins, based on electron microscopy. Thus a careful purification protocol was developed to discriminate clearly between tyrosinases and the closely related haemocyanins. The physicochemical properties of haemocyanins and tyrosinases are different with respect to electronegativity and hydrophobicity. The hexameric nature of arthropod tyrosinases suggests that these proteins were the ideal predecessors from which to develop the oxygen-carrier protein haemocyanin, with its allosteric and co-operative properties, later on.
Animals↗
All hierarchical levels are involved in conformational transitions of the 4 x 6-meric tarantula hemocyanin upon oxygenation.
The respiratory protein of the tarantula Eurypelma californicum is a 4 x 6-meric hemocyanin that binds oxygen with high cooperativity. This requires the existence of different conformations which have been confirmed by small angle X-ray scattering (SAXS). Here we present reconstructed 3D-models of the oxy- and deoxy-forms of tarantula hemocyanins, as obtained by fitting small angle X-rays scattering curves on the basis of known X-ray structures and electron microscopy of related hemocyanins. For the first time, the involvement of movements at all levels of the quaternary structure was confirmed for an arthropod hemocyanin upon oxygenation. The two identical 2 x 6-meric half-molecules of the native 4 x 6-mer were shifted in the oxy-state along each other compared with the deoxy-state by about 14 A. In addition, the angle between the two 2 x 6-meric half-molecules increased by 13 degrees. Within these 2 x 6-mers the two hexamers were rotated against each other by about 26 degrees with respect to the deoxy-state. In addition, the distance between the two trimers of each hexamer increased upon oxygenation by about 2.5 A. These strongly coupled movements are based on the particular hierarchical structure of the 4 x 6-mer. It also shows a concept of allosteric interaction in hierarchically assembled proteins to guarantee the involvement of all subunits of a native oligomer to establish very high Hill coefficients.
Animals↗
Nested MWC model describes hydrolysis of GroEL without assuming negative cooperativity in binding.
Folding assistance and ATPase activity of GroEL are based on the existence of different conformations. In order to characterise these conformations, published data on steady state ATPase activity in the absence of GroES were reanalysed simultaneously in terms of the Nested MWC model. This model is a hierarchical extension of the symmetry-model of Monod et al. [J. Mol. Biol. 12 (1965) 88]. An unique set of GroEL specific parameters was obtained. This set was supported by comparison of predictions arising from this set of values with experimental data for hydrolysis of ATP in the presence of ADP and ATPgammaS, binding of ATPgammaS and ADP to GroEL in the absence of ATP, and binding of ATP as monitored by fluorescence labelling. Thus, for the first time, multiple data sets for the interaction of nucleotides with GroEL are described quantitatively by an allosteric model. A noteworthy feature of our model is that no negative cooperativity in ATP binding occurs in accordance to experimental observations. Furthermore, the model also includes the existence of a conformation with very high ATPase activity. Such a conformation might be of importance at a certain stage in the folding cycle.
Adenosine Diphosphate↗
Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis.
In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the alpha/beta hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the alpha/beta hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.
Acetonitriles↗