1,721,070 research outputs found
Crystal Structure of the beta;Ser178 -> Pro Mutant of Tryptophan Synthase - A Knock-out allosteric enzyme
No full textThe catalytic activity of the pyridoxal 5-phosphate-dependent tryptophan synthase α2β 2 complex is allosterically regulated. The hydrogen bond between the helix βH6 residue βSer178 and the loop αL6 residue Gly181 was shown to be critical in ligand-induced intersubunit signaling, with the α- β communication being completely lost in the mutant βSer178 -> Pro (Marabotti, A., De Biase, D., Tramonti, A., Bettati, S., and Mozzarelli, A. (2001) J. Biol. Chem. 276, 17747-17753). The structural basis of the impaired allosteric regulation was investigated by determining the crystal structures of the mutant βSer178 -> Pro in the absence and presence of the α-subunit ligands indole-3-acetylglycine and glycerol 3-phosphate. The mutation causes local and distant conformational changes especially in the β-subunit. The ligand-free structure exhibits larger differences at the N-terminal part of helix βH6, whereas the enzyme ligand complexes show differences at the C-terminal side. In contrast to the wild-type enzyme loop αL6 remains in an open conformation even in the presence of α-ligands. This effects the equilibrium between active and inactive conformations of the α-active site, altering kcat and Km, and forms the structural basis for the missing allosteric communication between the α- and β-subunits
Crystal structures of a new class of allosteric effectors complexed to tryptophan synthase
No full textTryptophan synthase is a bifunctional alpha(2)/beta(2) complex catalyzing the last two steps of L-tryptophan biosynthesis. The natural substrates of the alpha-subunit indole-3- glycerolphosphate and glyceraldehyde-3-phosphate, and the substrate analogs indole-3-propanolphosphate and DL-alpha- glycerol-3-phosphate are allosteric effectors of the beta- subunit activity. It has been shown recently, that the indole- 3-acetyl amino acids indole-3-acetylglycine and indole-3- acetyl-L-aspartic acid are both alpha-subunit inhibitors and beta-subunit allosteric effectors, whereas indole-3-acetyl-L- valine is only an alpha-subunit inhibitor (Marabotti, A., Cozzini, P., and Mozzarelli, A. (2000) Biochim. Biophys. Acta 1476, 287-299). The crystal structures of tryptophan synthase complexed with indole-3-acetylglycine and indole-3-acetyl-L- aspartic acid show that both ligands bind to the active site such that the carboxylate moiety is positioned similarly as the phosphate group of the natural substrates. As a consequence, the residues of the alpha-active site that interact with the ligands are the same as observed in the indole 3- glycerolphosphate-enzyme complex. Ligand binding leads to closure of loop alphaL6 of the alpha-subunit, a key structural element of intersubunit communication. This is in keeping with the allosteric role played by these compounds. The structure of the enzyme complex with indole-3-acetyl-L-valine is quite different. Due to the hydrophobic lateral chain, this molecule adopts a new orientation in the alpha-active site. In this case, closure of loop alphaL6 is no longer observed, in agreement with its functioning only as an inhibitor of the a- subunit reaction
Novel allosteric effectors of the tryptophan synthase alpha2beta2 complex identified by computer-assisted molecular modeling
No full textBiochemistry of hemoglobin
No full textHuman hemoglobin A (Hb) is the main protein component of red blood cells, making up to 97 % of their dry content. Hb plays a crucial role in vertebrates, as it carries oxygen from the lungs to the tissues for their oxidative metabolism
Proteomics of Meat Products
No full textProcessed meat, representing about 22% of per capita meat consumption, undergoes technological transformations, such as salting, drying, cooking, or frying. Proteomic methods have been applied to monitor the effects of the processing on meat from cow, yak, pig, lamb, goat, and chicken with the aim of correlating protein pattern with meat quality as well as of identifying adulterations
Structural and functional studies of hemoproteins from polar marine organisms
No full textAntarctica, more than any other habitat on Earth, represents a unique natural laboratory for fundamental research on the processes that have produced biological diversity in extreme environments and, at the same time, offers potential biotechnological opportunities.
One of the most interesting models, within vertebrates, to study the biological responses to cold is provided by Notothenioidei, a group of related species constituting the dominant suborder of teleosts living in Antarctica. To preserve biological activity, five high-Antarctic families of this suborder, living in a stable, extremely cold, and well-oxygenated marine environment, have evolved unique specialisations, including modification of hematological features, having reduced hemoglobin concentration and multiplicity. On the contrary, the three remaining small basal sub-Antarctic families exhibit high hemoglobin multiplicity, probably as a response to temperature differences and fluctuations of temperate waters, and provide an excellent opportunity for understanding the processes involved in cold adaptation.
In this thesis the structure and function of the oxygen-transport system of two sub-Antarctic notothenioids, Eleginops maclovinus (family Eleginopidae) and Dissostichus eleginoides (family Nototheniidae), were described and compared with respect to high-Antarctic species, and in particular to the hemoglobin of Trematomus bernacchii (family Nototheniidae). In contrast to high-Antarctic notothenioids, the hemoglobins of E. maclovinus and D. eleginoides show high oxygen affinity and cooperativity, and marked Root effect. In addition, in the major component of E. maclovinus, a strong stabilization of the low affinity T quaternary state and some peculiar features at the level of the tertiary and quaternary structures have been identified.
The study of these fish and their adaptations is also interesting in the production of new treatments for blood-related diseases and syndromes, including anaemia, hemoglobinopathies and thalassemias or in the development of hemoglobin-based oxygen carriers, a novel pharmaceutical class used in surgery or emergency medicine. Conjugation of human and animal hemoglobins with polyethylene glycol (PEG) has been widely explored as one the most promising strategies to develop blood substitutes. In fact, PEGylation of human hemoglobin led to products with significantly different oxygen-binding properties with respect to the unmodified tetramer and high NO dioxygenase reactivity, known causes of toxicity.
In this context, hemoglobins from Notothenioidei are particularly interesting as they show peculiar features, namely exceptionally low oxygen affinity, little or no dissociation of the tetramer into dimers, absence of cysteine β93, that make them potentially less sensitive to the undesirable effects of PEGylation. The action of PEGylation on properties of the oxygen-transport system of Antarctic fish, and in particular of T. bernacchii hemoglobin, was investigated and compared with that of PEGylated human hemoglobin, confirming that these PEGylated Antarctic hemoglobins potentially meet the functional requirements of blood substitutes.
A remarkable restriction to the research on Antarctic fish is the current lack of genomic sequence data. In contrast, this is not a problem in microorganisms with their small genomes, and which represent a remarkable source for the discovery of new potential biotechnological products.
The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) displays many features that make it an organism of choice for fundamental, environmental and biotechnological studies. The genome sequence shows that PhTAC125 copes with the high oxygen solubility, due to cold temperature, by increasing production of oxygen-scavenging enzymes and deleting entire metabolic pathways which generate reactive oxygen species as side products. The presence of multiple genes encoding 2/2 hemoglobins (annotated as PSHAa0030, PSHAa0458, PSHAa2217) and a flavohemoglobin gene (PSHAa2880), that seem be involved in the protection of the cells from nitrosative and oxidative stress, can also be seen in this perspective. While the flavohemoglobin is widely recognised to be involved in the NO detoxification, the role of the 2/2 hemoglobins is still not clear. Recent in vivo results demonstrated that the inactivation of the gene PSHAa0030 encoding the globin Ph-2/2HbO makes the mutant bacterial strain sensitive to high oxygen levels, hydrogen peroxide, and nitrosating agents. On this basis, the physiological role of the globin Ph-2/2HbO was investigated by in vivo and in vitro experiments. The results confirm its involvement in the protection of cells against the toxic effects of NO and related reactive nitrogen species
From protein structure to function via single crystal optical spectroscopy
No full textThe more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic "artifacts," including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5'-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms
Oxygen delivery via allosteric effectors of hemoglobin, blood substitutes and plasma expanders
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