ABSTRACT
Spinae are tubular surface appendages broadly found in Gram-negative bacteria. Little is known about their architecture, function or origin. Here, we report structural characterization of the spinae from marine bacteria Roseobacter sp. YSCB. Electron cryo-tomography revealed that a single filament winds into a hollow flared base with progressive change to a cylinder. Proteinase K unwound the spinae into proteolysis-resistant filaments. Thermal treatment ripped the spinae into ribbons that were melted with prolonged heating. Circular dichroism spectroscopy revealed a dominant beta-structure of the spinae. Differential scanning calorimetry analyses showed three endothermic transformations at 50-85°C, 98°C and 123°C, respectively. The heating almost completely disintegrated the spinae, abolished the 98°C transition and destroyed the beta-structure. Infrared spectroscopy identified the amide I spectrum maximum at a position similar to that of amyloid fibrils. Therefore, the spinae distinguish from other bacterial appendages, e.g. flagella and stalks, in both the structure and mechanism of assembly.
Subject(s)
Cell Surface Extensions/ultrastructure , Roseobacter/metabolism , Calorimetry, Differential Scanning , Cell Surface Extensions/chemistry , Cell Surface Extensions/physiology , Circular Dichroism , Cryoelectron Microscopy , TemperatureABSTRACT
Documenting the modes of interaction of uranyl (UO(2)2+) with large biomolecules, and particularly with proteins, is instrumental for the interpretation of its behavior in vitro and in vivo. The gathering of three-dimensional information concerning uranyl-first shell atoms from two structural databases, the Cambridge Structural Databank and the Protein Data Bank (PDB) allowed a screening of corresponding topologies in proteins of known structure. In the computer-aided procedure, all potentially bound residues from the template structure were granted full flexibility using a rotamer library. The Amber force-field was used to loosen constraints and score each predicted site. Our algorithm was validated as a first stage through the recognition of existing experimental data in the PDB. The coherent localization of missing atoms in the density map of an ambiguous uranium/uranyl-protein complex exemplified the efficiency of our approach, which is currently suggesting the experimental investigation of uranyl-protein binding site.
Subject(s)
Proteins/chemistry , Uranium/chemistry , Computers , Kinetics , Protein Binding , SoftwareABSTRACT
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Subject(s)
Adult , Humans , Aged , Male , Female , Middle Aged , Rural Population , Primary Health Care , Spain , Prospective Studies , Adenocarcinoma , Tomography, X-Ray Computed , Magnetic Resonance Imaging , Stroke , Syndrome , Motor Neurons , Motor Neuron Disease , Human T-lymphotropic virus 1 , HTLV-I Infections , Brain Neoplasms , Ovarian NeoplasmsABSTRACT
Chromogranin A (CgA), a major protein of chromaffin granules, has been described as a potential marker for neuroendocrine tumours. Because of an extensive proteolysis which leads to a large heterogeneity of circulating fragments, its presence in blood has been assessed in most cases either by competitive immunoassays or with polyclonal antibodies. In the present study, 24 monoclonal antibodies were raised against native or recombinant human CgA. Their mapping with proteolytic peptides showed that they defined eight distinct epitopic groups which spanned two-thirds of the C-terminal part of human CgA. All monoclonal antibodies were tested by pair and compared with a reference radioimmunoassay (RIA) involving CGS06, one of the monoclonal antibodies against the 198-245 sequence. It appears that CgA C-terminal end seems to be highly affected by proteolysis and the association of C-terminal and median-part monoclonal antibodies is inadequate for total CgA assessment. Our new immunoradiometric assay involves two monoclonal antibodies, whose contiguous epitopes lie within the median 145-245 sequence. This assay allows a sensitive detection of total human CgA and correlates well with RIA because dibasic cleavage sites present in the central domain do not seem to be affected by degradation. It has been proved to be efficient in measuring CgA levels in patients with neuroendocrine tumours.
Subject(s)
Antibodies, Monoclonal/immunology , Chromogranins/metabolism , Immunoradiometric Assay/methods , Neuroendocrine Tumors/metabolism , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Chromogranin A , Chromogranins/chemistry , Chromogranins/isolation & purification , Humans , Metalloendopeptidases/metabolism , Mice , Mice, Inbred BALB C , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
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