Biomedical subjects

A L Shaw

Publications and source records attributed to A L Shaw.

9 recordsLinked to original sources

PubMed2001

Trypsin cleavage stabilizes the rotavirus VP4 spike.

Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5(*) and VP8(*) in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8(*) portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5(*), at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.

Amino Acid Sequence↗

PubMed2000

Molybdate-dependent expression of dimethylsulfoxide reductase in Rhodobacter capsulatus.

Expression of the dimethylsulfoxide respiratory (dor) operon of Rhodobacter is regulated by oxygen, light intensity and availability of substrate. Since dimethylsulfoxide reductase contains a pterin molybdenum cofactor, the role of molybdate in the regulation of dor operon expression was investigated. In this report we show that the molybdate-responsive transcriptional regulator, MopB, and molybdate are essential for maximal dimethylsulfoxide reductase activity and expression of a dorA::lacZ transcriptional fusion in Rhodobacter capsulatus. In contrast, mop genes are not required for the expression of the periplasmic nitrate reductase or xanthine dehydrogenase in R. capsulatus under conditions of molybdenum sufficiency. This is the first report demonstrating a clear functional difference between the ModE homologues MopB and MopA in this bacterium. The results suggest that MopA is primarily involved in the regulation of nitrogen fixation gene expression in response to molybdate while MopB has a role in nitrogen fixation and dimethylsulfoxide respiration.

Bacterial Proteins↗

PubMed1999

Characterization of DorC from Rhodobacter capsulatus, a c-type cytochrome involved in electron transfer to dimethyl sulfoxide reductase.

The dorC gene of the dimethyl sulfoxide respiratory (dor) operon of Rhodobacter capsulatus encodes a pentaheme c-type cytochrome that is involved in electron transfer from ubiquinol to periplasmic dimethyl sulfoxide reductase. DorC was expressed as a C-terminal fusion to an 8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity chromatography. The DorC protein had a subunit Mr = 46,000, and pyridine hemochrome analysis indicated that it contained 5 mol heme c/mol DorC polypeptide, as predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at 551.5 nm (alpha-band), 522 nm (beta-band), and 419 nm (Soret band). Redox potentiometry of the heme centers of DorC identified five components (n = 1) with midpoint potentials of -34, -128, -184, -185, and -276 mV. Despite the low redox potentials of the heme centers, DorC was reduced by duroquinol and was oxidized by dimethyl sulfoxide reductase.

Cytochrome c Group↗

PubMed1996

Cloning and sequence analysis of the dimethylsulfoxide reductase structural gene from Rhodobacter capsulatus.

The dimethylsulfoxide reductase structural gene (dorA) of Rhodobacter capsulatus was cloned from a lambda expression library. The nucleotide sequence of the dorA gene was determined and it was found to encode a protein of 825 amino acids. Comparison of the deduced amino-acid sequence of DorA with N-terminal sequence of purified dimethylsulfoxide reductase from Rhodobacter capsulatus showed that the pre-protein possesses a 41-amino-acid N-terminal signal polypeptide. All of the conserved segments which have been described in bacterial enzymes which bind molybdopterin guanine dinucleotide (Berks, B.C., Ferguson, S.J., Moir, J.W.B. and Richardson, D.J. (1995) Biochim, Biophys. Acta 1232, 97-173) were identified in Rhodobacter capsulatus dimethylsulfoxide reductase.

Amino Acid Sequence↗

PubMed1996

The structure of aquareovirus shows how the different geometries of the two layers of the capsid are reconciled to provide symmetrical interactions and stabilization.

BACKGROUND: Aquareoviruses are important pathogens of aquatic animals and have severe consequences in aquaculture. These viruses belong to the family Reoviridae. A structural feature common to members of the Reoviridae is a multilayered capsid, formed by several concentric icosahedral shells with different protein compositions. How these proteins, which often are present in unequal stoichiometries, interact between icosahedral layers to stabilize the capsid is not well understood. RESULTS: We have determined the three-dimensional structure of aquareovirus to 23 A resolution using electron cryomicroscopy and computer image analysis. The protein capsid is composed of two structurally distinct icosahedral layers: an outer layer approximately 100 A thick, with incomplete T=13 left-handed symmetry, surrounds an inner layer 600 A in diameter that has T=1 symmetry and is perforated by channels near the fivefold axes. There are 120 subunits, arranged in dimers, in the inner layer, each of which interacts with two of the 600 subunits in the outer layer. A separate set of closely interacting proteins forms the fivefold axes of the virus structure, forming continuous density throughout both layers of the capsid. Comparison of full and empty (lacking RNA) virus structures reveals an RNA shell that lies directly beneath the inner layer. CONCLUSIONS: Our aquareovirus structure displays marked similarity to the mammalian reovirus intermediate subviral particles, suggesting a close evolutionary relationship. However, the noticeable distinction is that aquareovirus lacks the hemagglutinin spike observed in reovirus. The T=1 inner layer organization observed in the aquareovirus appears to be common to other members of the Reoviridae. Such organization may be of fundamental significance in the endogenous transcription of the genome in these viruses.

Animals↗

PubMed1996

Rotavirus structure: interactions between the structural proteins.

Structural studies on rotavirus using electron cryomicroscopy and computer image analysis have permitted visualization of each shell in the triple-layered rotavirus structure. Biochemical results have aided our interpretation of the structural organization of these layers and protein interactions seen in the three-dimensional structure, and have provided a better understanding of the structure-function relationships of the rotavirus structural proteins.

Capsid↗

PubMed1993

Three-dimensional visualization of the rotavirus hemagglutinin structure.

Three-dimensional structures of a native simian and reassortant rotavirus have been determined by electron cryomicroscopy and computer image processing. The structural features of the native virus confirm that the hemagglutinin spike is a dimer of VP4, substantiated by in vivo radiolabeling studies. Exchange of native VP4 with a bovine strain equivalent results in a poorly infectious reassortant. No VP4 spikes are detected in the three-dimensional reconstruction of the reassortant. The difference map between the two structures reveals a novel large globular domain of VP4 buried within the virion that interacts extensively with the intermediate shell protein, VP6. Our results suggest that assembly of VP4 precedes that of VP7, the major outer shell protein, and that VP4 may play an important role in the receptor recognition and budding process through the rough endoplasmic reticulum during virus maturation.

Animals↗

PubMed1992

Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids.

Cell-to-cell spread of tobacco mosaic virus (TMV) is presumed to occur through plant intercellular connections, the plasmodesmata. Viral movement is an active process mediated by a specific virus-encoded P30 protein. P30 has at least two functions, to cooperatively bind single-stranded nucleic acids and to increase plasmodesmatal permeability. Here, we visualized P30 complexes with single-stranded DNA and RNA. These complexes are long, unfolded, and very thin (1.5 to 2.0 nm in diameter). Unlike TMV virions (300 x 18 nm), the complexes are compatible in size with the P30-induced increase in plasmodesmatal permeability (2.4 to 3.1 nm), making them likely candidates for the structures involved in the cell-to-cell movement of TMV. Mutational analysis using single and double deletion mutants of P30 revealed three regions potentially important for the protein function. Amino acid residues 65 to 86 possibly are required for correct folding of the active protein, and the regions between amino acid residues 112 to 185 and 185 to 268 potentially contain two independently active single-stranded nucleic acid binding domains designated binding domains A and B, respectively.

Amino Acid Sequence↗

PubMed1987

Thoracic aortic aneurysm--subtle but serious: a case report.

A case is reported of a 55-yr-old man complaining of interscapular pain, lethargy, anorexia with weight loss and shallowness of breath following a recent traumatic blow to the abdomen. Radiographs revealed a marked and extensive aneurysm of the thoracic aorta. The importance of performing X-ray studies of the thoracic region is emphasized for all cases where histories of recent or past significant chest trauma is suspected and with older patients having hypertension and atherosclerotic heart disease.

Aorta, Thoracic↗