Biomedical subjects

Seán McSweeney

Publications and source records attributed to Seán McSweeney.

5 recordsLinked to original sources

PubMed2005

Automatic structure determination based on the single-wavelength anomalous diffraction technique away from an absorption edge.

The phasing of macromolecular structures based on the use of the single-wavelength anomalous diffraction method has recently enjoyed a revival. Here, additional evidence is provided that the method may be successfully applied at wavelengths remote from the absorption edge of interest and that it is in principle applicable to a large number of systems. This opens up the possibility of rapid and reliable automatic de novo structure determination using simple experimental configurations with no need for wavelength tunability or absorption-edge scanning. The method should therefore be exploitable at most synchrotron beamlines. The effects of data completeness and multiplicity on the quality of the phases obtained are discussed as are the prospects for the automation of macromolecular structure solution based on the experimental protocols described.

Alkaline Phosphatase↗

PubMed2004

Development of a bacterial biosensor for nitrotoluenes: the crystal structure of the transcriptional regulator DntR.

The transcriptional regulator DntR, a member of the LysR family, is a central element in a prototype bacterial cell-based biosensor for the detection of hazardous contamination of soil and groundwater by dinitrotoluenes. To optimise the sensitivity of the biosensor for such compounds we have chosen a rational design of the inducer-binding cavity based on knowledge of the three-dimensional structure of DntR. We report two crystal structures of DntR with acetate (resolution 2.6 angstroms) and thiocyanate (resolution 2.3 angstroms), respectively, occupying the inducer-binding cavity. These structures allow for the construction of models of DntR in complex with salicylate (Kd approximately or = 4 microM) and 2,4-dinitrotoluene that provide a basis for the design of mutant DntR with enhanced specificity for dinitrotoluenes. In both crystal structures DntR crystallises as a homodimer with a "head-to-tail" arrangement of monomers in the asymmetric unit. Analysis of the crystal structure has allowed the building of a full-length model of DntR in its biologically active homotetrameric form consisting of two "head-to-head" dimers. The implications of this model for the mechanism of transcription regulation by LysR proteins are discussed.

Base Sequence↗

PubMed2004

Expression, purification, crystallization and preliminary crystal structure analysis of the Deinococcus radiodurans organic hydroperoxide-resistance protein.

The organic hydroperoxide-resistance protein (DR1857) from Deinococcus radiodurans has been expressed, purified and crystallized. The crystals are suitable for X-ray analysis, diffract to at least 2.3 A resolution, have unit-cell parameters a = 45.7, b = 59.6, c = 49.7 A, beta = 90.43 degrees and belong to space group P2(1). The calculated Matthews coefficient of 2.1 A(3) Da(-1) coupled with a calculated solvent content of approximately 42% is consistent with the presence of a homodimer in the asymmetric unit. Here, the methods used in the overexpression and purification of the protein are described and details of crystallization conditions and preliminary X-ray diffraction are provided.

Bacterial Proteins↗

PubMed2004

The structure of the organic hydroperoxide resistance protein from Deinococcus radiodurans. Do conformational changes facilitate recycling of the redox disulfide?

The three-dimensional structure of the organic hydroperoxide resistance protein (OHRP) from Deinococcus radiodurans as determined using single crystal xray diffraction techniques is reported. Comparison of the structure with that obtained for OHRP from Pseudomonas aeruginosa reveals that the polypeptide chain of OHRPs can adopt two significantly different conformations ("in" and "out") in the region of the active site disulfide moiety. It is postulated that the closed configuration is consistent with efficient catalysis of the reduction of organic hydroperoxides, whereas the open form is required for enzyme recycling. Comparison of the structures of OHRP and that of the osmotically induced protein C (OsmC) from Mycoplasma pneumoniae shows that OHRPs and OsmCs are structurally homologous, perhaps indicating related functions for the two families of proteins.

Amino Acid Sequence↗

PubMed2003

The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation.

Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).

Animals↗