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Biomedical subjects

I Manfield

Publications and source records attributed to I Manfield.

5 recordsLinked to original sources
PubMed1997

A novel method for the purification of recombinant subunit I of the Dolichos biflorus seed lectin.

Lectins are carbohydrate-binding proteins that are ubiquitous in nature. Their ability to specifically bind carbohydrates has been used as a means of purification mainly through affinity chromatography techniques. Plant lectins are one of the most thoroughly studied class of lectins, however, details of their in situ function remains elusive. Recent advances in recombinant DNA techniques have been used in several laboratories to study the function of these lectins by heterologous overexpression. The larger subunit of the Dolichos biflorus seed lectin was described by Chao et al. in 1994 and purification through affinity chromatography techniques was described. Here we report on a new method for the purification of this recombinant protein with techniques that are not dependent on the ability of the lectin to bind sugars. This method may have uses in the purification of mutant proteins that may not bind carbohydrates. Characterization of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy shows that the lectin is over 99% pure with a molecular weight of 27,090 +/- 16.17 Da, and hemagglutination assays confirm that the lectin retains its biological activity.

Chromatography, Affinity↗
PubMed1994

The Met repressor-operator complex: DNA recognition by beta-strands.

The crystal structure of the E. coli met repressor in complex with a synthetic 19-base pair oligonucleotide reveals two dimeric repressor molecules bound to adjacent sites on the DNA. The oligonucleotide contains two adjacent repeats of an 8-mer known as a met-box, which represents the consensus of the met operator sites. Each met repressor dimer is centered on a met box and interacts with the adjacent dimer through antiparallel alpha-helices, which explained the observed cooperative nature of the binding. DNA binding takes place through the insertion of a beta-ribbon into the major groove of B-form DNA, representing a novel DNA binding motif. Sequence specificity arises from direct interactions between side chains of the beta-strands and the edges of the bases in the major groove. The local DNA conformation confers additional specificity through interactions between protein and the phosphate backbone. The repressor is activated through binding of S-adenosyl methionine (SAM), the corepressor, to the face opposite to that used for DNA binding. The lack of significant conformational change upon SAM binding, together with electrostatic calculations, suggests that DNA binding enhancement occurs through long-range electrostatic interactions.

Bacterial Proteins↗
PubMed1992

Probing met repressor-operator recognition in solution.

The three-dimensional crystal structure of the Escherichia coli methionine repressor, MetJ, complexed with a DNA operator fragment is described in an accompanying article. The complex exhibits several novel features of DNA-protein interaction. DNA sequence recognition is achieved largely by hydrogen-bond contacts between the bases and amino-acid side chains located on a beta-ribbon, a mode of recognition previously hypothesized on the basis of modelling of idealized beta-strands and DNA, and mutagenesis of the Salmonella phage P22 repressors Arc and Mnt. The complex comprises a pair of MetJ repressor dimers which bind to adjacent met-box sites on the DNA, and contact each other by means of a pair of antiparallel alpha-helices. Here we assess the importance of these contacts, and also of contacts that would be made between the C-helices of the protein and DNA in a previous model of the complex, by studying mutations aimed at disrupting them. The role of the carboxy-terminal helix face in operator binding was unclear, but we demonstrate that recognition of operator sequences occurs through side chains in the beta-strand motif and that dimer-dimer interactions are required for effective repression.

Base Sequence↗
PubMed1989

Cooperative tandem binding of met repressor of Escherichia coli.

We present biochemical and genetic data to support the hypothesis that the Escherichia coli met repressor, MetJ, binds to synthetic and natural operator sequences in tandem arrays such that repression depends not only on the affinity of the DNA-protein interaction, but also on protein-protein contacts along the tandem array. This represents a novel form of regulatory switch. Furthermore, there seems to be homology between the organization of the met and trp operators.

Apoproteins↗