Crystal structure of the integral membrane diacylglycerol kinase.

Diacylglycerol kinase catalyses the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria. For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology, folding, assembly and stability. Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

A fast, simple and robust protocol for growing crystals in the lipidic cubic phase.

A simple and inexpensive protocol for producing crystals in the sticky and viscous mesophase used for membrane protein crystallization by the in meso method is described. It provides crystals that appear within 15-30 min of setup at 293 K. The protocol gives the experimenter a convenient way of gaining familiarity and a level of comfort with the lipidic cubic mesophase, which can be daunting as a material when first encountered. Having used the protocol to produce crystals of the test protein, lysozyme, the experimenter can proceed with confidence to apply the method to more valuable membrane (and soluble) protein targets. The glass sandwich plates prepared using this robust protocol can further be used to practice harvesting and snap-cooling of in meso-grown crystals, to explore diffraction data collection with mesophase-embedded crystals, and for an assortment of quality control and calibration applications when used in combination with a crystallization robot.

Development of an immunosensor for the detection of testosterone in bovine urine.

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) substrate system, at +100 mV. The EC50 values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL(-1) and 960 pg mL(-1), respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL(-1) to 40 ng mL(-1); while that in urine ranged from 0.03 ng mL(-1) to 1.6 ng mL(-1). The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL(-1) and 1.8 pg mL(-1). Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV<13%, n=3) and repeatability (CV<9%, n=3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 degrees C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.