Waterston, Claire Louise
Structural studies of putative general stress and related proteins from Deinococcus radiodurans.
PhD thesis, University of Glasgow.
Full text available as:
This study describes the cloning, expression, purification, biophysical characterisation and crystallisation of DR_1146; a putative general stress protein from the extremophilic bacterium Deinococcus radiodurans (R1).
The extraordinary ability of D. radiodurans to resist mutation or apoptosis on exposure to high does of ionising radiation has formed the basis of a structural genomics project underway at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The work presented in this study forms part of the ESRF’s D. radiodurans initiative, and was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and the ESRF as an Industrial Cooperative Award in Science and Engineering (CASE) PhD studentship. A period of one-year was spent on secondment at the ESRF, working within the Macromolecular Crystallography Group.
Several constructs of the dr_1146 gene have been successfully overexpressed in E. coli cells to give high yields of target protein. Purification by immobilised metal affinity chromatography (IMAC) was facilitated by the incorporation of a 6xHis tag and supplemented by a final gel filtration step. Although high purity levels were achieved, imaging by SDS-PAGE analysis identified that DR_1146 was susceptible to stringent proteolysis. It is thought that initial crystallisation trials were unsuccessful due to inhomogeneity of the sample caused by reported degradation of the target protein.
Biophysical characterisation of DR_1146 by isothermal titration calorimetry (ITC) and fluorescence spectroscopy (FS) identified a moderate affinity of 4-11 μM for the flavin molecules, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Differential scanning calorimetry (DSC) and circular dichroism (CD) experiments demonstrated an increase in chemical and thermal stability of the protein on binding to the flavin molecule, FMN.
Analytical ultracentrifugation (AUC) and Nuclear magnetic resonance (NMR) spectroscopy were employed to investigate the solution behaviour of DR_1146 in the presence of FMN. AUC results uncovered a monomer-dimer equilibrium; with DR_1146 self-associating to form a dimer at a concentration of 7.67 μM. NMR spectroscopy depicted that global changes occur within the structure of DR_1146 on binding to FMN. The high quality of spectra obtained showed potential for 3-D structure determination by NMR if ordered crystals could not be obtained for X-ray diffraction. Interestingly, analysis of NMR spectra proved to be integral to identifying a homogenous sample for successful crystallisation of DR_1146. By monitoring chemical shifts it was possible to determine the time needed for degradation of DR_1146 to cease, and the amount of FMN needed to ensure saturation of binding sites. From this particular sample, a stable 28 kDa fragment was isolated by gel filtration. Automated sitting-drop vapour-diffusion experiments resulted in the growth of yellow DR_1146-FMN crystals for which, although poor in quality, X-ray diffraction was obtained. Overall this study reflects the importance and advantage of incorporating information gained from biophysical characterisation into the strategies employed for successful protein crystallisation.
The characterisation of DR_1146 as a flavoprotein points towards a possible role in electron transfer due to the extensive redox capacity of flavin. This could implicate the protein in the production of damaging reactive oxygen species (ROS) as a result of irradiation, contributing to oxidative stress levels. Alternatively, if DR_1146 is identified as a FMN-binding pyridoxine 5'-phosphate oxidase (PNPOx) enzyme, as sequence homology suggests, it could play a role in detoxification and stress response through production of pyridoxal 5'-phosphate (PLP), a known scavenger of ROS. Only further characterisation and elucidation of a 3-D structure would confirm or dispel these functional hypotheses and ultimately provide a greater understanding of how D. radiodurans is able to deal with such oxidising conditions.
Simultaneously, experiments were carried out on other soluble and membrane protein targets from D. radiodurans and their corresponding homologues from Streptococcus pneumoniae (TIGR4). The aim of comparable studies was to identify key structural or functional differences between the two Gram-positive bacterial strains. Identification of features unique to D. radiodurans, but unconserved in S. pneumoniae, could contribute to further understanding of bacterial radioresistance.
SP_1651 is a thiol peroxidase which forms part of the Mn-ABC transport system in S. pneumonia. Its homologue from D. radiodurans, DR_2242 is a putative thiol-specific antioxidant protein, the structure of which has been solved by Dr. Dave Hall as part of the ESRF’s structural genomics project (unpublished). The aim of this part of the project was to elucidate the structure of SP_1651 so that a comparison with DR_2242 could be made.
The sp_1651 gene (psaD) was successfully expressed and purified to homogeneity by IMAC and gel filtration. After the proteolytic removal of a 6xHis tag, the purified protein was crystallised by sitting-drop vapour-diffusion. Preliminary diffraction with a resolution limit of 3.2 Å was obtained, however data showed high mosaic spread. Unfortunately, attempts to reproduce initial crystals failed and hence, structural comparisons with DR_2242 could not be made.
DR_0463 is a 108 kDa maltooligosyltrehalose synthase (MTSase) which has been shown to catalyse the breakdown of maltooligosaccharide (or starch) into the disaccharide, trehalose. The full length gene was expressed in BL21(DE3)pLysS cells, producing large yields of insoluble target protein. DR_0463 was solubilised with 8 M Urea and then purified by IMAC in the presence of the denaturant. The low affinity of DR_0463 for the Ni2+ matrix of the HisTrap column proved to be problematic when trying to obtain homogeneity. However, by sequentially repeating IMAC purification up to three times with the same protein sample, a large proportion of impurities were removed.
SP_1648 (PsaB) is an ATP-binding protein that forms part of the Mn-ATP transport system in S. pneumoniae and its homologue from D. radiodurans, DR_2284 is predicted to share similar function. Purification of soluble SP_1648, expressed in B834(DE3) cells, was complicated by an inability to bind the protein to the column matrix for IMAC. In the case of DR_2284, expression trials yielded only a minute amount of insoluble protein in BL21-AI competent cells. The bottlenecks in early expression and purification stages provided valuable experience in dealing with problematic proteins.
As an introduction to molecular cloning, two genes predicted to encode integral membrane proteins from D. radiodurans, were cloned for preliminary expression trials. This work was carried out at the ESRF and contributed to an extension of the structural genomics project, to incorporate membrane protein targets from D. radiodurans. Full length forms of the genes thought to encode an undecaprenyl diphosphatase (UDP) and a diacylglycerol kinase (DGKA) were successfully cloned in to pET-28b, with incorporation of separate N- and C- terminal 6xHis tags.
Actions (login required)