Browsing by Author "Clark, Alice Rosemary"
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- ItemThe filamin A actin binding domain structure and function: implications for a gain-of-function mechanism for the otopalatodigital syndrome: a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 2010) Clark, Alice RosemaryThe filamin family act as scaffolding proteins associating with actin filmanents, acting through a highly conserved actin binding domain (ABD). The ABD of the filamins is homologous to that found in other F-actin binding proteins such as dystrophin. Mutations in the filamin A gene cause a wide range of disease symptoms in humans reflecting the diversity of the roles that filamin A has in cell structure and signalling pathways. The diseases fall into two separate phenotypic groups. Periventricular nodular heterotopia (PVNH) generally results from the complete loss of filamin A protein, and affects the central nervous system. The clinically separate otopalatodigital disorders (OPD) spectrum disorders are skeletal disorders and were hypothesised to be gain of function phenotype diseases. At the beginning of this work, there was very little structural data available for the human filamins, and none for the crucial highly conserved actin binding domain. This lack of structural data limited the interpretation of the biochemical and genetic data and constrained our understanding of the disease associated mutations that cluster in this domain. These studies aimed to provide insights into the structure and mechanism of actin binding domains, and thus provide a better understanding of the diseases caused when this domain is mutated. A secondary structural analysis and crystal structures of the wildtype and OPD2 associated mutant ABDs were obtained. The overall fold of the three proteins was equivalent as determined by circular dichroism spectroscopy and x-ray crystallography. The ABD from filamin A E254K showed 3.7 fold increased F-actin affinity, accompanied by a reduced thermostability (of 5.6 °C). Western blotting of OPD2, frontometaphyseal dysplasia (FMD) and PVNH patient fibroblast lysates showed similar levels of filamin A compared to the control cells. In addition the OPD and PVNH patient fibroblasts were able to adhere to fibronectin and migrate with an equivalent rate to control cells. Together these results have allowed correlations to be developed between structure, protein stability, actin affinity, cellular phenotype and the overall clinical phenotype. Showing that, at least in one example, OPD2 may be due to an increased actin affinity providing further evidence for a gain of function mechanism of OPD2.
- ItemInvestigation of a novel intein-based Escherichia coli expression system for human methylmalonyl CoA mutase : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry(Massey University, 2005) Clark, Alice RosemaryHuman methylmalonyl CoA mutase (hMCM) is a 78 kDa homodimeric mitochondrial matrix enzyme. hMCM catalyses the conversion of 2R-methylmalonyl CoA to succinyl CoA in the metabolism of propionyl groups, and requires the vitamin B12 -derived cofactor adenosylcobalamin (AdoCbl). The mechanism of catalysis involves homolytic cleavage of AdoCbl's unusual C-Co bond, to generate radicals. Dysfunctional hMCM results in the rare, potentially fatal metabolic disorder methylmalonic acidemia. An experimentally determined structure of hMCM would add to the understanding of both the mechanism of catalysis and the molecular basis of some of the mutations underlying methylmalonic acidemia. The structure of the bacterial orthologue from Propionibacterium shermanii has been solved by x-ray crystallography, enabling the development of structural models of hMCM. Critical differences, however, between these two enzymes, mean that some regions of the models could be inaccurate. There is no x-ray crystal structure of hMCM. Purification of native hMCM for crystallization trials is complicated by ethical problems, low yields, and heterogeneity generated by the cofactor. To provide a more convenient source of pure, active human methylmalonyl CoA mutase for x-ray crystallography, an expression system for recombinant hMCM is required. Other researchers have expressed hMCM in Escherichia coli as (i) insoluble inclusion bodies, (ii) soluble fusion protein that cannot be separated efficiently from the fusion tag, or (iii) in low quantities. This research aimed to develop an E. coli expression system for the production of active human methylmalonyl CoA mutase, to enable x-ray crystallography structural studies. Based on the results of previous expression systems, four novel expression vectors were developed utilising the maltose binding protein and thioredoxin as solubility tags. It was hoped that conventional protease cleavage, to remove these solubility tags, could be circumvented by the use of intein-mediated cleavage. Intein-mediated cleavage was successful, and soluble active hMCM was recovered in low yields from a C-terminal thioredoxin solubility tag construct. hMCM was insoluble when expressed with MBP at the C-terminus.