Monolayer crystallization of membrane proteins. Membrane proteins comprise 25-40% of all proteins and conduct a myriad of finely tuned reactions in every cell. Despite their importance and diversity only ~40 membrane protein structures have been solved, due to the difficulty of producing high quality 2D and 3D crystals. We propose to develop and use the new monolayer crystallization technique, which employs a lipid monolayer as a crystallization template for 2D crystal production. A number of ....Monolayer crystallization of membrane proteins. Membrane proteins comprise 25-40% of all proteins and conduct a myriad of finely tuned reactions in every cell. Despite their importance and diversity only ~40 membrane protein structures have been solved, due to the difficulty of producing high quality 2D and 3D crystals. We propose to develop and use the new monolayer crystallization technique, which employs a lipid monolayer as a crystallization template for 2D crystal production. A number of important membrane proteins are available for these structural studies including ABC transporters, Caveolin-3 and the NS1 protein of Dengue virus, all of which are difficult to crystallize using conventional techniques.Read moreRead less
Function and regulation of the Na+,K+-ATPase. The Na+,K+-ATPase is the major energy-consuming enzyme of animal cells. Its ion pumping is essential for numerous physiological functions (e.g. heart, kidney, brain). Molecular detail of its pumping mechanism is, however, lacking and its regulation is still unclear. We will use rapid reaction methods on purified enzyme in vitro to locate the rate-determining step of the enzyme cycle, determine its mechanism, investigate its regulation by sodium conce ....Function and regulation of the Na+,K+-ATPase. The Na+,K+-ATPase is the major energy-consuming enzyme of animal cells. Its ion pumping is essential for numerous physiological functions (e.g. heart, kidney, brain). Molecular detail of its pumping mechanism is, however, lacking and its regulation is still unclear. We will use rapid reaction methods on purified enzyme in vitro to locate the rate-determining step of the enzyme cycle, determine its mechanism, investigate its regulation by sodium concentration, phosphorylation and membrane composition, and isolate its charge-transporting steps. The results will have immediate impact on the understanding of the enzyme's mechanism, its metabolic control and its role in disease.Read moreRead less
Three-dimensional structures, substrate specificities and catalytic mechanisms of polysaccharide synthases. Plant wall polysaccharides are extensively used for food and fibre, and are important dietary components in human nutrition. A precise knowledge of mechanisms used by plants to synthesize these polysaccharides is unavailable, but would reveal potential routes to manipulate their biosynthesis in important crop species. For example, the levels or structures of polysaccharides might be modif ....Three-dimensional structures, substrate specificities and catalytic mechanisms of polysaccharide synthases. Plant wall polysaccharides are extensively used for food and fibre, and are important dietary components in human nutrition. A precise knowledge of mechanisms used by plants to synthesize these polysaccharides is unavailable, but would reveal potential routes to manipulate their biosynthesis in important crop species. For example, the levels or structures of polysaccharides might be modified to improve their efficacy as anti-cancer agents in human diets, to enhance digestibility of animal stock feeds, or to synthesise pharmologically valuable compounds. Thus, economic, social and environmental benefits will flow to both producers and consumers. Read moreRead less
Role of 3'-phosphorylated phosphoinositides in neurosecretion. Neurons communicate through the release of neurotransmitter by synaptic vesicles. Minute changes underlie normal processes such as memory and modifications of neurotransmitter level contribute to a number of neurological diseases. I am interested in deciphering the role of phosphoinositides, an inner membrane-based lipid, during steps leading to the fusion of a synaptic vesicle with the plasma membrane. I have recently discovered tha ....Role of 3'-phosphorylated phosphoinositides in neurosecretion. Neurons communicate through the release of neurotransmitter by synaptic vesicles. Minute changes underlie normal processes such as memory and modifications of neurotransmitter level contribute to a number of neurological diseases. I am interested in deciphering the role of phosphoinositides, an inner membrane-based lipid, during steps leading to the fusion of a synaptic vesicle with the plasma membrane. I have recently discovered that phosphatidylinositol-3 phosphate production was critical for the vesicle to acquire the competence to fuse with the plasma membrane. This project aim to understand by which mechanism this lipid interacts with the release machinery to promote such priming step.Read moreRead less
How cholesterol optimises ion pump function in animal membranes. This project aims to determine how cholesterol optimises ion pump function in animal membranes and to identify the major effects of cholesterol and its derivatives on membranes’ physical properties. All animal cells need high levels of cholesterol in the plasma membrane for survival. Insufficient cholesterol biosynthesis leads to severe birth defects. The need for cholesterol is likely linked to its acceleration of sodium pump acti ....How cholesterol optimises ion pump function in animal membranes. This project aims to determine how cholesterol optimises ion pump function in animal membranes and to identify the major effects of cholesterol and its derivatives on membranes’ physical properties. All animal cells need high levels of cholesterol in the plasma membrane for survival. Insufficient cholesterol biosynthesis leads to severe birth defects. The need for cholesterol is likely linked to its acceleration of sodium pump activity, essential to physiological processes including cell division, nerve, muscle and kidney activity. An expected benefit of the project is knowledge on the molecular origin of diseases associated with inhibition of cholesterol production, and a more complete understanding of the crucial role played by cholesterol via its effect on ion pumping towards the healthy functioning of vital organs, particularly in heart muscle and nerves.Read moreRead less
Lipid-protein interplay in the mechanism of the sodium pump. The sodium pump is the major energy-consuming enzyme of animal cells. Its ion pumping is essential to numerous physiological processes (e.g. nerve, muscle and kidney activity and the maintenance of cell volume). Because of its importance in so many cell functions, the enzyme must be able to respond to cellular conditions. Using measurements of the enzyme's activity in isolated membrane fragments and comparison with its behaviour in liv ....Lipid-protein interplay in the mechanism of the sodium pump. The sodium pump is the major energy-consuming enzyme of animal cells. Its ion pumping is essential to numerous physiological processes (e.g. nerve, muscle and kidney activity and the maintenance of cell volume). Because of its importance in so many cell functions, the enzyme must be able to respond to cellular conditions. Using measurements of the enzyme's activity in isolated membrane fragments and comparison with its behaviour in living cells, this project aims to determine how sodium pump activity is modulated by transmembrane electric potential and intramembrane electric field strength. Our project could provide fundamental new knowledge on how membrane protein function in general can be controlled by electrical properties of their lipid surroundings.Read moreRead less
Mammalian histidine kinase: its characterisation and role in hepatic cellular proliferation. Protein kinases regulate all aspects of cellular metabolism, but there is a class of protein kinase, histidine kinases, which exists in mammalian cells, about which remarkably little is understood. This project will be the first detailed characterisation of such an enzyme and investigation of its cellular function. The enzyme to be characterised is hepatic and it has been implicated in the regulation of ....Mammalian histidine kinase: its characterisation and role in hepatic cellular proliferation. Protein kinases regulate all aspects of cellular metabolism, but there is a class of protein kinase, histidine kinases, which exists in mammalian cells, about which remarkably little is understood. This project will be the first detailed characterisation of such an enzyme and investigation of its cellular function. The enzyme to be characterised is hepatic and it has been implicated in the regulation of DNA replication in regenerating liver. We shall obtain a detailed picture of the role of this histidine kinase in liver cell biology and an insight into the more general biological role of this class of enzyme.Read moreRead less
A global approach to structure, assembly and function of yeast mitochondrial ATP synthase. ATP synthase is responsible for the synthesis of the ubiquitous energy substance ATP in living cells. This multisubunit enzyme complex is a molecular rotatory motor whose mechanism relies on functional dynamic interactions of the component protein subunits of the complex. Using a combination of powerful and sophisticated molecular biology techniques the functional and dynamic contacts made by subunits wi ....A global approach to structure, assembly and function of yeast mitochondrial ATP synthase. ATP synthase is responsible for the synthesis of the ubiquitous energy substance ATP in living cells. This multisubunit enzyme complex is a molecular rotatory motor whose mechanism relies on functional dynamic interactions of the component protein subunits of the complex. Using a combination of powerful and sophisticated molecular biology techniques the functional and dynamic contacts made by subunits within the complex, or between ATP synthase and other enzyme complex involved in energy production (including other ATP synthase complexes) will be investigated. New and significant insights into one of the most fundamental and challenging enzymes of life will be obtained.Read moreRead less
The Structure Function and Biology of Serpins. The serpin family of proteins control protease activity, in a variety of biological systems, via a dramatic conformational change. We are 4 leaders in the serpin reseacrh field using an integrated approach of biochemistry, biophysics, molecular biology, cell biology, structural biology and bioinformatics to analyse every facet of serpin structure, function and biology. We will apply and develop novel technologies that will determine how these confor ....The Structure Function and Biology of Serpins. The serpin family of proteins control protease activity, in a variety of biological systems, via a dramatic conformational change. We are 4 leaders in the serpin reseacrh field using an integrated approach of biochemistry, biophysics, molecular biology, cell biology, structural biology and bioinformatics to analyse every facet of serpin structure, function and biology. We will apply and develop novel technologies that will determine how these conformational changes are achieved and how they are controlled. Our increased understanding of this inhibitor - enzyme interaction will have implications to other proteins which undergo conformational change and biotechnology.Read moreRead less
Structural and functional studies on prokaryote serpins. Proteins are the machines of life, many of which undergo complex movements to achieve function. The basic research described in this proposal will result in major outcomes in understanding how proteins fold, undergo complex changes in conformation, and misfold to dysfunctional states. As a result, the study will contribute fundamental knowledge that will underpin research in the fields of structural biology, protein design and protein fo ....Structural and functional studies on prokaryote serpins. Proteins are the machines of life, many of which undergo complex movements to achieve function. The basic research described in this proposal will result in major outcomes in understanding how proteins fold, undergo complex changes in conformation, and misfold to dysfunctional states. As a result, the study will contribute fundamental knowledge that will underpin research in the fields of structural biology, protein design and protein folding. Through providing insight into protein misfolding, the work will will contribute to our understanding of degenerative misfolding pathologies that affect the ageing population and thus targets the 'ageing well ageing productively' priority goal.Read moreRead less