Over 100 years ago, X-ray crystallography gave scientists a window to the atomic world with varied applications in biology, chemistry and physics among other subjects. Macromolecular crystallography is now considered an essential tool for solving the three-dimensional structure of proteins and understanding their physiological role at the atomic level. As crystal growth remains a bottleneck in crystallography, various other techniques are often employed to help understand the protein structure and function. These methods range from simple analysis of the protein sequence to experiments such as dynamic light scattering, isothermal titration calorimetry, activity assays, and analytical ultracentrifugation. The additional knowledge gained about proteins from these methods can then assist in the modification of the protein to facilitate its crystallisation. The structural biology of proteins belonging to two diverse families; Galectins and Sirtuins, both involved in the regulation of cancer, was studied in this thesis. Galectins are evolutionarily conserved and ubiquitously present animal lectins with a high affinity for -galactose containing oligosaccharides. To date, 15 mammalian galectins have been identified. Their involvement in cell–cell and cell–matrix interactions has highlighted their importance in signal transduction and other intracellular processes. Human Galectin-7 (hGal-7) is a 16 kDa prototype galectin which is involved in the stimulation and development of cancer. The crystal structure of native hGal-7 and its complex with galactose and lactose have been reported. In this study, cross-linking of hGal-7 by glycodendrons and the resulting clustering and lattice formation have been studied. For this purpose, the high resolution X-ray structures of hGal-7 in complex with carbohydrate-based multivalent dendrons have been elucidated and analysed. Also discussed in this thesis are preliminary binding affinity results obtained using isothermal calorimetry. Supramolecular assembly formation was also assessed using dynamic light scattering. These experiments reveal how multivalent glycodendendrons interact with and form cross-links with hGal-7 molecules. Understanding how these dendrimeric compounds interact with hGal-7 would help in the design of new tools to investigate the recognition of multivalent carbohydrates by lectins and their resulting role in aggregation processes in tumour embolisation and survival. Sirtuins are NAD+-dependent deacylases that are involved in the regulation of diverse biological functions such as ageing, metabolism and stress resistance, in normal cellular physiology. Their role in ageing and ageing-related diseases, including cancer and neurodegenerative diseases among others, has received much attention and sirtuins have been extensively studied to help in extension of human lifespan. Seven members of the sirtuin family (SIRT1-7) are known, which are diversely sub-localised in the cell. A myriad of questions regarding their deacylation activity, their interplay and their role in various diseases still remain unanswered. In this study, structural biology techniques have been used to understand the role of SIRT1, SIRT2 and SIRT7. The cloning, expression, purification and crystallisation of these sirtuins are presented in this thesis. Various supporting techniques used to confirm the identity and activity of the proteins are also discussed. A brief discussion of the methods that can be employed to overcome various barriers in structural biology is also presented in this thesis. Elucidating the structure of full length sirtuins would help in the development of highly selective modulators of sirtuins to aid in the understanding of their role in ageing and ageing-related diseases.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:669041 |
| Date | January 2015 |
| Creators | Ramaswamy, Sneha |
| Contributors | Acharya, K-Ravi |
| Publisher | University of Bath |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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