Over 100 years ago, X-ray crystallography gave scientists a window to theatomic world with varied applications in biology, chemistry and physics amongother subjects. Macromolecular crystallography is now considered an essentialtool for solving the three-dimensional structure of proteins and understandingtheir physiological role at the atomic level. As crystal growth remains abottleneck in crystallography, various other techniques are often employed tohelp understand the protein structure and function. These methods range fromsimple analysis of the protein sequence to experiments such as dynamic lightscattering, isothermal titration calorimetry, activity assays, and analyticalultracentrifugation. The additional knowledge gained about proteins from thesemethods can then assist in the modification of the protein to facilitate itscrystallisation. The structural biology of proteins belonging to two diversefamilies; Galectins and Sirtuins, both involved in the regulation of cancer, wasstudied in this thesis.Galectins are evolutionarily conserved and ubiquitously present animal lectinswith a high affinity for -galactose containing oligosaccharides. To date, 15mammalian galectins have been identified. Their involvement in cell–cell andcell–matrix interactions has highlighted their importance in signal transductionand other intracellular processes. Human Galectin-7 (hGal-7) is a 16 kDaprototype galectin which is involved in the stimulation and development ofcancer. The crystal structure of native hGal-7 and its complex with galactoseand lactose have been reported. In this study, cross-linking of hGal-7 byglycodendrons and the resulting clustering and lattice formation have beenstudied. For this purpose, the high resolution X-ray structures of hGal-7 incomplex with carbohydrate-based multivalent dendrons have been elucidatedand analysed. Also discussed in this thesis are preliminary binding affinityresults obtained using isothermal calorimetry. Supramolecular assemblyformation was also assessed using dynamic light scattering. These experimentsreveal how multivalent glycodendendrons interact with and form cross-linkswith hGal-7 molecules. Understanding how these dendrimeric compounds interact with hGal-7 would help in the design of new tools to investigate therecognition of multivalent carbohydrates by lectins and their resulting role inaggregation processes in tumour embolisation and survival.Sirtuins are NAD+-dependent deacylases that are involved in the regulation ofdiverse biological functions such as ageing, metabolism and stress resistance, innormal cellular physiology. Their role in ageing and ageing-related diseases,including cancer and neurodegenerative diseases among others, has receivedmuch attention and sirtuins have been extensively studied to help in extensionof human lifespan. Seven members of the sirtuin family (SIRT1-7) are known,which are diversely sub-localised in the cell. A myriad of questions regardingtheir deacylation activity, their interplay and their role in various diseases stillremain unanswered. In this study, structural biology techniques have been usedto 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 theproteins are also discussed. A brief discussion of the methods that can beemployed to overcome various barriers in structural biology is also presented inthis thesis. Elucidating the structure of full length sirtuins would help in thedevelopment of highly selective modulators of sirtuins to aid in theunderstanding of their role in ageing and ageing-related diseases.
|Date of Award||9 Mar 2015|
|Supervisor||Ravi Acharya (Supervisor)|