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oai:researchcommons.waikato.ac.nz:10289/35192017-12-08T00:47:40Zcom_10289_2222col_10289_2223

Protein Dynamics and its Correlation to Protein Activity and Stability Clement, David The aim of this thesis is to investigate the role of fast protein dynamics (picosecond timescale) in enzyme activity and stability, and specifically to test the hypothesis that enzyme activity and stability are inversely related by their internal dynamics. Activity Dynamics (flexibility) 1/Stability In order to test this hypothesis, the well known anti-cancer drug: methotrexate was used as an informative ligand in the network established between these properties. A multidisciplinary approach combining neutron scattering, circular dichroism, UV absorption, isothermal titration calorimetry and X-ray crystallography was undertaken to examine the current paradigm using the enzyme: dihydrofolate reductase as a model. As inferred by neutron spectroscopy, the binding of MTX influences the dynamical behavior of DHFR. Macromolecular dynamics such as the resilience: lt;kgt; (i.e. structural rigidity) was found to be increased and, inversely, the flexibility decreased upon MTX binding. In addition, as revealed by circular dichroism, this dynamical dependency upon MTX binding was correlated with an enhanced thermal stability. Compared to the free enzyme, the melting temperature was found to be increased by 13.8 C in the presence of MTX. The inhibitory power of MTX was also examined by steady state kinetics and isothermal titration calorimetry. The Ki for MTX was found to be in the nanomolar range Ki= 10.9 nM. Using isothermal titration calorimetry, the binding thermodynamic signature between MTX and DHFR was characterized. The binding event was found to be largely favourable (DGb=-12.1 Kcal mol-1), enthalpy driven (DHb= -16.8 Kcal mol-1) with an unfavourable entropy DSb=-15.6 cal K-1mol-1. In conclusion, the modulation of the macromolecular dynamics may reflect how specific conformations are favoured for subsequent protein function in response of the binding of specific ligand and how conformational substates approach to protein function. In this context the unprecedented power of transition state analogs such as MTX on protein function might therefore be dependent on fast protein dynamics. The University of Waikato 2009 Thesis en https://hdl.handle.net/10289/3519 https://researchcommons.waikato.ac.nz/bitstreams/452d2017-10ff-4614-9d4d-b12e2801790e/download 7c9416b40c969392ccf79c4b4dc6e8f8 https://researchcommons.waikato.ac.nz/bitstreams/154c9bee-b554-4c67-9418-d4654509f8df/download 45a9c5de19ef0d3018862ecfe554f4c5 All items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated. Incoherent neutron scattering circular dichroism isothermal titration calorimetry steady state kinetics dihydrofolate reductase methotrexate