{"id":{"repo_id":"waikato-masters","oai_identifier":"oai:researchcommons.waikato.ac.nz:10289/3519"},"canonical_url":"https://search.dev.ndltd.org/etd/waikato-masters/oai:researchcommons.waikato.ac.nz:10289/3519","repository":{"repo_id":"waikato-masters","name":"University Waikato","base_url":"https://researchcommons.waikato.ac.nz/server/oai/request"},"display":{"title":"Protein Dynamics and its Correlation to Protein Activity and Stability","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Clement, David"],"institution":"The University of Waikato","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2009,"date_issued":"2009","date_published":"2009","updated_at":"2026-07-24T05:57:30Z","subjects":["Incoherent neutron scattering","circular dichroism","isothermal titration calorimetry","steady state kinetics","dihydrofolate reductase","methotrexate"],"languages":[],"rights":["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."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":null,"outbound_label":null,"outbound_source":null},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Clement, David"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2009"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["The University of Waikato"]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://hdl.handle.net/10289/3519"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Incoherent neutron scattering","circular dichroism","isothermal titration calorimetry","steady state kinetics","dihydrofolate reductase","methotrexate"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["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."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://researchcommons.waikato.ac.nz/bitstreams/452d2017-10ff-4614-9d4d-b12e2801790e/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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. 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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."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["7c9416b40c969392ccf79c4b4dc6e8f8","45a9c5de19ef0d3018862ecfe554f4c5"]},{"key":"dc:title","label":"Title","values":["Protein Dynamics and its Correlation to Protein Activity and Stability"]}]}],"canonical_facts":{"dc:creator":["Clement, David"],"dc:date.issued":["2009"],"dc:description.abstract":["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. 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