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      Protein Dynamics and its Correlation to Protein Activity and Stability

      Clement, David
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      Clement, D. (2009). Protein Dynamics and its Correlation to Protein Activity and Stability (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/3519
      Permanent Research Commons link: https://hdl.handle.net/10289/3519
      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.
      Date
      2009
      Type
      Thesis
      Degree Name
      Doctor of Philosophy (PhD)
      Publisher
      The University of Waikato
      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.
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