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.
