Proteins are flexible and dynamic molecules, which serve crucial
functions in essentially all biological events in living cells. An important
example is allostery, the coupling between ligand binding and protein
conformational change. The primary focus of the research in this dissertation
is to elucidate the detailed mechanism of large scale (main-chain) structural
changes of specific proteins where conformational flexibility is essential for
function.
The functional states of proteins can be viewed as a minimum in the free
energy landscape. Conformational exchanges between structures within this
native (folded) minimum occur with rates controlled by the height of the
energy barrier between them. The distribution of the conformational
substates is highly complex and the dynamics of transitions between
these substates are generally controlled by relatively low probability
conformational ensembles. The main challenge is to describe the transition
state ensembles at the residue level, giving site specific description of
the transition mechanism.
To address this important issue I developed an analytical model that
accommodates the free energy minima relevant to transition between two
particular well-folded conformations. The free energy surface of the
protein is approximated using a reference Hamiltonian that corresponds
to a polymer in a non-uniform external field that harmonically constrains
the fluctuations of the monomers to average positions, uniformly
interpolating between two meta-stable native structures. The free energy
surfaces are parameterized by conformational flexibility of each residue.
Transition routes and the site-resolved structure of the transition state
ensembles are determined by constrained minima of the variational free
energy surface. I mainly focus on two separate proteins with flexibility
determined allosteric transitions to illustrate the model: Calmodulin
(CaM) and the N-terminal receiver domain of nitrogen regulatory protein
C (NtrC).
CaM is a flexible protein and plays an essential role in calcium-mediated
eukaryotic cellular signaling. This signal transduction is accomplished primarily through a calcium-induced open/closed conformational change of the CaM
domains. I investigate this conformational change of the two domains of CaM independently. Our study illustrates that inherent flexibility is the key determinant
of the transition mechanism of the two domains. In particular, our
results reveal that C-terminal domain of CaM which is inherently less
flexible than its homologous and structurally similar N-terminal
domain unfolds partially and refolds during the transition. These
findings are also in harmony with molecular dynamics simulations, as
well as nuclear magnetic resonance (NMR) measurements characterizing
the slow conformational dynamics of the CaM domains. Furthermore,
these observations might have some significance on the diverse
functions of CaM.
NtrC of enteric bacteria is a response regulator and plays a central
role in the control of genes involved in nitrogen metabolism.
Phosphorylation (activation) of the inactive NtrC, results
in large structural changes. NMR studies suggested that allostery in
this protein occurs by shifting the preexisting population from the
inactive to active state upon phosphorylation. From the folding study
of NtrC, I explore that different folding mechanisms of
the two states are mainly due to the stabilization of the active
conformation upon phosphorylation.
I also investigate the mechanism of phosphorylation induced inactive/active
conformational change of NtrC. Our results show significant
decrease in the flexibility of this protein upon activation due to a large
entropic contribution in consistent with the NMR experiments.