Research
My
research interests lie
in the general area of molecular biological physics. The main focus of
my present research is to explore the interplay between
protein
structural transitions and functions. The three-dimensional structure
of a folded protein often gives molecular level insight to its
biological function: structure determines function. At the same time,
it is also clear that the key to understand a protein's function often
lies in its dynamics and
conformational flexibility. Complex conformational transitions,
induced by
ligand binding
for example, are often essential to proteins
participating in regulatory networks or enzyme catalysis. More
generally, a protein's ability to sample a variety of conformational
sub-states implies that proteins have an intrinsic flexibility
and mobility that influences their function. While experimental
measurement can offer direct dynamical information about specific
residues, uncovering the detailed mechanisms controlling conformational
transitions between two meta-stable states are often elusive.
More specifically, experimental studies using nuclear magnetic
resonance (NMR) and X-ray provide only a few local structural
changes of protein and have not been able to capture the molecular
details necessary to fully understand the mechanism of conformational
transitions. Whereas atomistic simulations can potentially
bridge the gap on time scale up to microsecond, this time scale
falls orders of magnitude short for slow protein dynamics
(millisecond
to second).
Also, the use of atomistic approaches
becomes
computationally
inefficient with the increased size of a system. To overcome
the problems associated with all-atom simulations, many studies
have demonstrated the use of low-resolution (coarse-grained) protein
models with simplified representations, such as, only alpha-carbons
as point masses and simplified energy functions. Such models
require much less computational cost, making them practical to describe
the conformational transitions of even large proteins.
Conformational Flexibility and Functional Transitions
To study the protein conformational transitions, we have developed a coarse-grained analytical model. Our approach is inspired by a variational model previously developed to characterize protein folding [PRL, 81, 5237, 1998]. The present model accommodates two meta-stable folded conformations as minima of the calculated free energy surface. The natural order parameters of this model are very well suited to describe partially ordered ensembles essential to the conformational dynamics of flexible protein like calmodulin (CaM) and may be an ideal model system to illustrate how conformational flexibility and mobility are a key determinant of its diverse biological functions. Upon binding calcium, each domain of CaM undergoes a ``closed'' (apo) to ``open'' (holo) conformational transition that exposes a large hydrophobic surface responsible for molecular recognition of various cellular targets such as myosin light chain kinase. Our studies revealed that the inherent flexibility play a crucial role in the apo/holo conformational transition mechanism of the CaM domains. Results from our studies are in very good qualitative agreement with NMR and X-ray studies for our estimated conformational flexibility of the CaM domains. In addition, from our studies predicted apo/holo transition mechanism of each residue can be compared with the site-directed mutagenesis experiments in future.
Interplay among Topology, Plasticity and Stability
The other related issues which we recently investigated are the interplay between topology, structural plasticity and energetics of the proteins from their functional transitions. CaM domains may be a very good system for this, since, the two CaM domains are topologically similar and homologous. However, the two domains also exhibits different conformational flexibility, binding affinity and melting temperatures. Interestingly, from our study we found that the closed to open conformational transition mechanism of the CaM domains are distinct predominantly due to ``cracking" or partial local unfolding and refolding in the C-terminal CaM domain (cCaM). This result is also in harmony with other experimental and simulation studies of the CaM domains. To analyze cracking in more detail we calculated strain energy for each residue of the CaM domains using the elastic network model (ENM) approach. Curiously, the calculated strain energy agrees very well with the change in flexibility of the corresponding CaM domains along the closed to open conformational change. The above findings further confirm that functional transitions in proteins are predetermined due to their intrinsic ``plasticity" which is encoded in the amino acid sequence network (contact map). Currently, we are focusing on exploring the complex inter-relationship between stability and conformational flexibility of the proteins. For this purpose other than the CaM domains, we are also investigating the large-scale structural change in several other proteins. Specifically, we are changing the conformational transition temperatures of these proteins to see how it may affect the flexibility and stability of each residue in the proteins mainly by characterizing the ensemble of intermediate conformational states. Since, a proper balance between stability and flexibility is essential for the protein-ligand binding mechanism.
Protein-Ligand Binding and `Fly-Casting' Mechanism
We are also extending our conformational transition model to study protein-ligand binding. Our initial attempt is to explore protein-ligand interactions for metal ions and small drug molecules. We are mainly focusing to understand the influence of conformational flexibility on structural transition kinetics, binding affinity, and the kinetics of binding. Perhaps the most extreme example of conformational change due to interactions with other molecules is folding upon binding or the `fly-casting' mechanism, when folding and binding are concomitant. Here, the protein molecule is largely unfolded until it interacts with its binding partner. The molecular surface that induces folding can be another folded protein, DNA, or a fluid membrane. In near future we will be interested in deeper understanding of the kinetic and thermodynamic advantages associated with rapidly growing list of natively unfolded proteins.
Conformational Flexibility and Functional Transitions
To study the protein conformational transitions, we have developed a coarse-grained analytical model. Our approach is inspired by a variational model previously developed to characterize protein folding [PRL, 81, 5237, 1998]. The present model accommodates two meta-stable folded conformations as minima of the calculated free energy surface. The natural order parameters of this model are very well suited to describe partially ordered ensembles essential to the conformational dynamics of flexible protein like calmodulin (CaM) and may be an ideal model system to illustrate how conformational flexibility and mobility are a key determinant of its diverse biological functions. Upon binding calcium, each domain of CaM undergoes a ``closed'' (apo) to ``open'' (holo) conformational transition that exposes a large hydrophobic surface responsible for molecular recognition of various cellular targets such as myosin light chain kinase. Our studies revealed that the inherent flexibility play a crucial role in the apo/holo conformational transition mechanism of the CaM domains. Results from our studies are in very good qualitative agreement with NMR and X-ray studies for our estimated conformational flexibility of the CaM domains. In addition, from our studies predicted apo/holo transition mechanism of each residue can be compared with the site-directed mutagenesis experiments in future.
Interplay among Topology, Plasticity and Stability
The other related issues which we recently investigated are the interplay between topology, structural plasticity and energetics of the proteins from their functional transitions. CaM domains may be a very good system for this, since, the two CaM domains are topologically similar and homologous. However, the two domains also exhibits different conformational flexibility, binding affinity and melting temperatures. Interestingly, from our study we found that the closed to open conformational transition mechanism of the CaM domains are distinct predominantly due to ``cracking" or partial local unfolding and refolding in the C-terminal CaM domain (cCaM). This result is also in harmony with other experimental and simulation studies of the CaM domains. To analyze cracking in more detail we calculated strain energy for each residue of the CaM domains using the elastic network model (ENM) approach. Curiously, the calculated strain energy agrees very well with the change in flexibility of the corresponding CaM domains along the closed to open conformational change. The above findings further confirm that functional transitions in proteins are predetermined due to their intrinsic ``plasticity" which is encoded in the amino acid sequence network (contact map). Currently, we are focusing on exploring the complex inter-relationship between stability and conformational flexibility of the proteins. For this purpose other than the CaM domains, we are also investigating the large-scale structural change in several other proteins. Specifically, we are changing the conformational transition temperatures of these proteins to see how it may affect the flexibility and stability of each residue in the proteins mainly by characterizing the ensemble of intermediate conformational states. Since, a proper balance between stability and flexibility is essential for the protein-ligand binding mechanism.
Protein-Ligand Binding and `Fly-Casting' Mechanism
We are also extending our conformational transition model to study protein-ligand binding. Our initial attempt is to explore protein-ligand interactions for metal ions and small drug molecules. We are mainly focusing to understand the influence of conformational flexibility on structural transition kinetics, binding affinity, and the kinetics of binding. Perhaps the most extreme example of conformational change due to interactions with other molecules is folding upon binding or the `fly-casting' mechanism, when folding and binding are concomitant. Here, the protein molecule is largely unfolded until it interacts with its binding partner. The molecular surface that induces folding can be another folded protein, DNA, or a fluid membrane. In near future we will be interested in deeper understanding of the kinetic and thermodynamic advantages associated with rapidly growing list of natively unfolded proteins.