Date of Award

Winter 2020

Project Type


Program or Major

Chemical Engineering

Degree Name

Doctor of Philosophy

First Advisor

Harish Prof. Vashisth

Second Advisor

Nivedita Prof. Gupta

Third Advisor

Kang Prof. Wu


In this thesis, I have studied two classes of systems using molecular dynamics (MD) simulations as a primary approach. The first class of systems is responsible for transport of small molecules. Specically, I have investigated diffusion pathways of inhibitory gas CO within the enzyme FeFe-hydrogenase as well as water transport in articial water channels. The second class of systems are biological proteins and their interactions with ligands. I have focused on Regulators of G-protein Signaling (RGS) proteins and their binding to thiadiazolidinone (TDZD) based small-molecule inhibitors as well as phosphosdiesterase 6 (PDE6) binding with cyclic guanosine monophosphate (cGMP).

Specically, I have studied the thermodynamics of CO diffusion in the Clostridium pasteurianum CpI FeFe-hydrogenase, which is sensitive to the inhibitory gas CO that deactivates this enzyme. Given that the active site is deeply buried in the FeFe-hydrogenase structure, it is highly likely that mutagenesis of specic amino acids lining gas diffusion pathways can improve the tolerance of this enzyme to inhibitory gases. To achieve that goal, the first step is to have detailed knowledge on diffusion network of CO inside the protein matrix. Therefore, I used advanced sampling methods to reveal the three-dimensional diffusion network of CO. I discovered that one of the minima in the vicinity of the active site suggests a high affnity for CO. I proposed several potential candidate residues located in the vicinity of the free-energy barriers for disrupting the CO diffusion network and for providing guidance to future experimental studies. In addition, comparisons between the diffusion networks of two inhibitory gases CO and O2 suggested several residues of the mutations of which can simultaneously improve the tolerance of the enzyme to both inhibitory gases CO and O2.

I have also studied the conformational dynamics in an artificial peptide-appended pillar[5]arene (PAP) water channel with perturbations from the surrounding membrane and other channels. Specically, I have incorporated multiple PAP channels in a lipid membrane matrix or a block copolymer membrane matrix to probe the channel-channel and channel-membrane interactions. MD simulations showed clustering of PAP channels only in a lipid membrane matrix, while enhanced sampling simulations showed a thermodynamically favored dimeric state of PAP channels in both membrane matrices. I discovered that the free-energy barrier for the dissociation of dimerized channels was ~4 kcal/mol higher in the BCP membrane than in the lipid membrane. While the water permeability values of all PAP channels are at the same order of magnitude, the results suggested that the water permeability of PAP channels correlated with the flexibility of PAP channels: a higher flexibility leads to a lower permeability. Collectively, the channel-channel and channel-membrane interactions governed the structural and functional water transport characteristics of PAP channels.

Among second class of systems, I studied RGS proteins that bind to Gα subunits of G-proteins to terminate signaling by G-protein coupled receptors (GPCRs) by accelerating hydrolysis of GTP. The pathways in which RGS proteins participate are implicated in various diseases including cancer, cardiovascular diseases, and central nervous system disorders. The binding of RGS proteins to G-proteins can be terminated by inhibitors that allosterically bind to cysteine residues in RGS proteins and inhibit the RGS/Gα interaction. TDZD inhibitors are potential drugs that covalently bind to cysteine residues in RGS proteins. However, TDZD inhibitors have shown different potencies and selectivities toward different RGS proteins. Protein dynamics is an important approach to explain the similarities and differences in behavior toward allosteric inhibition originating at a conserved cysteine in RGS proteins (Cys95 at RGS4). To probe this, I studied the dynamics in five homologous proteins of the RGS family (RGS4, RGS8, RGS9, RGS17, and RGS19) using MD simulations which revealed differences in structural dynamics, allosteric communication and pathways, and salt-bridging interactions. This study probed the allosteric pathways originating at the conserved cysteine residue.

Besides the conserved cysteine residue, RGS proteins contain other cysteine residues that can be targeted with covalent inhibitors. It is expected that targeting one of cysteine residues may lead to different allosteric perturbations in the RGS/Gα interface. Therefore, dissecting the role of each cysteine is of importance to evaluate differences in perturbations. In addition, inhibitor binding to one cysteine can perturb other unbound cysteines such as increasing their exposure window. Therefore, I have further investigated RGS8 protein with two cysteine residues that were chosen as a model system to dissect the role of individual cysteines when different TDZD inhibitors are bound. These studies revealed different roles for each cysteine, their synergistic inhibitory effect, and the effect of different TDZD inhibitorson perturbations in the protein-protein interface.

In final set of studies reported here, I have employed MD simulations to probe the conformational dynamics of cone PDE6 GAFab domain, which plays a critical role in phototransduction process, with and without the ligand cyclic guanosine monophosphate (cGMP). A stable crystal structure of cone PDE6, which I used in my work, was recently obtained. I observed different conformational dynamics in two identical subunits in both apo (unbound) and cGMP-bound PDE6. I also observed allosteric communication between the GAFa domain and the GAFb domain through the GAFb β1/β2 loop.