Date of Award

Spring 2019

Project Type


Program or Major

Chemical Engineering

Degree Name

Doctor of Philosophy

First Advisor

Harish Vashisth

Second Advisor

Russell T. Carr

Third Advisor

Nivedita Gupta


In this thesis work, I conducted large-scale molecular dynamics (MD) simulation studies of interactions of enzymes and signaling proteins with inhibitory ligands. Specifically, I have studied three classes of proteins: the first part of my thesis reports studies on the hydrogen-producing [FeFe]-hydrogenase enzyme, the second part reports on studies of regulatory proteins from the G-protein coupled receptor (GPCR) family, and the third part reports on studies of the phosphodiesterase (PDE) enzyme family.

In the first part, I studied the problem of [FeFe]-hydrogenase sensitivity to the presence of inhibitory gases oxygen (O2) and carbon monoxide (CO) that cause irreversible damage to the active site of this enzyme. Therefore, a detailed knowledge of the diffusion pathways of these inhibitory gases is necessary to develop strategies for designing novel enzymes that are tolerant to these gases. Specifically, I studied the diffusion pathways of O2 and CO in the CpI [FeFe]-hydrogenase from Clostridium pasteurianum. I used several enhanced sampling and free-energy simulation methods to reconstruct a three-dimensional free-energy surface for diffusion of each gas which revealed free-energy minima forming an interconnected network of pathways. I discovered multiple pathways of minimal free-energy as diffusion portals for O2 and CO, and observed that the global minimum in the free-energy surface is located in the vicinity of the active site metal cluster, the H-cluster. Among potential residues that I propose as candidates for future mutagenesis studies to increase the tolerance of this enzyme to both inhibitory gases, 11 residues are shared between O2 and CO. I hypothesize that these shared candidate residues are potentially useful for designing new variants of this enzyme that are tolerant to both inhibitory gases.

In the second part, I have studied the interplay of protein conformational dynamics and effects of small-molecule inhibitors in a class of signaling proteins, known as the Regulators of G protein signaling (RGS) proteins, that negatively modulate signaling in GPCRs. Recently discovered thiadiazolidinone(TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for several known RGS proteins, thereby suggesting intrinsic differences in dynamics of these proteins upon binding of these compounds. I characterized the effect of binding of several small-molecule inhibitors on perturbations and dynamical motions in RGS4.

Specifically, I studied two conformational models of RGS4 in which a buried cysteine residue is solvent-exposed due to side-chain motions or due to flexibility in neighboring helices. I found that TDZD compounds with aromatic functional groups perturb the RGS4 structure more than compounds with aliphatic functional groups. Moreover, small-molecules with aromatic functional groups but lacking sulfur atoms only transiently reside within the protein and spontaneously dissociate to the solvent. I further probed the salt-bridges forming across isoforms of RGS proteins, resulting in a hypothesis that differences in salt-bridges between a pair of helices in RGS proteins are responsible for differences in flexibility and potency among isoforms.

In the final part of my thesis, I evaluated differences in binding interactions of phosphodiesterase 4 (PDE4) inhibitors within the PDE4 catalytic domain. From residues within 5 Å of the ligand binding site, five residues revealed significant differences in non-bonded interaction energies that could account for the differential binding affinities of inhibitors. I found one site (Phe506 in human PDE4; Tyr253 in the C. elegans PDE4 catalytic domain) that alters the binding conformation of roflumilast and zardaverine (human PDE 4 inhibitors) into a less energetically favorable state. These results support the feasibility of designing the next-generation of anthelmintics/nematicides that could selectively bind to nematode PDEs. Overall, my thesis has resulted in enhancement of detailed mechanistic insights into several protein and inhibitory ligand interactions that are potentially useful in the developement of novel inhibitors targeting protein/protein and protein/ligand interactions.