Regulation of protein activity is essential for normal cell functionality. Many proteins are regulated allosterically, that is, with spatial gaps between stimulation and active sites. Biological stimuli that regulate proteins allosterically include, for example, ions and small molecules, post-translational modifications, and intensive state-variables like temperature and pH. These effectors can not only switch activities on-and-off, but also fine-tune activities. Understanding the underpinnings of allostery, that is, how signals are propagated between distant sites, and how transmitted signals manifest themselves into regulation of protein activity, has been one of the central foci of biology for over 50 years. Today, the importance of such studies goes beyond basic pedagogical interests as bioengineers seek design features to control protein function for myriad purposes, including design of nano-biosensors, drug delivery vehicles, synthetic cells and organic-synthetic interfaces. The current phenomenological view of allostery is that signaling and activity control occur via effector-induced changes in protein conformational ensembles. If the structures of two states of a protein differ from each other significantly, then thermal fluctuations can be neglected and an atomically detailed model of regulation can be constructed in terms of how their minimum-energy structures differ between states. However, when the minimum-energy structures of states differ from each other only marginally and the difference is comparable to thermal fluctuations, then a mechanistic model cannot be constructed solely on the basis of differences in protein structure. Understanding the mechanism of dynamic allostery requires not only assessment of high-dimensional conformational ensembles of the various individual states, including inactive, transition and active states, but also relationships between them. This challenge faces many diverse protein families, including G-protein coupled receptors, immune cell receptors, heat shock proteins, nuclear transcription factors and viral attachment proteins, whose mechanisms, despite numerous studies, remain poorly understood. This dissertation deals with the development of new methods that significantly boost the applicability of molecular simulation techniques to probe dynamic allostery in these proteins. Specifically, it deals with two different methods, one to obtain quantitative estimates for subtle differences between conformational ensembles, and the other to relate conformational ensemble differences to allosteric signal communication. Both methods are enabled by a new application of the mathematical framework of machine learning. These methods are applied to (a) identify specific effects of employed force fields on conformational ensembles, (b) compare multiple ensembles against each other for determination of common signaling pathways induced by different effectors, (c) identify the effects of point mutations on conformational ensemble shifts in proteins, and (d) understand the mechanism of dynamic allostery in a PDZ domain. These diverse applications essentially demonstrate the generality of the developed approaches, and specifically set the foundation for future studies on PDZ domains and viral attachment proteins.

Protein-ligand binding and protein allostery play a crucial role in cell signaling, cell regulation, and modern drug discovery. In recent years, experimental studies of protein structures including crystallography, NMR, and Cryo-EM are widely used to investigate the functional and inhibitory properties of a protein. On the one hand, structural classification and feature identification of the structures of protein kinases, HIV proteins, and other extensively studied proteins would have an increasingly important role in depicting the general figures of the conformational landscape of those proteins. On the other hand, free energy calculations which include the conformational and binding free energy calculation, which provides the thermodynamics basis of protein allostery and inhibitor binding, have proven its ability to guide new inhibitor discovery and protein functional studies. In this dissertation, I have used multiple different analysis and free energy methods to understand the significance of the conformational and binding free energy landscapes of protein kinases and other disease-related proteins and developed a novel alchemical-based free energy method, restrain free energy release (R-FEP-R) to overcome the difficulties in choosing appropriate collective variables and pathways in conformational free energy methods like umbrella sampling and metadynamics. / Chemistry

abstract: Traditionally, allostery is perceived as the response of a catalytic pocket to perturbations induced by binding at another distal site through the interaction network in a protein, usually associated with a conformational change responsible for functional regulation. Here, I utilize dynamics-based metrics, Dynamic Flexibility Index and Dynamic Coupling Index to provide insight into how 3D network of interactions wire communications within a protein and give rise to the long-range dynamic coupling, thus regulating key allosteric interactions. Furthermore, I investigate its role in modulating protein function through mutations in evolution. I use Thioredoxin and β-lactamase enzymes as model systems, and show that nature exploits "hinge-shift'' mechanism, where the loss in rigidity of certain residue positions of a protein is compensated by reduced flexibility of other positions, for functional evolution. I also developed a novel approach based on this principle to computationally engineer new mutants of the promiscuous ancestral β-lactamase (i.e., degrading both penicillin and cephatoxime) to exhibit specificity only towards penicillin with a better catalytic efficiency through population shift in its native ensemble.I investigate how allosteric interactions in a protein can regulate protein interactions in a cell, particularly focusing on E. coli ribosome. I describe how mutations in a ribosome can allosterically change its associating with magnesium ions, which was further shown by my collaborators to distally impact the number of biologically active Adenosine Triphosphate molecules in a cell, thereby, impacting cell growth. This allosteric modulation via magnesium ion concentrations is coined, "ionic allostery''. I also describe, the role played by allosteric interactions to regulate information among proteins using a simplistic toy model of an allosteric enzyme. It shows how allostery can provide a mechanism to efficiently transmit information in a signaling pathway in a cell while up/down regulating an enzyme’s activity. The results discussed here suggest a deeper embedding of the role of allosteric interactions in a protein’s function at cellular level. Therefore, bridging the molecular impact of allosteric regulation with its role in communication in cellular signaling can provide further mechanistic insights of cellular function and disease development, and allow design of novel drugs regulating cellular functions. / Dissertation/Thesis / Doctoral Dissertation Physics 2020