Investigating the Role of Conformational Dynamics in Allosteric Regulation PDF Download

Author: Dennis Sean Winston
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Languages : en
Pages : 0

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Book Description
Allosteric regulation, when a perturbation at one site in a macromolecule affects the function of a distant site, is widespread in biology. While the study of allostery historically has primarily focused on ligand binding, the concept applies more broadly to any site-specific perturbation including amino acid substitutions, protease cleavage, changes in oligomerization state, and post-translational modification. While there is sometimes a clear and elegant structural explanation for the mechanism of allostery, it is often mediated by conformational dynamics in a way that is not always intuitive. In this work, we focus on two enzyme systems where allosteric regulation is not well-understood: poliovirus 3CD and yeast chorismate mutase. We rely primarily on nuclear magnetic resonance (NMR) relaxation experiments to gain insight into the conformational dynamics of these proteins in order to better understand their allosteric regulation. In the case of poliovirus 3CD, the 3CD precursor is proteolytically processed to produce the 3C and 3D proteins. This production of multiple proteins from the same amino acid sequence is a strategy of expanding the number of protein functions available to viruses, which have very small genomes. 3C is the main protease responsible for cleaving the poliovirus polyprotein and 3D is the RNA-dependent RNA polymerase responsible for replicating the viral RNA. 3CD has different protease specificity from 3C, no polymerase activity, and participates in different protein-protein interactions from 3C and 3D. Structurally, 3CD appears to consist of 3C and 3D domains separated by a flexible linker with no clear structural changes to explain the differences in function. In Chapter 2, we show that there are differences in conformational dynamics between 3CD and its processed products in functionally important regions, suggesting that the function of the 3C and 3D domains of 3CD are allosterically affected by the presence of the 3D & 3C domains. In Chapter 3, we provide the first experimentally determined atomic-level details of poliovirus 3C-phosphoinositide interactions in a membrane context. Membrane binding may alter the conformational ensemble of 3C and 3CD, allowing for further functional regulation of poliovirus proteins over the course of infection. Yeast chorismate mutase (ScCM) catalyzes the conversion of chorismate to prephenate, a key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine. ScCM is allosterically inhibited by tyrosine and activated by tryptophan, the end product of the competing pathway. The allosteric regulation has been previously proposed to occur via the Monod-Wyman-Changeux (MWC) model of allostery, where the enzyme fluctuates between states of different activity with an equilibrium constant that is altered by effector binding. In Chapter 4, we use NMR to study conformational fluctuations of ScCM in the absence of effector and show that the MWC model does not fully describe the allosteric regulation by tyrosine and tryptophan binding. Overall, this dissertation contributes to our understanding of the role of conformational dynamics in allosteric regulation across two different proteins.