<p>Ligand binding can modulate function of proteins and nucleic acids by changing both the populations of functionally distinct conformational states and the timescales on which they interconvert. For this reason, both thermodynamic and kinetic details of coupling can be important to proper function. How tightly does ligand bind to the different conformational states? What effect does ligand binding have on the conformational equilibrium and conformational kinetics? On what timescales and in what order do binding and conformational change occur? Using a combination of stopped-flow kinetics, isothermal titration calorimetry, and x-ray crystallography, we determine the mechanisms of coupled binding and conformational change in protein (Bacillus subtilis RNase P protein) and RNA (DP17 biosensor) systems. </p><p>The results demonstrate that rigorous kinetic analysis can be used to estimate the equilibrium and rate constants for conformational changes, as well as the affinities of ligands for different conformational states. A single ligand can bind to different conformational states of the same protein or nucleic acid with affinities that differ by orders of magnitude. This binding shifts the conformational equilibrium towards the higher affinity state through a combination of increasing rate constants for the forward conformational change and decreasing rate constants for the reverse conformational change. Using a flux-based analysis of the mechanisms we show that molecular recognition is kinetically partitioned between a number of pathways that differ by the order in which binding and conformational change occur. The absolute and relative flux through these pathways varies with ligand concentration, the affinities of the ligand for the various conformational states, and the ability of ligand to accelerate the conformational change. Together, the results give insights into how biological function depends on the kinetic and thermodynamic details of coupled binding and conformational change.</p> / Dissertation

Thesis advisor: Evan R. Kantrowitz / The understanding of how cells regulate and control all aspects of their function is vital for our ability to intervene when these control mechanisms break down. Almost all modes of cellular regulation can be related in some manner to protein conformational changes such as the quaternary conformational changes of allosteric enzymes that alter enzyme activity to regulate metabolism. The control of metabolic pathways by allosteric enzymes is analogous to a molecular valve with "on" and "off" positions. In the "off" position, flow through the pathway is severely hindered, while in the "on" position the flow is normal. For a comprehensive understanding of allosteric regulation we must elucidate in molecular detail how the allosteric signal is transmitted to the active site to alter enzyme activity. In this work we use unnatural amino acid mutagenesis to introduce a fluorescent amino acid into the allosteric binding site of aspartate transcarbamoylase (ATCase), the enzyme responsible for regulation of pyrimidine nucleotide biosynthesis. The fluorescence from the amino acid is exquisitely sensitive to the binding of the allosteric effectors ATP, CTP, UTP, and GTP. In particular we show how the asymmetric nature of the allosteric sites of the enzyme are used to achieve regulatory sensitivity over a broad range of mixed heterotropic effector concentrations as is observed in the cell. Furthermore, employing the method of random sampling - high dimensional model representation (RS-HDMR) we derived a model for how ATCase is regulated when all four nucleotides are present at fluctuating concentrations, consistent with physiological conditions. We've discovered the fundamental requirements to induce the allosteric transition to the R state by showing that although ATCase can accept L-asparagine as an unnatural substrate, the transition to the R allosteric state requires the correct positioning of the alpha-carboxylate of its natural substrate L-aspartate. However, linking the functionalities of L-asparagine and carbamoyl phosphate into a single molecule is sufficient to correctly position the bi-substrate analog in the active site to induce the allosteric transition to the R-state. The cooperative nature of ATCase was further investigated through the isolation of a unique quaternary structure of ATCase consisting of two catalytic trimers linked covalently by disulfide bonds. By relieving the quaternary constraints imposed by the bridging regulatory subunits of the native holoenzyme, the flexibility of the c6 subunit significantly enhanced enzyme activity over the native holoenzyme. Unlike the native c3 catalytic subunit, the c6 species displays homotropic cooperativity for L-aspartate demonstrating that, when two catalytic trimers are linked, a binding event at one or more active sites can be transmitted through the molecule to the other active sites in the absence of regulatory subunits. The catalytic reaction of ATCase follows an ordered sequential mechanism that is complicated by the transition from the T state to the R state upon the binding of the second substrate L-aspartate. Acquiring X-ray crystal structures at each step along the pathway has advanced our understanding of the catalytic mechanism, yet R-state structures are difficult to obtain. Using a mutant version of ATCase locked in the R-allosteric state by disulfide bonds we captured crystallographic images of ATCase in the R state bound to the true substrates (CP and Asp), products (CA and Pi), and in the process of releasing the final product (Pi) prior to reversion of the molecule to the T state. These structures depict the steps in the catalytic cycle immediately before the catalytic reaction occurs, immediately after the reaction, and after the first product has been released from the active site. This work also focuses on developing allosteric inhibitors of the enzyme fructose-1,6-bisphosphatase (FBPase), one of the enzymes responsible for regulation of the gluconeogenesis pathway. Inhibitors of FBPase could serve as potential therapeutic agents against type-2 diabetes. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Proteins present at the synapse need to be multitasking in order to perform all vital functions in this limited space. In this thesis I have analyzed the function and evolution of such proteins, focusing on the PDZ domain and the paralemmin family. The PDZ domains bind to a wide variety of interaction partners. The affinity for each partner is regulated by residues at the binding site, but also through intradomain allostery. How this intradomain allostery is transferred to the binding site is not established. I here show that side chain interactions can explain all transfer of intradomain allostery in three analyzed PDZ domains. A circularly permuted PDZ domain has an identical set of amino acids as the original protein and a very similar structure with only a few perturbed side chains. By using the circular permutant I show that a slight alteration in the position of a side chain leads to a corresponding change in allosteric signal. I further study the folding of several PDZ domains and show that they all fold via a conserved folding mechanism, supporting the notion that the final structure has a part in deciding folding mechanism. The folding mechanism of the circularly permuted PDZ domain is conserved compared to the original protein illustrating how circular permutations can be tolerated through evolution. The multifunctionality of paralemmins probably lies in their highly flexible structures. I have studied the evolution of the paralemmins and found that the four mammalian paralemmins arose in the two whole-genome duplications that occurred early in the vertebrate evolution. The fact that all four paralemmins have survived evolution since the gene duplications suggests that they have important functions, possibly in the development of the nervous system. Synaptic proteins are crucial for many biological processes, and their misfolding implicated in many diseases. The results presented here shed light on the mechanisms of action of the synaptic proteins and will help us to understand how they generate disease.

Enzymes are nature’s wizards: balanced delicately on the margin of order and entropy, they perform chemical reactions and syntheses at rates and yields human chemists can only dream of. Many possess exquisite control mechanisms to keep the flow of metabolites through our cells precisely regulated. This work explores the regulation mechanism of α-isopropylmalate synthase (α-IPMS). The branched-chain amino acid biosynthetic pathways in bacteria are of interest as novel antibiotic targets. α-IPMS catalyses the first committed step in the pathway to form leucine, an essential amino acid. It performs the Claisen condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate (α-IPM). Almost all previously characterised α-IPMS enzymes are feedback regulated by leucine, the end-product of this pathway. This study uses the α-IPMS enzymes from two pathogenic species, Myco- bacterium tuberculosis and Neisseria meningitidis (MtuIPMS and NmeIPMS, respectively). These enzymes are homodimeric in solution, and have a catalytic dimer of (β/α)8 barrels. This is connected via two more subdomains to a dimerised C-terminal regulatory domain, where leucine binds. The crystal structures of MtuIPMS with and without leucine bound are almost identical. Thus, we do not yet fully understand the mechanisms by which leucine is recognised, nor how the allosteric signal is conducted ̴ 50 Å from the regulatory domain to the active site, and how this disrupts catalysis. Chapter 2 explores the residues responsible for recognising and binding leucine. We use insights from the partial crystal structure of a similar enzyme in Leptospira interrogans, citramalate synthase (CMS). CMS catalyses a similar reaction to α-IPMS: the condensation of AcCoA and α-ketobutyrate (α-KB) to form citramalate, as the first step in isoleucine production in this organism. CMS is feedback regulated by isoleucine just as α-IPMS is regulated by leucine. CMS also shares a very similar overall structure to α-IPMS, and four conserved residues in each enzyme were identified as being responsible for binding the allosteric effector. In previous work, Tyler Clarke1 mutated each of the four MtuIPMS residues to the corresponding residue from LiCMS in an attempt to make an isoleucine-regulated MtuIPMS. While one mutant did show an increased sensitivity to the related amino acid norvaline, none of these mutations by themselves were sufficient to create an isoleucine-sensitive MtuIPMS. This work found that by using certain combinations of these mutations, we were able to create isoleucine-inhibited α-IPMS enzymes. Dr. Wanting Jiao has been using molecular dynamics simulations to identify the residues important for allosteric signal propagation and disrupting catalysis in NmeIPMS . Chapter 3 details several of these residues which we have mutated, and presents the preliminary results of activity and inhibition studies on the mutant enzymes. Chapter 4 summarises our findings and outlines the work required to further our understanding of the allosteric control systems studied here. Adapting the power of enzymes to contribute to the development of green chemistry, biosensors, and new antibiotics may prove to be one of the greatest opportunities ahead of modern chemistry.

Allosteric regulation of important enzymes is a mechanism frequently employed by organisms to exert control over their metabolism. The shikimate pathway is ultimately responsible for the biosynthesis of the aromatic amino acids in plants, microorganisms and apicomplexans. Two enzymes of the pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) and chorismate mutase (CM) are located at critical positions along the aromatic amino acid biosynthetic pathway and are often tightly feedback regulated in order to control the flux of metabolites through the pathway. This research presents studies on the allosteric function of these two enzymes. These studies emphasise the complexity of the intersecting network of allosteric response, which alters the catalytic activity of each enzyme in response to metabolic demand for the aromatic amino acids.

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.

Glucokinase (GK) is an enzyme that catalyzes the ATP-dependent phosphorylation of glucose to form glucose-6-phosphate, and it is a tightly regulated checkpoint in glucose homeostasis. The monomeric enzyme possesses a highly exotic kinetic profile, with a sigmoidal dependence on glucose, which has been the source of vigorous investigation and debate in the last several decades. This unique regulatory behavior can be thought of as a remarkable glucose sensor, which may result in hyperglycemia when it is not active enough and hypoglycemia when it is too active. This interdisciplinary study, which draws on small angle X-ray scattering (SAXS) integrated with atomistic molecular dynamics simulations and experimental glucose binding thermodynamics, I reveal the critical regulation of the glucose sensor is due to a solvent controlled switch. Moreover, this solvent controlled switch manifests a regulatory mechanism of GK; a specific local conformational change that leads to an enzyme structure that has a much more favorable solvation energy than most of the protein ensemble. These findings have direct implications for the design of small molecule GK activators as anti- diabetes therapeutics as well as for understanding how proteins can be designed to have built-in regulatory functions via solvation energy dynamics.

GLUT1-mediated, facilitated sugar transport is proposed to be an example of transport by a carrier that alternately presents exofacial (e2) and endofacial (e1) substrate binding sites, commonly referred to as the alternating access carrier model. This hypothesis is incompatible with observations of co-existent exo- and endofacial ligand binding sites, transport allostery, and e1 ligand (e.g. cytochalasin B) induced GLUT1 sugar occlusion. The fixed-site carrier model proposes co-existent, interacting e2 and e1 ligand binding sites but involves sugar translocation by geminate exchange through internal cavities. Demonstrations of membrane-resident dimeric and tetrameric GLUT1 and of e2, e1 and occluded GLUT conformations in GLUT crystals of monodisperse, detergent-solubilized proteins suggest a third model. Here, GLUT1 is an alternating access carrier but the transporter complex is a dimer of GLUT1 dimers, in which subunit interactions produce two e2 and two e1 conformers at any instant. The crystallographic structures in different conformations can be utilized to further understand the transport cycle, ligand binding behavior and complex kinetics observed in GLUT1. Specifically, the GLUT1 crystal structure and homology models based upon related major facilitator superfamily proteins were used in this study, to understand inhibitor binding, ligand binding induced GLUT1 transport allostery and the existence of helix packing/oligomerization motifs and glycine induced flexibility. These studies suggest that GLUT1 functions as an oligomeric allosteric carrier where cis-allostery is an intramolecular behavior and trans-allostery is an intermolecular behavior. Additionally, mutations of a dynamic glycine affect the turnover of the transporter while mutations to helix packing motifs affect affinity.

The studies described in this dissertation examine the effects of F-2,6-P2 and AMP or phosphorylation on the kinetic mechanism of d-PFK. The effect of varied pH on the activation by F-2,6-P2 is also described.

cAMP receptors contain highly conserved cAMP binding pockets, in part responsible for allosteric activation, yet CBDs exhibit a wide array of cAMP binding affinities. While several cAMP:CBD crystallographic structures have been solved, they are insufficient to explain differences in cAMP:CBD affinities. We hypothesize that it is the position of the apo autoinhibitory equilibrium and/or a change in the state-specific association constants of the active and inactive CBD forms that are primarily responsible for modulating ~1000-fold difference in cAMP affinities. Interestingly, we discovered that PKARIα and HCN2 have comparable state-specific association constants, suggesting that the position of the apo autoinhibitory equilibrium is primarily responsible for the large difference in observed cAMP affinities in these systems. In addition, the individual components of the cAMP binding pocket (i.e. BBR, PBC, and lid) show functional variability across different CBDs. In RIα, both the BBR and lid are dispensable for high affinity cAMP binding, leaving the PBC as the key determinant of cAMP affinity. Interestingly, in addition the PBC:cAMP contact side-chains, non-contact side-chains are also important in modulating cAMP affinity (ie. L201 and Y205). Further dissection of the contributions arising from the apo pre-equilibrium and the cAMP binding pockets is required to better understand cAMP affinity and selectivity. / Thesis / Master of Science (MSc)