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Proteins are synthesised as linear polymeric chains. The subtle energetic interplay of interatomic interactions results in chain folding, through which proteins may acquire defined structures. This spatial organisation is encoded by the protein sequence itself; the so-called thermodynamic hypothesis formulated by Anfinsen in 1961. A defined structure is often considered a pre-requisite to protein function, but widespread existence of intrinsically disordered proteins (IDPs) has prompted a re- evaluation of the ways biological function may be encoded into polypeptide chains. Furthermore, proteins often exist as part of multi-component entities, where regulation of assembly is integral to their properties. The interplay between disorder, oligomerisation and function is the focus of this thesis. Some IDPs fold conditionally upon interacting with a partner protein; a process known as coupled folding and binding. What are the biophysical advantages and consequences of disorder in the context of these interactions? A common feature of IDPs is their sequence composition bias, with charged residues being often over-represented. It is therefore tempting to speculate that electrostatic interactions may play a major role in coupled folding and binding reactions. Surprisingly, the opposite was found to be true. Charge-charge interactions only contributed about an order of magnitude to the association rate constants of two contrasting model systems. The lack of pre-formed binding interfaces-a consequence of disorder-might preclude electrostatic acceleration from complementary patches. By looking at the role of the sequence, many studies have taken a protein-centric approach to understanding disorder. Yet there is paucity of data about the effect of extrinsic factors on interactions involving disordered partners. Investigating the role of co-solutes, it was discovered that the kinetic and thermodynamic profiles of coupled folding and binding reactions were sensitive to ion-types. This effect followed the Hofmeister series, and occurred at physiological concentrations of salt. The sensitivity of coupled folding and binding reactions-a consequence of the lack of stability of IDPs-might be advantageous. Given the role of ions in biology, this 'biophysical sensing' could be a mechanism of physiological relevance, allowing modulation of protein-protein interactions involving disordered partners in response to changes in their environments. In cells, signalling networks are often multi-layered, and involve competing protein-protein interactions. The interplay between the biophysical characteristics of the components, and the behaviour of the network were investigated in a model tripartite system composed of folded and disordered proteins. The BCL-2 family regulates the intrinsic pathway of apoptosis through control of mitochondrial outer-membrane permeabilisation; a result of BAK and BAX oligomerisation. Through a shared homology motif (termed BH3), the subtle balance of their interactions determines cellular fate at the molecular level. Characterisation of the model under simple biochemical conditions revealed large differences in affinities among binary interactions; the consequence of the lifetime of the complexes, not their speed of association. A membrane-like environment, re-created using detergents, allows the oligomerisation of BAK and BAX in vitro. Furthermore, investigation of the tripartite system under detergent conditions showed that regulation of the network was the result of competing hetero- and homo-oligomerisation events. Relationships to their biophysical properties were gained by probing their energy landscapes using protein folding techniques. The connection between the biophysical properties of the components of the network and their interactions provides a molecular explanation for the regulation of apoptosis. This thesis offers insights into the ways structured assemblies and environmentally responsive disorder elements may encode functions into proteins.

Molecular biophysics is a rapidly evolving field aimed at the physics-based investigation of the biomolecular processes that enable life. In this thesis, we explore two such processes: the thermodynamics of DNA bending, and the mechanism of the RecQ DNA helicase. A computational approach using a coarse-grained model of DNA is employed for the former; an experimental approach relying heavily on single-molecule fluorescence for the latter. There is much interest in understanding the physics of DNA bending, due to both its biological role in genome regulation and its relevance to nanotechnology. Small DNA bending fluctuations are well described by existing models; however, there is less consensus on what happens at larger bending fluctuations. A coarse-grained simulation is used to fully characterize the thermodynamics and mechanics of duplex DNA bending. We then use this newfound insight to harmonize experimental results between four distinct experimental systems: a 'molecular vise', DNA cyclization, DNA minicircles and a 'strained duplex'. We find that a specific structural defect present at large bending fluctuations, a 'kink', is responsible for the deviation from existing theory at lengths below about 80 base pairs. The RecQ DNA helicase is also of much biological and clinical interest, owing to its essential role in genome integrity via replication, recombination and repair. In humans, heritable defects in the RecQ helicases manifest clinically as premature aging and a greatly elevated cancer risk, in disorders such as Werner and Bloom syndromes. Unfortunately, the mechanism by which the RecQ helicase processes DNA remains poorly understood. Although several models have been proposed to describe the mechanics of helicases based on biochemical and structural data, ensemble experiments have been unable to address some of the more nuanced questions of helicase function. We prepare novel substrates to probe the mechanism of the RecQ helicase via single-molecule fluorescence, exploring DNA binding, translocation and unwinding. Using this insight, we propose a model for RecQ helicase activity.

Allergic reactions are mainly mediated by the interactions between the IgE and its ligands, amongst them CD23 and CD21 in what is termed the IgE network. CD23 is involved in upregulating IgE expression by forming a trimolecular complex with CD21 and IgE on the B-cell surface, resulting in the specific activation of IgE-positive B cells. CD21 also interacts with C3d and is a bridge between the innate and the immune system. A crystal structure of the interaction has been solved (Szakonyi et al., 2001) but was controversial because it contradicted previous biochemical analyses. The aims of this thesis were to use various biophysical techniques to study the interactions between the molecules in the IgE network and its possible inhibition. Part 1: Characterisation of a phage display-derived peptide that inhibit IgE binding to CD23 A peptide was previously derived using phage display technology and tested for binding ability to CD23 using SPR and ITC. Subsequent NMR experiments were performed to identify the binding site, followed by characterization of its derivatives. Crystallisation of CD23 with the peptide and soaking with its truncated tripeptide, NWP, were also attempted. Part 2: Characterisation of CD23 and its interaction with its ligands X-ray crystallography was undertaken to solve the structure of derCD23 in complex with a phage display-derived peptide (Part1) followed by crystal soaking with a truncated tripeptide, NWP. However, a reproducible, high-resolution wild type derCD23 structure was determined at 1.9 Å. A comparison of the binding behaviour between the monomeric derCD23 and a trimeric CD23 construct was carried out in order to see the effect of oligomerisation upon IgE binding. Using the known interaction map as well as a crystal structure, the possible interacting residues between CD23 and IgE were examined. The characterisation of the CD23/CD21 interaction was continued from previous efforts in order to confirm that the binding epitope of CD23 for CD21 lies within the C-terminus of CD23. Characterisation of the interactions of CD23/IgE/FcεRI was performed to examine these multimolecular interactions and possible regulatory mechanisms in mast cell degranulation. It was shown that CD23 can form multimeric complexes with IgE-Fc that bind to FcεRI with higher apparent affinity than IgE-Fc alone, which may lead to increases in mast cell degranulation. It was also found that the IgE bound on FcεRI still binds to CD23 although with a lower binding capacity, presumably due allosteric changes. The binding of CD23 with a monoclonal antibody IDEC-152 was also characterised using SPR and NMR spectroscopy. It was proposed that IDEC-152 might interfere with the trimerisation site of CD23 thus reducing its affinity for IgE. A thermofluor assay was developed and optimised for potential screening of compounds that bind to derCD23 using a qPCR machine, which may be useful to screen compounds that bind to CD23 as part of future drug discovery project. Crystallisation of the derCD23/CD21 and IgE/triCD23/CD21 complexes was also attempted as part of ongoing crystallisation projects. Part 3: The interaction between C3d and CD21 The interaction between C3d and CD21 is believed to be a bridge between the innate and adaptive immune response, and is thought to be pivotal in the initiation of autoimmune disease. Following from previous studies on this interaction, further characterisations were performed using NMR and ITC to confirm the involved sites on CD21 (SCR1-2) in binding to C3d. Several potential salt bridges have been identified so far, allowing a high-resolution docked structure of the C3d/CD21 complex.

Molecular motors hydrolyze ATP to produce mechanical work by stepping along the cytoskeleton network and carrying cargos. Dynein has many cellular roles which require its minus end-directed motility and force generation along the microtubules (MTs). All dynein activity needs to be tightly regulated by its many associated factors. Lis1 is the only associated factor that directly binds to dynein’s ATP hydrolyzing AAA ring, and it is involved in most, if not all, cellular processes that require dynein activity. In my thesis work, working with both mammalian and yeast proteins, I showed how dynein motility and force generation is regulated by Lis1 and its yeast homolog Pac1 (both Lis1 from here on). Mammalian dynein is mostly autoinhibited, that is, it cannot take many steps before detaching from the microtubules, a property that is essential for dynein-mediated cargo transportation. For processive motility, dynein needs to be relieved from this autoinhibition and needs to bind to dynactin and a cargo adapter. Using protein engineering, single molecule motility, optical trapping, and biophysical characterization assays, we have shown that Lis1 relieves dynein from autoinhibition, thus allowing the formation of dynein dynactin cargo adapter complex (DDX). Through the same mechanism, Lis1 increases the copy number of dynein in DDX complexes which enables faster motility and higher force generation. However, even after the formation of these complexes Lis1 can remain bound to dynein. In that case, we see an inhibitory effect of Lis1. In my thesis, I have shown the mechanism by which Lis1 binding affects dynein motility I switched my research to S. cerevisiae cytoplasmic dynein which has inherently processive motility without needing any cofactors, unlike mammalian dynein. I showed that Lis1 binding to the motor domain slows down dynein motility thus confirming previous studies done on yeast dynein and Lis1. Through multicolor TIRF colocalization assays, I have demonstrated that binding of individual Lis1 molecules causes dynein to pause or stop, and its unbinding restores dynein velocity. I have made three discoveries: 1. Lis1 binding to dynein has been proposed to inhibit or slow dynein motility by tethering dynein to the microtubule. I ruled out this model by showing that Lis1 only weakly interacts with the microtubule lattice, and this interaction does not slow dynein motility. 2. Lis1 binding has been proposed to block the force-generating conformational changes of the dynein linker domain. Using optical trapping, we ruled out this model by showing that Lis1 does not reduce the dynein stall force. 3. I observed that Lis1 binding decreases the asymmetry in detachment kinetics of force-induced detachment of dynein from the microtubule. Mutations that disrupt Lis1’s interactions with dynein’s stalk (an anti-parallel coiled-coil that leads to dynein’s microtubule-binding domain) partially restore the asymmetry. Because dynein’s stalk “slides” or changes its coiled-coil registry in a nucleotide-dependent manner, my data suggest that Lis1’s interaction with the dynein stalk interferes with the stalk sliding mechanism. I propose that this is what leads to slowing the detachment of dynein from the microtubule under force. These results are compatible with studies of Lis1 in live cells and provide a mechanistic explanation for why Lis1 needs to dissociate from dynein for efficient minus-end-directed motility. They also suggest an additional regulatory role for Lis1, such as anchoring dynein to the microtubule in order to facilitate the proper assembly of dynein with dynactin. I believe that the studies presented in this thesis will be broadly interesting to biophysicists studying the mechanics of motor proteins in vitro, cell biologists interested in the mechanism and regulation of intracellular transport, and neurobiologists who study the molecular basis of neurodevelopmental disorders.

Biological macromolecular assemblies play crucial roles in most cellular processes. The determination of their structures, thermodynamics, and kinetics is essential to understand their function, evolution, modulation, and design. Determining such models, however, remains challenging. One particularly powerful approach to constructing models in general is integrative modeling. Integrative modeling aims to maximize the accuracy, precision, and completeness of models, by simultaneously utilizing all available information, including experimental data, physical principles, statistical analyses, and other prior models. The goal of this thesis is to expand the scope of integrative modeling to the inference of spatial, thermodynamic, and kinetic aspects of macromolecular assemblies. In Chapter I, I introduce the integrative modeling framework for spatiotemporal modeling of biological macromolecular assemblies. In Chapter II, I demonstrate how the synergy between multi-chemistry cross-linking mass spectrometry and integrative modeling can map the structural dynamics of macromolecular assemblies, by application to the human Cop9 signalosome complex. In Chapter III, I present a method for determining structures, free energy differences, and kinetic rates of macromolecular assemblies along their functional cycle, mainly from negative stain electron microscopy (EM). We apply the method to the yeast Hsp90 to estimate the free energy differences and kinetic parameters along its nucleotide hydrolysis cycle, which includes open and closed states of Hsp90. In Chapter IV, I describe a validation of stochastic sampling in integrative modeling. The remaining chapters describe applications of integrative modeling to assemblies of various sizes and scales, using various sources of information, thus illustrating the flexibility of the integrative modeling approach. Specifically, I apply integrative modeling to the human ECM29-Proteasome assembly under oxidative stress (Chapter V), the yeast nuclear pore complex (NPC) cytoplasmic mRNA export platform (Chapter VI), the major membrane ring component of the yeast NPC (Chapter VII), the entire yeast NPC (Chapter VIII), and the reconstruction of 3D structures of MET antibodies (Chapter IX).

The forces and energetic principles that guide molecular structure and dynamics in cellular lipid bilayers are poorly understood, largely due to limited familiarity with physical chemistry outside of water. We strive to enhance our fundamental chemical intuition concerning membrane proteins because their biochemical behavior within lipid membranes governs critical cellular processes and dictates health or disease. Likewise, this intuition paired with the ability manipulate membrane protein structure and function using computational modeling, theoretical calculations, and molecular design are instrumental to the engineering of new therapeutics. The work described here focuses on using protein design and integrative computational approaches to dissect the fundamental biophysical principles underlying protein interactions and folding within the lipid environment, spanning studies of both natural and totally synthetic proteins. Chapter 1 describes the design and experimental characterization of a family of simple synthetic model membrane proteins that rely entirely on the steric packing of lipid-embedded apolar sidechains for their stabilization and folding. This work reveals new fundamental principles governing van der Waals forces for proteins in lipid which should dictate protein structure, function, and evolution. Chapter 2 reports discovery of an evolutionarily conserved interaction motif within the transmembrane (TM) domains of integrin family proteins regulating their biochemical function. This discovery was driven by molecular modeling and all-atom dynamics simulations paired with co-workers’ optical tweezer force spectroscopy experiments. Chapter 3 details the development and application of software to design lipid-soluble peptides that target the transmembrane domains of proteins. We report several molecules that activate integrin α5β1 in human endothelial cell culture, and one with binding specificity for the β1 subunit. Chapter 4 outlines a second campaign with similar software to engineer small synthetic transmembrane proteins that bind and inhibit signal-amplifying erythropoietin receptor (EpoR), a canonical cytokine family receptor. Here, we demonstrate computational design can be used to target membrane-soluble molecules to specifically adopt custom, non-native binding modes to engage targeted regions of protein embedded in lipid. Thus, the efforts described represent leaps in our capacity to compute designer tool molecules entirely from scratch useful for manipulating membrane-spanning regions. These works together define progress in our fundamental biophysical understanding of protein interactions and folding in lipid, as well as our ability to dissect membrane protein structure and function with in silico molecular modeling algorithms and novel chemical biology tools.

Electrostatic and geometric factors are critical to modeling the interactions and solvation effects of biomolecules in the aqueous environments of biological cells as they respectively influence the polar and non-polar components of the associated free energies. Conventional protocols use a hard-sphere model of atoms to devise and study the underlying thermodynamics. But this traditional model tends to overlook some of the important biophysical aspects at the cost of oversimplification of the representation of the solute-solvent environments. Here an alternative and physically appealing model of atoms – a Gaussian-based model, is presented which replaces the hard-sphere model with a smooth density-based description of atoms. This dissertation explains the derivation of a physically appealing dielectric distribution from the Gaussian schematic to model the electrostatics of biomolecules using the implicit-solvent/Poisson-Boltzmann (PB) formalism. It also demonstrates the advantages of using it for computing geometric properties of a molecule such as its volume and surface area (SA) for estimating non-polar portions of the free energy. While highlighting the qualitative importance of the Gaussian-based model, it offers conceptual proofs towards its validity through computational investigations of explicit solvent simulations. It also reports the key features of the Gaussian-based model, which impart to it the capacity of accurately capturing the crucial biophysical factors that characterize biomolecular properties, namely – the effect of intrinsic conformational flexibility and salt distribution. The non-triviality of these factors and their portrayal through the Gaussian models are meticulously discussed. A major theme of this work is the implementation of the Gaussian model of dielectric distribution and volume/SA estimation into the PB solver package called Delphi. These developments illustrate the manner in which the utility of Delphi has been expanded and its reputation as a popular tool for modeling solvation effects with appreciable time-efficacy and accuracy has been enhanced.

Viral DNA packaging is a required step in the lytic cycle of many DNA viruses. A DNA polymer is packaged into a pre-formed capsid to a near crystalline density state by a powerful molecular motor that is fueled by ATP hydrolysis. To accomplish this task, the motor must generate enough force to match the resistance forces that arise from tight polymer confinement. These forces are due to entropy loss, bending energy, and electrostatic repulsion. Despite recent advances in the fields of viral DNA packaging and ejection, there are still many questions that remain unanswered. Does the packaged DNA undergo non-equilibrium dynamics? Do partly attractive DNA-DNA interactions as mediated by polyamines enhance or inhibit packaging? What affect does the conformational history of the packaged DNA have on the subsequent packaging dynamics? Which residues of the motor are responsible for generating force? And finally, is the force driving viral DNA ejection similar in magnitude to the package force? In this work, I present experimental studies that shed light on these questions. The packaging of single DNA molecules into single empty pro-capsids was measured using a custom-built dual optical tweezers system. Using the bacteriophage phi29 and T4 systems, I and my research collaborators pioneered new techniques in optical tweezers for investigating the dynamics of DNA packaging and force generation of a viral packaging motor. Using these techniques, we discovered many new findings in the areas of polymer confinement, non-equilibrium dynamics, DNA condensation, and enzyme kinetics.