Doctor of Philosophy

dissertationThe unstable expansion of the polyglutamine (polyQ) tract is a critical factor in the pathogenic pathway of at least ten neurodegenerative diseases, including Huntington's disease, spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and seven spinocerebellar ataxias, all of which are termed as polyglutamine diseases. One less understood but common feature of polyQ diseases is polyQ protein aggregation. This dissertation explores the protein folding, hydrogen bonding, and water accessibility changes which are induced by the enlargement of the polyQ tract using advanced informatics and computational methods, including protein 3D structure modeling and molecular dynamics simulations. This dissertation also demonstrates that these state-of-the-art computational and informatics methods are powerful tools to provide useful insights into protein aggregation in polyQ diseases. The enlargement of polyQ segments affects both local and global structures of polyQ proteins as well as their water-accessibility, hydrogen bond patterns, and other structural characteristics. Results from both isolated polyQ and polyQ segments in the context of ataxin-2 and ataxin-3 show that the polyQ tracts increasingly prefer self-interaction as the lengths of the tracts increase, indicating an increased tendency toward aggregation among larger polyQ tracts. These results provide new insights into possible pathogenic mechanisms of polyQ diseases based solely on the increased propensity toward polyQ aggregation and suggest that the modulation of solvent-polyQ interaction may be a possible therapeutic strategy for treating polyQ diseases. The analysis pipeline designed and used in this study is an effective way to study the molecular mechanism of polyQ diseases, and can be generalized to study other diseases associated with the protein conformation changes, such as Parkinson's disease, Alzheimer's disease, and cancer

Combining experiments and simulation to characterise structural and dynamical properties of intrinsically disordered peptides and regions

Intrinsically disordered proteins and regions play important roles in the regulation of protein dynamics and protein-protein interactions. In this thesis two IDPs, both of which have been implicated in neurodegenerative diseases, are explored using fully atomistic molecular dynamics simulations. The first is the N-terminal fragment of the huntingtin protein, which controls the protein’s localisation and function in vivo. The second is the disordered pro domain of the proNGF dimer, which antagonises NGF in the brain. Huntingtin is the causative agent of Huntington’s disease, which is a progres- sive neurodegenerative disease, characterised by CAG repeats in the first exon of Huntingtin, which are translated into a polyglutamine (polyQ) tract, responsible for protein aggregation and subsequent neuron death. Huntingtins poly-Q tract is preceded by a 17-residue regulatory fragment (Htt1-17), which is intrinsically dis- ordered in aqueous environments but forms an amphipathic helix in the presence of TFE or DPC micelles. Htt1-17 regulates localisation and function of the full-length protein and is subject to multiple post-translational modifications in the cell. I used molecular dynamics simulations with a novel enhanced sampling method, to study the effect of phosphorylation, phosphomimetic substitutions and acetylation on the secondary structure of Htt1-19. ProNGF is the precursor to the neurotrophin NGF, and is involved in apoptotic signalling in the brain. A disturbed proNGF:NGF was shown to lead to Alzheimer’s disease. A high-resolution structure of the pro domain has been missing so far. I modelled the proNGF dimer by combining experimental data with long MD simu- lations

Estudios estructurales y computacionales de amiloides y plegamientos nocivos en biomoléculas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de lectura: 20-11-2015El amiloide es un tipo de estructura de proteínas consistente en láminas beta muy largas y altamente estables, responsables de más de veinte enfermedades mortales en humanos, incluyendo Alzheimer, Parkinson y Esclerosis Lateral Amiotrófica (ELA). Paradójicamente los amiloides no siempre son patológicos, sino que también son esenciales en procesos vitales tales como la regulación del ARN o la consolidación de la memoria. A pesar de los avances en el campo, todavía hay muchos detalles desconocidos acerca de los aspectos energéticos y la formación de estas estructuras. En esta tesis presento resultados sobre la formación y los factores que estabilizan el amiloide del heptapéptido GNNQQNY de Sup35, un prion de levaduras. Mis resultados computacionales y experimentales muestran que el intermedio clave en la amiloidogénesis es una lámina beta paralela curvada compuesta por tres hebras. Esta lámina es capaz de dimerizar expulsando fácilmente las moléculas de agua de solvatación y así producir la estructura llamada columna beta cruzada (cross-β spine). La presencia de enlaces de hidrógeno híper-estables es crucial para la estabilidad tanto de este intermedio, como de oligómeros más largos y fibras de Sup35. Con estos datos, se propone un modelo para la formación de este núcleo que plantea la cuestión de si los oligómeros tóxicos son una consecuencia de columnas beta cruzadas en un plegamiento anómalo. La TDP-43 (Proteína de unión a ADN de respuesta transactiva de 43 kDa) es particularmente fascinante, puesto que su región C-terminal forma agregados de tipo amiloide implicados en la ELA, además de un hipotético amiloide funcional esencial para el transporte y la regulación del ARN. Puesto que existe un debate acerca de la naturaleza amorfa o amiloide de los agregados de TDP-43, en esta tesis se presentan una serie de ensayos bioquímicos y espectroscópicos que revelan la naturaleza de tipo amiloide de estos agregados. Entre ellos se incluyen experimentos de unión a cromóforos y fluoróforos, reconocimiento por anticuerpos conformacionales, dicroísmo circular, difracción de rayos-X, resonancia magnética nuclear (RMN) en estado líquido y sólido, así como microscopía electrónica. Mediante métodos espectroscópicos y computacionales, hemos investigado conformaciones permitidas para el posible amiloide patológico, elaborando modelos atómicos para el monómero constituyente. La estructura consiste en horquillas beta alineadas en una topología de giro beta y empaquetadas en paralelo para formar una estructura cuasi-amiloide con un plegamiento novedoso. Por otra parte, el dominio N-terminal (DNT) de la TDP-43 juega papeles clave en la regulación de la agregación funcional y patológica de la región C-terminal. La estructura de alta resolución del DNT ha sido obtenida por RMN y consiste en una hélice alfa y seis hebras beta, con un núcleo hidrófobo bien empaquetado, dos grupos de residuos cargados negativamente y dos cisteínas expuestas y distantes entre sí. La estabilidad conformacional, determinada por intercambio de hidrógeno/deuterio, es de 4 kCal/mol a pH 4 y 25 °C. Las propiedades dinámicas han sido elucidadas usando métodos de RMN y muestran que la hélice alfa y cinco de las seis hebras beta son rígidas. Una de las hebras del borde y los giros son más flexibles. Estos descubrimientos avanzan nuestra comprensión del DNT y proveen un medio para estudiar su interacción con la región C-terminalAmyloid is a class of protein structures composed of very long and highly stable β-sheets. Amyloid-like oligomers cause over twenty mortal human diseases, including Alzheimer’s, Parkinson’s diseases and Amyotrophic Lateral Sclerosis (ALS). Paradoxically, other amyloids are key to vital physiological processes, including RNA regulation and memory consolidation. Despite advances, many details on the formation and energetics of amyloids are still unknown. In this thesis, I present results on the formation and factors that stabilize the amyloid formed by the yeast prion Sup35 heptapeptide GNNQQNY. My computational and experimental results revealed that the key intermediate in amyloidogenesis is a twisted, three-stranded, parallel β-sheet. This sheet dimerizes with facile release of water molecules to yield the cross-β spine structure. Hyper-stable H-bonds are crucial to the stability of this intermediate and to that of larger Sup35 oligomers and amyloids. A model for this nucleus’ formation is proposed, raising the question of whether noxious oligomers are a consequence of misfolded cross-β spines. TDP-43 (Transactive response DNA-binding protein 43 kDa) is particularly fascinating because its C-terminal region forms amyloid-like aggregates implicated in ALS yet also a putative functional amyloid vital to RNA regulation and transport. Since a debate existed as to whether TDP-43 aggregates are amyloid-like or amorphous, a series of biochemical and spectroscopic assays were performed, which revealed the amyloid-like nature of these aggregates. This includes fluorophore and cromophore binding, recognition by conformational antibodies, circular dichroism, X-ray diffraction, solution and solid-state NMR, and electron microscopy. Using spectroscopic and computational methods, the conformation of the putative pathological amyloid was investigated and structural models for the monomeric amyloidogenic intermediate and amyloid-like oligomers were determined. The structure consists of β-hairpins aligned in a “β-turn” topology and packed in parallel to form an amyloid-like structure whose topology is novel. The N-terminal domain (NTD) of TDP-43 plays key roles in regulating the functional and pathological aggregation of the C-terminal region. The structure of the NTD has been solved at high resolution by NMR methods, and it consists of an α-helix and 6 β-strands featuring a well-packed hydrophobic core, two exposed clusters of negatively charged residues and two separated, exposed cysteine residues. The conformational stability, determined by hydrogen/deuterium exchange, is 4 kcal/mol at pH 4 and 25 °C. The dynamic properties were elucidated using NMR methods, and show that the α-helix and 5 of 6 β-strands are rigid. One edge strand and the loops are more flexible. These findings advance our understanding of the NTD and provide the means to study its interaction with the C-terminal regio

Intrinsically disordered energy landscapes.

Analysis of an intrinsically disordered protein (IDP) reveals an underlying multifunnel structure for the energy landscape. We suggest that such 'intrinsically disordered' landscapes, with a number of very different competing low-energy structures, are likely to characterise IDPs, and provide a useful way to address their properties. In particular, IDPs are present in many cellular protein interaction networks, and several questions arise regarding how they bind to partners. Are conformations resembling the bound structure selected for binding, or does further folding occur on binding the partner in a induced-fit fashion? We focus on the p53 upregulated modulator of apoptosis (PUMA) protein, which adopts an α-helical conformation when bound to its partner, and is involved in the activation of apoptosis. Recent experimental evidence shows that folding is not necessary for binding, and supports an induced-fit mechanism. Using a variety of computational approaches we deduce the molecular mechanism behind the instability of the PUMA peptide as a helix in isolation. We find significant barriers between partially folded states and the helix. Our results show that the favoured conformations are molten-globule like, stabilised by charged and hydrophobic contacts, with structures resembling the bound state relatively unpopulated in equilibrium.The authors thank Prof. Jane Clarke, Dr. Chris Whittleston, Dr. Joanne Carr, Dr. Iskra Staneva and Dr. David de Sancho for helpful discussions. Y.C. and A.J.B. acknowledge funding from the EPSRC grant number EP/I001352/1, D.C. gratefully acknowledges the Cambridge Commonwealth European and International Trust for financial support and D.J.W. the ERC for an Advanced Grant.This is the final version. It was first published by NPG at http://www.nature.com/srep/2015/150522/srep10386/full/srep10386.html?WT.ec_id=SREP-639%2C638-20150526#abstract

Enhanced Sampling Techniques Reveal Key Information about Membrane Active Peptides

Membrane active peptides (MAPs) show promise in terms of future drug development. Whether it be adapting macrocycles for use in targeting protein-protein interactions or adopting cell-penetrating peptides (CPPs) to cause lysis in target cells, the field is burgeoning with possibilities for designer drugs. The following work encompasses the exploration of three such peptides. The pH-Low Insertion Peptide (pHLIP) is a membrane-active peptide that spontaneously folds into a transmembrane $\alpha$-helix upon acidification. This activity enables pHLIP to potentially act as a vector for drugs related to diseases characterized by acidosis such as cancer or heart ischemia. First, we explored the conformational space sampled by pHLIP while in bulk solution via constant pH molecular dynamics (MD) simulations. It was determined that pHLIP\u27s acidic residues are similar to single-residue-in-solution values and the P20G maintains a higher helicity in solution than wt-pHLIP. The next study was on the 17 N-terminal residues of the huntingtin (htt) protein (Nt17). Nt17 is essential for the patheoogenesis of Huntington\u27s Disease through its role in both htt aggregation and membrane association. We investigated Nt17 and its association with three model membranes with a consistent headgroup and tails with varying degrees of unsaturation and length. We found no correlation between the effect of lipid vesicles on aggregation and the degree of htt-lipid complexes formed, supporting that the properties of the membrane have direct influence on the aggregation mechanism. We also determined that Nt17-membrane association is regulated by complimentarily-sized hydrophobic residues in Nt17 and defects in the lipid bilayer. Finally, we developed a high-throughput assay for determining the permeability cyclic peptides. Targeting protein-protein interactions with traditional small-molecule drugs can be challenging when the binding pocket is too large. However, cyclic peptides are the key to targeting these interactions: passive membrane permeability and more consistent structure to prevent off targeting. Using a library of peptides with known permeability, we performed Gaussian accelerated molecular dynamics (GaMD) simulations on roughly 200 peptides of the library in octanol and water to estimate their permeability. Initially, we did not directly replicate the permeability from the experimental results, however, we did replicate the trend that correlates N-methylation and permeability. After using PCA to determine the peptides with biggest difference in populations between octanol and water, we more successfully reproduced the permeability data from experiment for those peptides

Coarse-Grained Molecular Dynamics Simulations of Peptide Aggregation on Surfaces

Protein aggregation involves self-assembly of normally soluble proteins or peptides into supramolecular structures. This process is particularly important due to its involvement in several amyloid diseases, such as Parkinson's, Alzheimer's, and Type II diabetes. Several fibrillization mechanisms have been proposed, including a condensation-ordering mechanism where ordered fibril structures emerge from disordered oligomers and a dock-lock mechanism where a growing fibril induces attached polypeptides to organize individually into fibril-compatible conformations.We present a series of computational studies using a coarse-grained peptide aggregate model that exhibits a rich diversity of structures: amorphous/disordered aggregates, beta-barrels, multi-layered fibrils, and aggregates of mixed type. Our model has a tunable backbone stiffness that governs the propensity to form fibrils in bulk solution. In this work, we investigate how this beta-sheet propensity couples with the properties of a surface template to influence the mechanism of aggregation. Here, we focus on peptide aggregation in the presence of three templates: a solid surface, the surface of a pre-existing aggregate seed, and a lipid bilayer. Aggregation on solid hydrophilic or hydrophobic surfaces frequently occurs in many experimental setups. We find that the solid surface strongly biases toward the formation of fibrillar aggregates. Peptide-peptide interactions and surface attraction couple cooperatively on a solid surface to influence the binding/aggregation transition. Aggregation and binding occur almost simultaneously since the surface's crystal symmetry enforces a preferred direction of bound fibril growth, thus accelerating the process.Seeding peptides with compatible aggregates removes the nucleation barrier for aggregation. We find that the aggregation mechanism is strongly dependent on the beta-sheet propensity of both the seed and bulk peptides. Additionally, bulk peptides that exhibit polymorphism can have multiple pathways to aggregation depending on which class of aggregate they initially form. We find that a fibrillar seed can induce amorphous-prone peptides into fibrillar structures via a condensation-ordering mechanism, thus sequestering potentially cytotoxic oligomers into a more inert form.We simulate aggregation on lipid bilayers in an effort to approximate the complexity of the cellular milieu. While aggregation in vivo would occur in the presence of membrane surfaces, few simulation studies have been conducted on this combined system due to its computational complexity. We have determined that a membrane surface, like a crystal surface, biases toward fibrillar aggregates. However, membrane undulations disturb multi-layered fibrils into non-planar beta-sheet structures, such as beta-barrels. The presence of fibrils on the membrane also affects its fluid properties, creating a hexagonally packed lipid ordering underneath the fibrils, locally increasing its bending modulus and aligning lipid tilt to the orientation of the peptides. Thus peptide aggregation and membrane fluidity affect each other's structure and dynamics.The key general features of a surface that control its modulation of peptide aggregation are its structural order and fluidity. An ordered, rigid template biases more strongly toward fibrillar structures and restricts the set of aggregation pathways and morphologies. The dynamic nature of a fluid surface biases less toward fibrils and enhances the range of aggregation dynamics

Next-generation coarse-grained models for molecular dynamics simulations of fluid phase equilibria and protein biophysics using the relative entropy

Coarse grained models for molecular dynamic simulations of liquid structure and protein folding and self-assembly have been the subject of decades of research efforts. Although such models enable probing into longer length and time scales, they are still limited in accuracy and scale poorly to thermodynamic conditions or chemical entities beyond the ones at which they are developed. In this work, I leverage a powerful coarse graining framework based on an information theoretic metric known as the relative entropy to design novel coarse grained models for equilibrium phase behavior in liquid mixtures that correctly address the relevant manybody physics critically involved in phase behavior and thus, can provide structurally accurate descriptions of macroscopic phase separation across a large range of mixture compositions. I also develop protein models that are “next- generation” in the sense that they are systematically extendable in complexity while depending minimally on experimental data. These protein models offer remarkable predictive accuracy for folding of single proteins and insights into large scale protein self-assembly commonly seen in neurodegenerative pathologies such as Alzheimer’s and prion diseases. This dissertation develops and applies powerful coarse-graining techniques to meet longstanding challenges in the field and elevate coarse grained models from being “toy” systems to accurate, “production-level” tools for the development of biotechnologies and advanced materials

Using Molecular Constraints and Unnatural Amino Acids to Manipulate and Interrogate Protein Structure, Dynamics, and Self-Assembly

Protein molecules can undergo a wide variety of conformational transitions occurring over a series of time and distance scales, ranging from large-scale structural reorganizations required for folding to more localized and subtle motions required for function. Furthermore, the dynamics and mechanisms of such motions and transitions delicately depend on many factors and, as a result, it is not always easy, or even possible, to use existing experimental techniques to arrive at a molecular level understanding of the conformational event of interest. Therefore, this thesis aims to develop and utilize non-natural chemical modification strategies, namely molecular cross-linkers and unnatural amino acids as site-specific spectroscopic probes, in combination with various spectroscopic methods to examine, in great detail, certain aspects of protein folding and functional dynamics, and to manipulate protein self-assemblies. Specifically, we first demonstrate how strategically placed molecular constraints can be used to manipulate features of the protein folding free energy landscape, thus, allowing direct measurement of key components via temperature-jump kinetic studies, such as folding from a transition-state structure or the effect of internal friction on the folding mechanism. Secondly, we utilize a photolabile non-natural amino acid, Lys(nvoc), to probe the mechanism of protein misfolding in a β-hairpin model and identify an aggregation gatekeeper that tunes the aggregation propensity. We further develop a method where the induced-charge produced by photocleavage of Lys(nvoc) can be used to target and destabilize hydrophobic regions of amyloid fibril assemblies, resulting in complete disassembly, Finally, we highlight new useful properties of a site-specific spectroscopic probe, 5-cyanotryptophan (TrpCN), by demonstrating (1) how the frequency and linewidth of the infrared nitrile stretching vibration is sensitive to multiple hydrogen bonding interactions and solvent polarity, (2) that the fluorescence emission, quantum yield, and lifetime is extremely sensitive to hydration, and serves as a convenient fluorescence probe of protein solvation status, and (3) that the unique characteristics of TrpCN can be used to target the structure, local environment, and mechanism of the tryptophan gate in the M2 membrane proton channel of the influenza A virus