Molecular Dynamics Study of Single Stranded Peptide Nucleic Acids

A PNA molecule is a DNA strand where the sugar-phosphate backbone has been replaced by a structurally homomorphous pseudopeptide chain consisting of N(2-aminoethyl)-glycine units. PNA binds strongly to both DNA and RNA. However, an analysis of the X-ray and NMR data show that the dihedral angles of PNA/DNA or PNA/DNA complexes are very different from those of DNA:DNA or RNA:RNA complexes. In addition, the PNA strand is very flexible. One way to improve the binding affinity of PNA for DNA/RNA is to design a more pre-organized PNA structure. An effective way to rigidify the PNA strand is to introduce ring structures into the backbone. In several experimental studies, the ethylenediamine portion of aminoethylglycine peptide nucleic acids (aegPNA) has been replaced with one or more (S,S)- trans cyclopentyl (cpPNA) units. This substitution has met with varied success in terms of DNA/RNA recognition. In the present work, molecular modeling studies were performed to investigate PNA and modified PNA analogs. Molecular dynamics (MD) simulations is a principal tool in the theoretical study of biological molecules. This computational method calculates the time dependent behavior of a molecular system and provides detailed information on the fluctuations and conformational changes. The MD simulation uses an empirical parameterized energy functions. These parameters play an important role in the quality of the simulations. Therefore, novel empirical force field parameters were developed for cyclopentane modified PNA analogs. We demonstrate that our parameterization can accurately reproduce high level quantum mechanical calculations. Detailed investigations on the conformational and dynamical properties of single stranded aegPNA and cpPNA were undertaken to determine how the cyclopentane ring will improve binding and to determine the contributions of both entropy and dihedral angle preference to the observed stronger binding. The effects of single and multiple modifications of the PNA backbone were also analyzed to understand changes in conformational and dynamical properties

Structure and Activity of Antimicrobial Peptoids

This thesis concerns complementary experimental and computational investigations into the relationship between the primary sequence and secondary structure of peptoids. Peptoids are a class of peptide mimetic molecules with applications as novel antimicrobial agents. The antimicrobial properties of peptoids are linked to their interactions with lipid bilayers in cell membranes, which in turn are linked to their helical secondary structure, making understanding sequence to structure relationships crucial to the design of functional sequences. Here we investigate a library of linear, cationic peptoid sequences with structural variations in the proportion and positioning of helix inducing residues and the chemical nature of the cationic side chains. We use circular dichroism spectroscopy to characterise the peptoids in aqueous and organic solvent and also to investigate structural changes upon binding to lipid bilayers designed to mimic mammalian and bacterial membranes. We present a new set of force field parameters, derived from GAFF and quantum mechanical calculations, that accurately capture the backbone torsional preferences of peptoids. Subsequently we use the modified force field to perform atomistic MD simulations of our library of peptoid sequences, using Hamiltonian replica exchange to improve sampling at less computational expense than traditional replica exchange methods. The CD spectra reveal that the peptoids adopt characteristically helical secondary structures with variations depending on primary sequence. The intensity of helical features increases upon increasing the proportion of helix inducing residues, switching from an aqueous to an organic environment and as extra methylene groups are added to the cationic side chains, increasing their length. The length and proportion of cationic side chains also influences the folded hydrophobicity of the peptoids, though this does not correlate to their antimicrobial activity. Modelling the binding of the peptoids to lipids as a two state system enables us to estimate, in some cases, the free energy of transfer into the bilayer, where the length of the cationic side chain is also influential. MD simulations do not reveal a clear distinction in peptoid backbone conformation depending on cationic side chain length however it is clear that the peptoid backbone is more flexible and deviates more from a perfect helical conformation in aqueous than organic solvent. Ultimately these findings may aid in the rational design of new sequences

Multi-scale Modelling of Allostery in Protein Homodimers

Allostery is a form of signalling within biomolecules such that ligand binding to a protein affects its activity at a second site. Allostery was described by early models to be driven by structural changes in the protein. However, more recently there has been increasing evidence that dynamics can contribute to or even drive allostery. The protein studied in this thesis, the Catabolite Activator Protein (CAP), is an allosteric protein homodimer that has been shown to exhibit negatively cooperative binding of the ligand cyclic Adenosine Monophosphate (cAMP) to each of its monomers. Interestingly, CAP is a protein whose allostery is believed to be driven by dynamics rather than a conformational change. In this thesis, a number of coarse grained models are employed to investigate this dynamic allostery in CAP. One family of models, termed Super Coarse Grained (SCG) models explore the global properties of the dynamics of the CAP dimer that cause it to exhibit negatively cooperative allostery. It is shown through these models that changes in protein interactions can provide a basis for changing cooperativity. A second family of coarse grained models called Elastic Network Models (ENM) are studied. These are used to show that adjusting the interactions between specific residues can affect cooperative binding of cAMP to CAP. A number of atomistic approaches are also used to study the cAMP-CAP system, including Molecular Dynamics (MD) and Normal Mode Analysis (NMA). The efficacy of using such approaches for studying the thermodynamics of the allostery in CAP is investigated. The motion observed within the protein is also studied closely to identify potential allosteric pathways. X-ray crystallography and Isothermal Titration Calorimetry (ITC) are finally used to investigate how accurately computational methods can describe the cooperative binding of cAMP to CAP. They are also used to try and determine whether the allostery in CAP can be manipulated experimentally without any observed changes to its structure

X-ray crystallographic studies on adenylosuccinate synthetase from Escherichia coli

Adenylosuccinate synthetase catalyzes the first committed step in de novo biosyntheses of AMP from IMP and aspartate, using GTP as the energy source. Metal cations, ligand-induced conformational transitions and metabolic effectors significantly influence the catalytic potential of the synthetase. The Mg2+, Mn2+, or Ca 2+ bind to the same site, coordinating alpha-, beta-, and gamma-phosphoryl groups of GTP. The level of catalysis supported by each cation is linked to its influence on the basicity of Asp13, a residue which abstracts the proton from N1 of IMP. Zn2+, a potent inhibitor of the synthetase, coordinates the beta- and gamma-phosphoryl groups of GTP and His41, stabilizing a dead-end complex of the synthetase. The role of metal cations in the synthetase and the related G-proteins contrasts sharply. Hydrogen bond formation between the 5\u27-phosphate of IMP and Asn38 drives large conformational changes as far as 30 A away. Indeed the 5\u27-phosphoryl group of IMP, even though it is not directly involves in catalysis, is as important to the stability of the transition state as essential protein side chains directly involved with catalysis. Guanosine 5\u27diphosphate 3\u27-diphosphate (ppGpp), a pleiotropic effector of the stringent response, potently inhibits the synthetase. The combination of ppGpp with crystals of the synthetase, however, reveals guanosine 5\u27 -diphosphate 2\u27:3\u27-cyclic monophosphate, at the GTP Pocket. The sythetase itself may catalyze the formation of the cyclic inhibitor, leading to a tight ligand-enzyme complex. In fact, stringent effectors could modulate synthetase activity by way of several mechanisms, including the formation of a 6-phosphoryl-IMP and cyclic nucleotide complex

Minimalistic Peptide-Based Supramolecular Systems Relevant to the Chemical Origin of Life

All forms of life are based on biopolymers, which are made up of a selection of simple building blocks, such as amino acids, nucleotides, fatty acids and sugars. Their individual properties govern their interactions, giving rise to complex supramolecular structures with highly specialized functionality, including ligand recognition, catalysis and compartmentalization. In this thesis, we aim to answer the question whether short peptides could have acted as precursors of modern proteins during prebiotic evolution. Using a combination of experimental and computational techniques, we screened a large molecular search space for peptide sequences that are capable of forming supramolecular complexes with adenosine triphosphate (ATP), life’s ubiquitous energy currency, and uridine triphosphate (UTP). Our results demonstrate that peptides as short as heptamers can form dynamic supramolecular complexes, adapt their structure to a ligand upon binding, undergo phase-separation into spatially confined compartments and catalyze nucleotide-hydrolysis

Structure and dynamics of the NO sensing domain of the human soluble Guanylate Cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric heme-protein composed of two subunits called α and β [1-5]. The most common heterodimeric form is the combination of α1 with β1 subunits [1]. The α1 (80KDa) and β1 (70 KDa) subunits are 690 and 619 amino acids in length respectively, and are encoded by the genes, GUCY1A2 and GUCY1A3 respectively [3].(...

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field

Study of conductive polymers, biomolecules and their hybrids through computational approaches

In this thesis, different theoretical approaches designed to study a wide interval of length- and/or time-scales have been used to examine the microscopic properties of chemical systems with varying degrees of complexity and size. Firstly, the conformational preferences and optical properties of a tripeptide derived from the RGD sequence, the unit of a cell adhesive activity domain in adherent proteins, have been analyzed. Calculations on this peptide, which contains an exotic amino acid bearing a 3,4-ethylenedioxythiophene (EDOT) ring as side group, have been performed using DFT and time depending DFT methods. Results indicate that the bioactive characteristics of the RGD sequence become unstable in presence of the new residue because of both the steric hindrance caused by the EDOT side group and the repulsive interactions between the oxygen atoms belonging to the backbone amide groups and the EDOT side group. This information has been used to propose some chemical changes oriented to improve the bioadhesive properties. The interaction of a cell penetrating tetrapeptide, RPAR, adsorbed onto a gold substrate and the deposition of a pre-assembled peptide-polymer conjugate, cyc[(L-Gln-D-Ala-L-Lys-D-Ala)2] coupled with two poly(n-butyl acrylate) blocks, onto a mica substrate have been examined through molecular dynamics (MD). Results indicate that RPAR binds both the (100) and (111) gold surface facets. The conformation of the adsorbed peptide differs considerably from the bioactive conformation. However, the new conformations are not stabilized by strong intramolecular interactions. Accordingly, gold nanoparticles can be considered as suitable vehicles for the transport and targeted delivery of this CendR peptide. For the pre-assembled peptide-polymer conjugate, a theoretical approach that simulates the selective and progressive desolvation of the nanotube-like assembly has been used, demonstrating that the solvent presence during deposition is the main responsible for the unexplained conformational preferences of the acrylate blocks. When the proportion of solvent drops, the loss of many attractive solute-solvent interactions induces a meaningful increase in the number of torsions. MD has been also used to understand the impact of the solvation medium and the action of a detergent in the structure of a representative outer membrane protein (OMP). Calculations show the destabilization of the protein in water, while in presence of detergent molecules in solution or the bilayer induce a partial and complete protective effect, respectively. Combining stochastic algorithms and MD simulations the atomistic details of polymer coatings deposited over metal surfaces have been modeled to reproduce the experimentally observed topographic features of poly(3,4-ethylenedioxythiophene), PEDOT, deposited onto stainless steel. Results have provided an excellent model system to test the developed modeling strategy. Multi-phasic simulations have been conducted to explain the influence of protein···polymer interactions in the antimicrobial biocapacitors activity of lysozyme (LYZ)-containing PEDOT electrodes through atomistic MD calculations. Two models have been investigated: i) a biphasic system in which the protein was adsorbed onto the surface of PEDOT, PEDOT/LYZ; and ii) a biocomposite in which the LYZ was homogeneously distributed inside the PEDOT matrix, P(EDOT-LYZ). MD simulations have been performed in absence and presence of electric fields, the latter mimicking the one originated by the voltage cell difference in biocapacitors. In PEDOT/LYZ electrodes, the loss of biological activity has been attributed to the anisotropy of the PEDOT···LYZ electrostatic interactions. In contrast, anisotropic effects are minimized in P(EDOT-LYZ), conserving activity. The bactericidal activity of PEDOT/LYZ and P(EDOT-LYZ) biocapacitors is independent of the electric field applied or supplied during charge-discharge processes.En esta tesis, diferentes métodos diseñados para estudiar un amplio intervalo de tamaños y/o escalas de tiempo han sido usados para examinar las propiedades microscópicas de sistemas químicos con diferentes grados de complejidad y magnitud. Primeramente, se han analizado las preferencias conformacionales y las propiedades ópticas de un tripéptido derivado de la secuencia RGD, el dominio de actividad adhesiva en proteínas adherentes. Los cálculos de este péptido, que contiene un aminoácido exótico que porta un anillo de 3,4-etilendioxitiofeno (EDOT) como grupo lateral, han sido llevados a cabo usando métodos de DFT y DFT dependiente del tiempo. Los resultados indican que las características bioactivas de la secuencia RGD se vuelven inestables en presencia del nuevo residuo debido tanto a los impedimentos estéricos causados por el EDOT de la cadena lateral como por las interacciones repulsivas entre los átomos de oxígeno de los grupos amida que pertenecen a la cadena principal del péptido y del grupo lateral de EDOT. Esta información ha sido utilizada para proponer algunos cambios en la composición química orientados a mejorar las propiedades bioadhesivas. La interacción de un tetrapéptido permeabilizador vascular, RPAR, que ha sido adsorbido sobre un sustrato de oro y la deposición de un conjugado péptido-polímero pre-ensamblado, cyc[(L-Gln-D-Ala-L-Lys-D-Ala)2] unido a dos poli(n-butil acrilatos), sobre un sustrato de mica han sido examinados a través de dinámica molecular (MD). Los resultados indican que RPAR se une tanto a las caras (100) y (111) del oro. La conformación del péptido adsorbido difiere considerablemente de la conformación bioactiva. Aún así, las nuevas conformaciones no son estabilizadas por fuertes interacciones intramoleculares. De acuerdo a estos resultados, las nanopartículas de oro pueden ser consideradas como vehículos indicados para el transporte y liberación selectiva de este péptido CendR. Para el conjugado péptido-polímero pre-ensamblado, se ha usado un método teórico que simula la desolvatación progresiva del nanotubo, demostrando que la presencia del solvente durante la deposición es el principal responsable para las preferencias conformacionales inexplicadas de los bloques acrílicos. Cuando la proporción de solvente cae, la pérdida de muchas interacciones atractivas entre soluto y solvente induce un significante aumento en el número de torsiones. También se ha usado MD para entender el impacto del medio solvente y la acción de un detergente en la estructura de una proteína de membrana exterior (OMP) representativa. Los cálculos muestran la desestabilización de la proteína en agua, mientras que la presencia de moléculas de detergente en solución o en bicapa inducen un efecto protector parcial y completo, respectivamente. Combinando algoritmos estocásticos y simulaciones de MD, se ha modelado los detalles a nivel atómico de los recubrimientos de poli(3,4-etilendioxitiofeno), PEDOT, sobre acero inoxidable para reproducir las propiedades topográficas observadas experimentalmente. Los resultados han proporcionado un modelo excelente para probar la estrategia de modelado desarrollada. Se han realizado simulaciones multifásicas para explicar la influencia de las interacciones proteína-polímero en la actividad antimicrobiana de los condensadores que contienen electrodos lisozima (LYZ)-PEDOT, a través de cálculos de MD atomísticos. Dos modelos se han investigado: i) un sistema bifásico en los que la proteína ha sido adsorbida sobre la superficie de PEDOT, PEDOT/LYZ; y ii) un biocompuesto en el que la LYZ ha sido distribuida homogéneamente dentro la matriz de PEDOT, P(EDOT-LYZ). Las simulaciones de MD se han realizado tanto en ausencia como en presencia de campos eléctricos, el último para simular los originados por la diferencia de voltaje en los biocondensadores. La iso- y aniso-tropía de las interacciones son claves para la conservaciónPostprint (published version