Dimerization of Protegrin-1 in Different Environments

The dimerization of the cationic β-hairpin antimicrobial peptide protegrin-1 (PG1) is investigated in three different environments: water, the surface of a lipid bilayer membrane, and the core of the membrane. PG1 is known to kill bacteria by forming oligomeric membrane pores, which permeabilize the cells. PG1 dimers are found in two distinct, parallel and antiparallel, conformations, known as important intermediate structural units of the active pore oligomers. What is not clear is the sequence of events from PG1 monomers in solution to pores inside membranes. The step we focus on in this work is the dimerization of PG1. In particular, we are interested in determining where PG1 dimerization is most favorable. We use extensive molecular dynamics simulations to determine the potential of mean force as a function of distance between two PG1 monomers in the aqueous subphase, the surface of model lipid bilayers and the interior of these bilayers. We investigate the two known distinct modes of dimerization that result in either a parallel or an antiparallel β-sheet orientation. The model bilayer membranes are composed of anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) in a 1:3 ratio (POPG:POPE). We find the parallel PG1 dimer association to be more favorable than the antiparallel one in water and inside the membrane. However, we observe that the antiparallel PG1 β-sheet dimer conformation is somewhat more stable than the parallel dimer association at the surface of the membrane. We explore the role of hydrogen bonds and ionic bridges in peptide dimerization in the three environments. Detailed knowledge of how networks of ionic bridges and hydrogen bonds contribute to peptide stability is essential for the purpose of understanding the mechanism of action for membrane-active peptides as well as for designing peptides which can modulate membrane properties. The findings are suggestive of the dominant pathways leading from individual PG1 molecules in solution to functional pores in bacterial membranes

Potentials of Mean Force as a Starting Point for Understanding Biomolecular Interactions.

Computer simulations on the molecular dynamics of biological molecules can be used to explore the behavior of large molecules with a level of detail not possible in experiments. It is necessary to be cautious, however, when designing and interpreting such simulations, as the techniques commonly used to improve efficiency of simulations can lead to unrealistic results. The work presented in this dissertation explores ways in which the accuracy of molecular dynamics simulations can be both improved and validated by experimental data, primarily through the use of potentials of mean force (PMFs). Experimental input was used in the development of an umbrella sampling protocol by fitting restraining potentials to an experimental PMF. The method was tested on a model peptide using a ”guiding” PMF from simulations and then validated using an experimental PMF from force manipulation studies on a mechanical protein. The results show that the experimentally guided umbrella sampling replicates the appropriate pathways for both systems, whereas naively chosen potentials fail to do so. Experimental findings were also used in the design of steered molecular dynamics simulations on the β domain of streptokinase. High-temperature simulations were used to smooth the energy surface and enable the system to explore alternate unfolding pathways. The results show three distinct pathways, in agreement with experimental evidence of three types of behavior under force. The simulations reveal that the source of the differences are hydrophobic interaction in the core of the protein. Multi-dimensional PMFs were calculated to describe these pathways energetically. All-atom simulations were also used to study a different type of system, the interactions between DNA and a Polyamidoamine dendrimer. Both the dendrimer and DNA were found to deform substantially upon binding. While the interactions were shown to be driven primarily by electrostatics, we also find that ordered waters extend the interaction distance beyond the range of direct electrostatics for one orientation of the dendrimer. These water effects contribute almost a third of the total interaction free energy of the system. PMFs calculated in these simulations were used to calculate force extension curves which agree with experiments on DNA condensed by dendrimers.Ph.D.BiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/75883/1/pisaster_1.pd

Développements et applications de méthodes computationnelles pour l'étude de l'agrégation des protéines amyloïdes

Les protéines sont au coeur de la vie. Ce sont d'incroyables nanomachines moléculaires spécialisées et améliorées par des millions d'années d'évolution pour des fonctions bien définies dans la cellule. La structure des protéines, c'est-à-dire l'arrangement tridimensionnel de leurs atomes, est intimement liée à leurs fonctions. L'absence apparente de structure pour certaines protéines est aussi de plus en plus reconnue comme étant tout aussi cruciale. Les protéines amyloïdes en sont un exemple marquant : elles adoptent un ensemble de structures variées difficilement observables expérimentalement qui sont associées à des maladies neurodégénératives. Cette thèse, dans un premier temps, porte sur l'étude structurelle des protéines amyloïdes bêta-amyloïde (Alzheimer) et huntingtine (Huntington) lors de leur processus de repliement et d'auto-assemblage. Les résultats obtenus permettent de décrire avec une résolution atomique les interactions des ensembles structurels de ces deux protéines. Concernant la protéine bêta-amyloïde (AB), nos résultats identifient des différences structurelles significatives entre trois de ses formes physiologiques durant ses premières étapes d'auto-assemblage en environnement aqueux. Nous avons ensuite comparé ces résultats avec ceux obtenus au cours des dernières années par d'autres groupes de recherche avec des protocoles expérimentaux et de simulations variés. Des tendances claires émergent de notre comparaison quant à l'influence de la forme physiologique de AB sur son ensemble structurel durant ses premières étapes d'auto-assemblage. L'identification des propriétés structurelles différentes rationalise l'origine de leurs propriétés d'agrégation distinctes. Par ailleurs, l'identification des propriétés structurelles communes offrent des cibles potentielles pour des agents thérapeutiques empêchant la formation des oligomères responsables de la neurotoxicité. Concernant la protéine huntingtine, nous avons élucidé l'ensemble structurel de sa région fonctionnelle située à son N-terminal en environnement aqueux et membranaire. En accord avec les données expérimentales disponibles, nos résultats sur son repliement en environnement aqueux révèlent les interactions dominantes ainsi que l'influence sur celles-ci des régions adjacentes à la région fonctionnelle. Nous avons aussi caractérisé la stabilité et la croissance de structures nanotubulaires qui sont des candidats potentiels aux chemins d'auto-assemblage de la région amyloïde de huntingtine. Par ailleurs, nous avons également élaboré, avec un groupe d'expérimentateurs, un modèle détaillé illustrant les principales interactions responsables du rôle d'ancre membranaire de la région N-terminal, qui sert à contrôler la localisation de huntingtine dans la cellule. Dans un deuxième temps, cette thèse porte sur le raffinement d'un modèle gros-grain (sOPEP) et sur le développement d'un nouveau modèle tout-atome (aaOPEP) qui sont tous deux basés sur le champ de force gros-grain OPEP, couramment utilisé pour l'étude du repliement des protéines et de l'agrégation des protéines amyloïdes. L'optimisation de ces modèles a été effectuée dans le but d'améliorer les prédictions de novo de la structure de peptides par la méthode PEP-FOLD. Par ailleurs, les modèles OPEP, sOPEP et aaOPEP ont été inclus dans un nouveau code de dynamique moléculaire très flexible afin de grandement simplifier leurs développements futurs.Proteins are at the center of life. They are formidable molecular nanomachines specialized and optimized during million years of evolution for well-defined functions in the cell. The structure of proteins, meaning the tridimensional setting of their atoms, is closely related to their function. Absence of structure for a subset of proteins is also recognized to be as crucial. Amyloid proteins is a striking example : they fold into an ensemble of various structures hardly observable experimentally that are associated with neurodegenerative diseases. This thesis, firstly, is on the study of the structural ensemble of the amyloid proteins amyloid-beta (Alzheimer) and huntingtin (Huntington) during their folding and aggregation. Our results describe in details, with an atomic resolution, the characteristic interactions present in the structural ensemble of these two proteins. Concerning the amyloid-beta protein (AB), our results show the structural differences between three of its physiological forms during its first aggregation steps in an aqueous environment. We have then compared these results with those obtained during the past few years by several other research groups using various experimental and simulation protocols. Clear trends come out of this comparison regarding the influence of AB physiological form on its structural ensemble during its first aggregation steps. Their distinct aggregation pathways are rationalized by the identified differences. For their part, the identified similarities offer targets for therapeutical compounds disrupting the aggregation of the neurotoxic oligomers. Concerning the huntingtin protein, we identify the structural ensemble of its functional region at its N-terminal in an aqueous environment and in a phospholipid membrane. In agreement with the available experimental results on the global structure of this region in aqueous solution, our results reveal the dominant interactions, at an atomic precision, in its structural ensemble as well as the influence of its neighboring regions. We have also characterized the stability and the growth of nanotube-like structures that could occur during the aggregation of the amyloid region of huntingtin. Moreover, we have developed, in collaboration with a group of experimentalists, a precise model describing the main membrane interactions of huntingtin N-terminal, which serves as a membrane anchor that controls the localization of huntingtin in the cell. Secondly, this thesis is on the refinement of a coarse-grained model (sOPEP) and on the development of a new all-atom model (aaOPEP) that are both based on the coarse-grained OPEP force field, commonly used to study protein folding and amyloid protein aggregation. The goal behind the optimization of these models is to improve the de novo structure prediction of the PEP-FOLD method. These three models -- OPEP, sOPEP and aaOPEP -- are now also implemented in a new molecular dynamics software that we have developed specifically to greatly ease their future developments

Molecular dynamics simulations of protein-protein interactions and THz driving of molecular rotors on gold

The scope of this work is to gain insight and a deeper understanding of exploring and controlling molecular devices like proteins and rotors by fine tuned manipulation via mechanical or electrical energies. I focus on three main topics. First, I investigate vectorial forces as a tool to explore the energy landscape of protein complexes. Second, I apply this method to a biologically important force transduction complex, the integrin-talin complex. Third, I use Terahertz electric fields to manipulate the energy landscape of a molecular rotor on a gold surface and drive their effective rotation bidirectionally. Force is by nature a vector and depends on its three parameters: magnitude, direction and attachment point. Here, the impact of different force protocols varying these parameters is shown for an antibody-antigen complex and the ribonuclease-inhibitor complex barnase-barstar. Antibodies are essential for our adaptive immune system in their function to bind specific antigens. Here, the binding of an antibody to a peptide is probed with varying attachment points. Different attachment points clearly change the dissociation pathways. The barriers identified using experimental atomic force microscopy (AFM) and molecular dynamics (MD) simulations are in excellent agreement. I determine the molecular interactions of two main barriers for each setup. This results in a common outer barrier of the complex and different inner barriers probed by AFM. The ribonuclease barnase and its inhibitor barstar form an evolutionary optimized complex. Different force protocols are shown to determine the hierarchy of relative stability within a protein complex. For the barnase-barstar complex, the internal fold of the barstar is identified to be less stable than the barnase-barstar binding interaction. High velocities probe the lability or barriers of the system while low velocities probe the stability or energy wells of this system. Forces impact biological life on totally different length scales which range from whole organisms to individual proteins. Integrins are the major cell adhesion receptors binding to the extracellular matrix and talin. Talin activates the integrins and creates the initial connection to the actin cytoskeleton of the cell. Here, I have chosen to investigate the integrin-talin complex as a biologically important force transduction complex. The force dependence of the system is probed by constant force MD simulations. The two main results include the activation of the complex and its force response. I demonstrate, that the binding of talin to integrin does not disrupt the integrin's transmembrane helix interactions sterically. Since, this disruption is necessary for integrin activation, a modified activation mechanism requiring a small force application is proposed. The response of the integrin-talin complex normal and parallel to the cell membrane is analyzed. The complete dissociation pathways generated for both directions identify a force-induced formation of a stabilizing beta strand between integrin and talin only for normal forces. Furthermore, the complex tries to rotate such that the external force aligns with the more force resistant axis of the complex. In nature, molecular rotors are essential building blocks of many molecular machines and brownian motors like the F1-ATPase or the flagellum of a bacterium. The direction of rotation often steers different processes in clockwise and counterclockwise directions. Rotation on the nanoscopic level in artificial devices is still very limited and requires a deeper understanding. In my last project, I study the switching and driving of a molecular diethylsulfid rotor on a gold (111) surface by Terahertz electric fields. The response of the rotational energy landscape to static and oscillation electric fields is analyzed. Varying the Terahertz driving frequency, the rotation direction and frequency are controlled. A theoretical framework is presented to describe the behavior of the molecular rotor. This can be seen as the first step into the direction of man-made controllable nano-devices driven and controlled by energy from the electric wall-socket.Proteine sind die molekularen Maschinen der Zelle. Sie gehören zu den essentiellen Grundbausteinen des Lebens und dynamische Protein-Protein Wechselwirkungen steuern das Leben auf zellulärer Ebene. Thema dieser Arbeit ist es mittels Computersimulationen ein besseres Verständnis von Proteinkomplexen und molekularen Rotoren zu erlangen. Hierbei konzentriere ich mich auf drei Schwerpunktthemen: Erstens trage ich dazu bei ein besseres Verständnis zur methodischen Untersuchung der Energielandschaften von Proteinen und Proteinkomplexen mittels der Anwendung vektorieller Kräfte zu erlangen. Am Beispiel eines Antikörper-Antigen und eines Ribonuclease-Inhibitor Komplexes werden die Auswirkungen verschiedener Kraftparameter (Betrag, Richtung, Angriffspunkt und Zuggeschwindigkeit) auf die Entwicklung des Systems unter Krafteinfluss untersucht. Hervorzuheben sind die exzellenten Übereinstimmungen zwischen experimentellen Ergebnissen der Atomaren Kraftmikroskopie mit den Molekular Dynamik Simulationen im Antikörper-Projekt. Zweitens studiere ich den Integrin-Talin Komplex, welcher die initiale kraftleitende Verbindung zwischen Zellinnerem und -äußerem schafft. Die zwei wichtigsten Ergebnisse sind die Erweiterung des Aktivierungsmechanismusses des Integrins um eine zusätzlich benötigte Kraftkomponente und die Entdeckung der kraftinduzierten Stabilisierung des Komplexes durch die Ausbildung eines stabilisierenden beta-Faltblatts zwischen Integrin und Talin. In meinem dritten Projekt untersuche ich einen diethylsulfid Rotor auf einer Gold (111) Oberfläche mittels MD Simulationen. Die Energielandschaft dieses Rotors kann mit elektrischen Feldern im Terahertzbereich so manipuliert werden, dass die effektive Rotationsrichtung und -frequenzen im Gigahertzbereich gesteuert werden kann. Eine theoretische Beschreibung dieses Phänomens und seine Abhängigkeit von der Struktur des Rotors werden behandelt. Dies kann ein erster Schritt zu einem Interface zwischen bekannten elektrischen Schaltungen und zukünftigen artifiziellen Nanomaschinen sein