Investigations of the Unique Role of Alanines in the 'Elastin Puzzle' by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations.

Ph.D. Thesis. University of Hawaiʻi at Mānoa 2017

Proteins in silico-modeling and sampling

Proteins are linear chain molecules made out of amino acids. Only when they fold to their native states, they become functional. This dissertation aims to model the solvent (environment) effect and to develop & implement enhanced sampling methods that enable a reliable study of the protein folding problem in silico. We have developed an enhanced solvation model based on the solution to the Poisson-Boltzmann equation in order to describe the solvent effect. Following the quantum mechanical Polarizable Continuum Model (PCM), we decomposed net solvation free energy into three physical terms– Polarization, Dispersion and Cavitation. All the terms were implemented, analyzed and parametrized individually to obtain a high level of accuracy. In order to describe the thermodynamics of proteins, their conformational space needs to be sampled thoroughly. Simulations of proteins are hampered by slow relaxation due to their rugged free-energy landscape, with the barriers between minima being higher than the thermal energy at physiological temperatures. In order to overcome this problem a number of approaches have been proposed of which replica exchange method (REM) is the most popular. In this dissertation we describe a new variant of canonical replica exchange method in the context of molecular dynamic simulation. The advantage of this new method is the easily tunable high acceptance rate for the replica exchange. We call our method Microcanonical Replica Exchange Molecular Dynamic (MREMD). We have described the theoretical frame work, comment on its actual implementation, and its application to Trp-cage mini-protein in implicit solvent. We have been able to correctly predict the folding thermodynamics of this protein using our approach

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

Biological applications of discrete molecular dynamics

[eng] Sequence, structure and dynamics are an indivisible tandem to understand protein function. Luckily, evolution imposed a hierarchical rational between that facilitates the analysis: dynamics are encoded in the structure, which in turn, is encoded in the sequence. Decipher the mechanisms governing protein function requires contributions from diverse fields, particularly to follow molecular motions. There are technological limitations to monitor local, elemental, protein movements, since they are too fast to be followed by current experimental set-ups. Theoretical models provide necessary assistance in this regard mainly through molecular simulations. But atomistically simulations of large functional motions make computations, currently, unaffordable. The problem is that large-scale motions are rooted in the very fast elemental ones; so, in order to observe a biological-functional conformational change we have to keep track of all the elemental motions occurring. The gap in the time scale of both extremes of motions is devastating: fast motions are over 1015 times faster than functional ones. In this Thesis, I present our contribution to extend the simulation time range, in an effort towards more predictive computational models. We explored alternative methods to retrieve molecular motions from the underlying physical forces governing proteins. The method used is named Discrete Molecular Dynamics and represents by itself a significant improvement in computational efficiency. In order to go further, we lower the resolution of protein models to a coarse-grained representation both in terms of number of particles and interaction functions. We benefited from several existing algorithms to simplify calculations keeping the models as much accurate as possible. Putting all this methodological innovations together, we developed models to follow conformational transitions of proteins, from local re-arrangements to motions changing drastically the protein structure. Also, we applied novel computational approaches to account for protein flexibility upon recognizing and binding other interacting proteins. In a second stage, we investigated the echo of protein flexibility and dynamics printed out in the sequence of the protein. We observed over the history of the sequence that instead of one single native structure, proteins were tuned to have several conformations. We exploited this flexibility signature in the sequence to predict protein motions and eventually alternative protein conformations. Finally, we use our efficient tools to move protein dynamics analysis to the proteome scale. We searched for all proteins having two known conformations, a symptom of a conformational transition, and then, we used those conformations to follow the motion from one state to the other. We analyzed and structured all that dynamical information of proteins and connected our results to the most detailed simulation methods available to dissect the fine details of proteins dynamical behavior when required.[spa] Secuencia, estructura y dinámica forman un trío un insoslayable en el funcionamiento de las proteínas. El proceso evolutivo codificó la dinámica en la estructura de las proteínas, que a su vez, está codificada en la secuencia. Descifrar los mecanismos que rigen el movimiento de las proteínas requiere la fusión de experimentos y modelos teóricos. Los modelos teóricos proporcionan asistencia necesaria a través de simulaciones moleculares, pero su costo computacional es tan elevado que puede impedir el estudio. El problema radica en que los movimientos biológicamente interesantes son la consecuencia de un cúmulo de movimientos de alta frecuencia, que es necesario seguir para comprender los movimientos funcionales. La brecha entre ambos tiempos asciende a un impresionante ratio de 1015. En esta Tesis, presento métodos para aumentar la eficacia de los cálculos moleculares con el objetivo de acortar la diferencia entre el tiempo de lo que es simulable a lo que es biológicamente interesante. El método utilizado es Discrete Molecular Dynarnics y representa por sí mismo una mejora significativa en la eficiencia computacional. En resumen, hemos desarrollado modelos para seguir transiciones conformacionales de proteínas, desde movimientos locales hasta otros que cambian radicalmente la forma de la proteína. Dichos métodos fueron aplicados tanto a transiciones conformacionales como a interacciones proteína-proteína. En una segunda etapa, buscamos la imprenta en la secuencia del patrón de flexibilidad de la proteína, con el objetivo de predecir los cambios de conformación. Finalmente, utilizando los métodos desarrollados hemos concluido un análisis a gran escala sobre la dinámica de las proteínas, simulando todas las transiciones cuyos dos extremos fueron determinados experimentalmente. Los resultados de dichas simulaciones fueron integrados con los métodos de simulación más fiables disponibles, para aumentar en nivel de detalle cuando sea necesario

Multiplexed single molecule observation and manipulation of engineered biomolecules

Molecular processes in organisms are often enabled by structural elements resilient to mechanical forces. For instance, the microbial and hierarchical cellulosome protein system comprises enzymes and the receptor-ligand complexes Cohesin-Dockerin (Coh-Doc), that act in concert for the efficient hydrolysis of plant polysaccharides. The Coh-Doc complexes can withstand remarkably high forces to keep host cells and enzymes bound to their substrates in the extreme environmental conditions the microorganisms frequently live in. This work focuses on the investigation of mechanical stability of such biomolecules on the single-molecule level. The highly symmetric binding interface of the Coh-Doc type I complex from Clostridium thermocellum, enables two different binding conformations withcomparable affinity and similar strength. I was able to show that both conformations exist in the wild-type molecules and are occupied under native conditions. I further characterized one of the strongest non-covalent protein complexes known, Coh-Doc type III from Ruminococcus flavefaciens by elucidating the pivotal role of the adjacent xModule domain for the mechanical stabilization of the whole complex and the role of the bimodal rupture force distribution. Such large forces impair accuracy of measured contour length increments in unfolding studies by inducing conformational changes in poly-ethylene glycol (PEG) linkers in aqueous buffer systems. This problemwas solved by introducing elastin-like polypeptides (ELP) as surface tethers. Having a peptide backbone similar to that of unfolded proteins, ELP linkers do not alter accuracy of the single-molecule force spectroscopy (SMFS) assay. To provide high throughput and precise comparability, I worked on a microfluidic platform for the in vitro protein synthesis and immobilization. The Coh-Doc system was hereby integrated as a binding handle for multiplexed measurements of mechanostability. Employing a single AFM probe to measure multiple different molecules facilitates force precision required to shed light onto molecular mechanisms down to the level of single amino acids. I also applied the Coh-Doc complex to a purely protein based single-molecule cut and paste assay for the bottom-up assembly of molecular systems for quick phenotyping of spatial arrangements. With this system, interactions in enzymatic synergies can be studied by defined positioning patterns on the single molecule level. To understand and design force responses of complex systems, I complemented the investigation of protein systems with SMFS studies on DNA Origami structures. The results of SMFS on DNA were compared to a simulation framework. Despite their difference in force loading rates, both methods agree well within their results, enabling better fundamental understanding of complex molecular superstructures.Molekulare Prozesse in Organismenwerden oft von Strukturelementen ermöglicht, die mechanischen Kräften standhalten können. Ein Beispiel hierfür ist das mikrobielle und hierarchisch aufgebaute Proteinsystem des Zellulosoms. Enzyme und die Rezeptor-Liganden Komplexe Cohesin-Dockerin (Coh-Doc) arbeiten hierbei für die effiziente Hydrolyse von pflanzlichen Polysacchariden zusammen. Die Coh-Doc Komplexe können bemerkenswerten Kräften standhalten, um in den extremen Umweltbedingungen, in denen die Mikroorganismen teilweise leben, die Wirtszellen und Enzyme an ihre Substrate binden zu können. Die vorliegende Arbeit untersucht den Einfluss von mechanischer Kraft auf solche Biomoleküle mittels Einzelmolekülmessungen. Die hohe Symmetrie des Bindeinterfaces des Coh-Doc Typ I Komplexes aus Clostridium thermocellum ermöglicht zwei verschiedene Konformationen, die vergleichbare Affinität und Stärke aufweisen. Im Rahmen dieser Arbeit konnte ich beide in denWildtyp-Molekülen und unter nativen Bedingungen nachweisen. Eines der stärksten bekannten nicht-kovalenten Rezeptor-Liganden Systeme, Coh- Doc Typ III aus Ruminococcus flavefaciens wurde charakterisiert, und die Kernrolle des benachbarten xModuls für die Stabilität des gesamten Komplexes sowie die Rolle der bimodalen Kraftverteilung untersucht. Solch hohe Kräfte vermindern die Genauigkeit der gemessenenKonturlängeninkremente von Proteinentfaltungen, indem sie Konformationsänderungen der Poly- Ethylenglykol (PEG) Oberflächenanker in wässrigen Puffersystemen verursachen. Mit Elastin-ähnlichen Polypeptiden (ELP) als Anker wurde dieses Problem gelöst: durch die Ähnlichkeit des Peptid-Rückgrates von ELPs mit dem entfaltener Proteine beeinflussen diese die Genauigkeit des Experiments nicht. Für die Optimierung von Messdurchsatz und Vergleichbarkeit entwickelte ich an einer Mikrofluidik-Plattform zur in vitro Proteinsynthese und -immobilisierung. Das Coh-Doc System wurde hierbei als Binde-Molekül für gemultiplexte Messungen integriert. Die dadurch ermöglichte Nutzung einer einzigen AFM Messsonde für die Messung verschiedener Moleküle erlaubt die nötige Kraftpräzision, um molekulare Mechanismen bis auf die Ebene einzelner Aminosäuren aufzuklären. Des weiteren habe ich den Coh-Doc Komplex in einem rein auf Proteininteraktionen basierten ’Cut and Paste’ Assay für den modularen Aufbau molekularer Systeme implementiert. Dieses ermöglicht schnelle Phänotypisierung geometrischer Anordnunungen und die Untersuchung von Wechselwirkung zwischen Enzymen mittels definierter Positionierung auf Einzelmolekülebene. Um die Kraftantwort komplexer Systeme besser verstehen und letztendlich gestalten zu können, ergänzte ich die Untersuchung von Proteinsystemen mit derer von DNA-Origami Strukturen. Die Ergebnisse der Kraftspektroskopie an DNA wurden mit Computersimulationen verglichen, und trotz des großen Unterschieds ihrer Ladungsraten stimmen beide Methoden gut überein. Dadruch legen sie die Grundlagen für ein besseres Verständnis komplexer molekularer Superstrukturen

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

Doctor of Philosophy

dissertationThe coiled-coil is a common protein tertiary structural motif that is composed of two or more alpha helices intertwined together to formed a supercoil. In biological systems, the coiledcoil motif often forms the oligomerization domain of various proteins including DNA binding proteins, structural and transport proteins, and cellular transport and fusion proteins. It was first described by Crick in the 1950s while describing the structure of α-keratin and has since that time been the subject of numerous engineering and mutation studies. This versatile motif has been adapted to a number of nonbiological applications including environmentally responsive hydrogels, crosslinking agents, the construction of self-assembling fibers for tissue engineering, and biosensor surfaces. In this dissertation, we test the applicability of computational methods to understand the underlying energetics in coiled-coils as we apply molecular modeling approaches in the development of pharmaceutics. Two studies are described which test the limits of modern molecular dynamic force fields to understand the structural dynamics of the motif and to use energy calculation methodologies to predict favorable mutations for heterodimer formation and specificity. The first study considers the increasingly common use of fluorinated residues in protein pharmaceutics with regard to their incorporation in coiled-coils. Many studies find that fluorinated residues in the hydrophobic core increase protein stability against chemical and thermal denaturants. Often their incorporation fails to consider structural, energetic, and geometrical differences between these fluorinated residues and their nonfluorinated counterparts. To consider these differences, several variants of Hodges' very stable parallel heterodimer coiledcoil were constructed to examine the effect of salt bridge lengths and geometries with mixed fluorinated and nonfluorinated packed hydrophobic cores. In the second study, we collaborated with an experimental laboratory in the development of a mutant Bcr monomer with designed mutations to increase specificity and binding to the oncoprotein Bcr-Abl for use as an apoptosis inducing agent in chronic myelogenous leukemia (CML) cells. The final chapters of this dissertation discuss challenges and limitations that were encountered using force fields and energetic methods in our attempts to use computational chemistry to model this protein motif