Academic literature on the topic 'Biomolecular motors'

Myosin II is a biomolecular machine that is responsible for muscle contraction. Myosin II motors act cooperatively: during muscle contraction, multiple motors bind to a single actin filament and pull it against an external load, like people pulling on a rope in a tug-of-war. We model the dynamics of actomyosin filaments in order to study the evolution of motor–motor cooperativity. We find that filament backsliding—the distance an actin slides backward when a motor at the end of its cycle releases—is central to the speed and efficiency of muscle contraction. Our model predicts that this backsliding has been reduced through evolutionary adaptations to the motor’s binding propensity, the strength of the motor’s power stroke, and the force dependence of the motor’s release from actin. These properties optimize the collective action of myosin II motors, which is not a simple sum of individual motor actions. The model also shows that these evolutionary variables can explain the speed–efficiency trade-off observed across different muscle tissues. This is an example of how evolution can tune the microscopic properties of individual proteins in order to optimize complex biological functions.

xiv, 171 p. ; ill. (some col.) A print copy of this title is available through the UO Libraries. Search the library catalog for the location and call number.
Biological molecular motors, which use chemical energy from ATP hydrolysis to generate mechanical force, are involved in a variety of important mechanical processes in eukaryotic cells, such as intracellular transport, cell division and muscle contraction. These motors, which produce motion on the nanoscale, operate in the presence of substantial thermal noise. In this dissertation, two approaches are used to model the physics of nanoscale motors: (1) A theoretically established type of Brownian motor called the "flashing ratchet" is studied. This motor transports diffusive particles in a preferred direction. (2) A coarse-grained mechanical model for the biological molecular motor myosin-V is developed, and used to study the role of Brownian diffusion, and the interaction between chemical and mechanical degrees of freedom, in the transport mechanism of this motor. In chapter III, Brownian dynamics simulations and analytical calculations demonstrate that the average velocity of rigid chains of particles in a flashing ratchet reverses direction in response to changing the size of the chain or the temperature of the heat bath. Recent studies have introduced policies for "closed-loop" control of a flashing ratchet, in which the system is controlled based on information about its internal state (such as the positional distribution of particles). In chapter IV, the effect of time delay on the implementation of closed-loop control of a flashing ratchet is investigated. For a large ensemble, a well-chosen delay time improves the ratchet performance (increasing the velocity) by synchronizing into a quasi-stable mode that takes advantage of the semi-deterministic nature of the time development of average quantities for a large ensemble. I n chapter V, a coarse-grained mechanical model is presented for the transport mechanism of myosin-V, which walks along intracellular filaments. The model is well constrained by experimental data on the mechanical properties of myosin V and on the kinetic cycle. An experimentally motivated model for the intramolecular coordination of the motor's steps is proposed and tested.
Adviser: Heiner Linke

Thesis (Ph. D.)--University of Oregon, 2008.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 164-171). Also available online in Scholars' Bank; and in ProQuest, free to University of Oregon users.

Neuerliche Fortschritte im Verständnis biomolekularer Motoren rücken ihre Anwendung als Nanomaschinen in den Bereich des Möglichen. So könnten sie zum Beispiel als Nanoroboter arbeiten, um in molekularen Fabriken kleine – aber dennoch komplizierte – Strukturen auf winzigen Förderbändern herzustellen, um Netzwerke molekularer Nanodrähte und Transistoren für elektronische Anwendungen zu assemblieren oder sie könnten in adaptiven Materialien patrouillieren und diese, wenn nötig, reparieren. In diesem Sinne besitzen biomolekulare Motoren das Potenzial, die Basis für die Konstruktion, Strukturierung und Wartung nanoskaliger Materialien zu bilden
Recent advances in understanding how biomolecular motors work have raised the possibility that they might find applications as nanomachines. For example, they could be used as molecule- sized robots that work in molecular factories where small, but intricate structures are made on tiny assembly lines, that construct networks of molecular conductors and transistors for use as electrical circuits, or that continually patrol inside “adaptive” materials and repair them when necessary. Thus biomolecular motors could form the basis of bottom-up approaches for constructing, active structuring and maintenance at the nanometer scale

Biomolecular motor based transport reconstituted in synthetic environment has been recently established as a promising component for the development of nanoscale devices. A minimal system consisting of microtubules propelled over a surface of immobilized kinesin motor proteins has been used to transport and manipulate cargo for molecular sorting, analyte detection, and other novel nanotechnological applications. Despite these achievements, further progress of the field and translation of the reported applications to a real-world setting require overcoming several key challenges, such as, development of effective cargo conjugation strategies and precise control of the transport directionality with the reconstituted biomolecular motor systems. The challenge of cargo conjugation is addressed in this thesis through the development of a robust bioorthogonal strategy to functionalize microtubules. The versatility of the developed method is demonstrated by covalently conjugating various types of cargos to microtubules. Further, the effect of the linker length on cargo attachment to microtubules is investigated by attaching cargo to microtubules via linkers of different lengths. By using kinesin-driven transport of microtubules that are covalently conjugated to antibodies, detection of various clinically relevant analytes is demonstrated as proof-of-principle applications for biosensing. Finally, the challenge of gaining control over transport directionality is addressed through topographical guiding of microtubules in nanostructures, and optimization of assay parameters to achieve successful guiding of microtubules. Spatio-temporal analyte concentration, using transport in these nanostructues, is also explored to make the biomolecular-motor based applications more suitable for use real-world point-of-care setting. Taken together, the experimental work in this thesis contributes to the field of nanotechnological applications of biomolecular motors. The developed microtubule functionalization method and understanding of the effect of cargo attachment via linkers provide useful design principles for efficient cargo loading to microtubules. Moreover, establishment of assay components for successful guiding of microtubules in nanostructures is a vital step forward for practical translation of future nanoscale devices.

A major challenge in nanotechnology is the controlled transport of cargo on the nanometer scale. A promising approach to this problem is the use of molecular motors of the cellular cytoskeleton. The aim of this work was to develop a method to characterize the behavior of filamentous nanoshuttles – specifically of motor protein-driven microtubules – in three dimensions (3-D). The main requirements to meet were low impact on the nanotransport system, high spatial and temporal resolution, and versatility. Furthermore, this method was intended to be used to address open questions in the field of nanotransport. In particular, it was firstly attempted to characterize cargo transport in a system currently favored by most studies in the field, where nanoshuttles are powered by the microtubule motor best understood so far – the plus-end-directed kinesin-1. Secondly, the goal was to further the understanding of potential counter-players of kinesin-1 in nanotransport applications - the much less well understood microtubule minus-end-directed motor proteins 22S dynein and the kinesin-14 non-claret disjunctional (ncd). A novel method to study the linear forward motion as well as the axial motion of filamentous nanoshuttles, which are driven by motors of the cell cytoskeleton, has been introduced. The method uses fluorescence interference-based 3-D nanometer tracking of quantum dots as optical probes that are attached to the nanoshuttles. While other recently reported 3-D tracking techniques based on dual-focus imaging offer similar sensitivity, the method here can be easily performed on any standard epi-fluorescence microscope, even with arc lamp illumination, and additionally holds the potential to retrieve absolute height values. It is strongly suggested that the ease of use might help to spread this valuable and versatile tool for a variety of applications, including studies of interactions between single molecules or even intramolecular changes. Specifically, 3-D tracking has been used to visualize and analyze the rotation of microtubules around their longitudinal axis when they are propelled on a motor protein-coated surface. This geometry called gliding assay is currently favored for most proof-of-principle studies that investigate the use of biomolecular motors for transport of nanoscale cargo with the goal to assemble and manipulate nanostructures. The suitability of the method has been proven for kinesin-1 gliding assays, where knowledge of properties of both, microtubules and kinesin-1, allowed a very precise prediction of microtubule rotation, which was matching the actual measured values very well. The microtubule rotation in kinesin-1 gliding assays has turned out to be robust against the attachment of small cargo in the shape of quantum dots (diameter ∼20 nm), but also against the reduction of electrostatic interactions between microtubules and kinesin-1 by cleavage of the tubulin E-hook. The situation was dramatically different when large cargo (beads with diameter of ∼3 µm) was attached to microtubules. In this case, filament rotation was stopped, but otherwise the impact on motility was surprisingly low. In particular, the velocity of the gliding microtubules only decreased to a negligible degree. This shows that in principle microtubules driven by processive motors like kinesin-1 can make flexible, responsive and effective molecular shuttles for nanotransport applications. In addition, the results might indicate that in vivo kinesin-1 molecules, which transport cargo along microtubules, can likewise flexibly respond to an axial force by deviating from their path parallel to the protofilament axes. Two microtubule minus-end-directed motors that might be employed to counteract kinesin-1 in engineered nanotransport systems are dynein and ncd. Both motors have been found to be capable of generating torque causing short-pitched microtubule rotation in gliding motility assays. The results for 22S dynein helped to resolve controversial findings of earlier reports about the ability of 22S dynein to generate torque. However, it turned out difficult to establish conditions where the movement of the dynein-driven nanoshuttles was homogeneous and reproducible. In contrast, motility in ncd gliding assays looks much more promising. The obtained results supported previous reports of torque generation by ncd. Moreover, a strong dependence of rotational pitches of gliding microtubules on ATP concentration was found. The reason could be that ncd motors in the nucleotide-free microtubule-bound state impede the forward movement of gliding microtubules stronger than the axial motion. To fully understand the nature of this effect, further research is required. Most likely, this will substantially contribute to the understanding of ncd function in vivo. Furthermore, the possibility of tuning the rotation of microtubules acting as nanoshuttles might provide a means to increase control of processes like cargo-loading and unloading
Eine große Herausforderung auf dem Gebiet der Nanotechnologie ist der kontrollierte und präzise Transport von nanoskaligen Objekten. Der Einsatz von molekularen Motoren des zellulären Zytoskeletts hat sich dabei als vielversprechender Ansatz erwiesen. Ziel der hier vorgelegten Arbeit war die Entwicklung einer Methode, um das Verhalten von filamentartigen Nanotransportern - speziell von Mikrotubuli, die durch Motorproteine über Oberflächen bewegt werden - in drei Dimensionen (3-D) zu charakterisieren. Die Hauptkriterien waren dabei eine geringe Störung des zu untersuchenden Systems, hohe räumliche und zeitliche Auflösungen sowie die generelle Anwendbarkeit für Einzelmolekülstudien. Ein weiteres Ziel war es, die entwickelte Methode zur Beantwortung offener Fragen bezüglich des Nanotransports mittels Zytoskelett-basierter Motoren einzusetzen. Insbesondere sollte das System aus Mikrotubuli und dem Motorprotein Kinesin-1, welches für die meisten aktuellen Studien zum Thema Nanotransport herangezogen wird, untersucht werden. Schließlich sollten neue Erkenntnisse über weniger gut erforschte Motorproteine, speziell über 22S Dynein und das Kinesin-14 „Non-claret disjunctional“ (Ncd), gewonnen werden. Beide Motoren könnten in Nanotransportsystemen als Gegenspieler von Kinesin-1 agieren. In der vorliegenden Arbeit wird eine neuartige, auf Fluoreszenz-Interferenz basierende 3-D Nanometertrackingmethode beschrieben. Auf deren Grundlage wird es möglich, die Bewegung von einzelnen fluoreszenten Partikeln nahe einer reflektierenden Oberfläche mit einer Genauigkeit im Nanometerbereich zu verfolgen. Im Vergleich zu anderen kürzlich vorgestellten 3-D Techniken, welche auf bifokaler optischer Mikroskopie basieren und ähnliche Genauigkeiten zulassen, ist die hier vorgestellte Methode mit deutlich geringerem Aufwand auf der Basis eines herkömmlichen Epi-Fluoreszenzmikroskops umsetzbar. Dabei kann die Fluoreszenzanregung wahlweise mit einer Bogenlampe oder einem Laser erfolgen. Weiterhin besteht die Möglichkeit, nicht nur Differenzwerte (wie bei bifokaler Mikroskopie), sondern absolute Werte in der Höhendimension zu messen. Im Ergebnis wurde ein mit geringem Aufwand umsetzbares, gleichwohl hochgradig genaues und vielseitig einsetzbares Werkzeug geschaffen, welches ideal für Studien der Interaktionen von Einzelmolekülen oder auch intramolekularer Dynamik geeignet ist. Mit Hilfe der hier vorgestellten 3-D Trackingmethode wurden die Rotationen von Mikrotubuli um ihre Längsachse während des Gleitens auf mit Motorproteinen besetzten Oberflächen analysiert. Diese Geometrie wird derzeit bevorzugt in Studien eingesetzt, welche den Einsatz von biomolekularen Motoren für den Transport von nanoskaligen Objekten untersuchen und das Ziel verfolgen, Nanostrukturen zu erzeugen und zu manipulieren. Die Ergebnisse zu Rotationen von Mikrotubuli, welche über mit Kinesin-1 besetzte Oberflächen bewegt werden, sind konsistent mit (i) der Eigenschaft von Kinesin-1 sich entlang der Protofilamente von Mikrotubuli zu bewegen und (ii) der Superhelixstruktur von in vitro rekonstituierten Mikrotubuli. Dies belegt die Eignung der Methode für die Charakterisierung von Nanotransportsystemen. Die Rotation von Mikrotubuli, welche durch Kinesin-1 angetrieben werden, hat sich sowohl beim Transport von kleinen Objekten in Form von Quantum Dots (Durchmesser ca. 20 nm) als auch bei der Reduktion elektrostatischer Wechselwirkungen zwischen Kinesin-1 und Mikrotubuli durch Verdau der Tubulin-C-Termini als stabil erwiesen. Ein vollkommen anderes Bild ergab sich für den Transport von großen Objekten (Durchmesser ca. 3 µm). In diesem Fall wurde die Rotation der Filamente angehalten. Unerwarteterweise war jedoch die Vorwärtsbewegung der Mikrotubuli und insbesondere deren Geschwindigkeit kaum betroffen. Dies zeigt, daß Mikrotubuli, welche von prozessiven Motoren wie Kinesin-1 angetrieben werden, das Potential zu responsiven, flexiblen und effektiven molekularen Shuttles besitzen. Außerdem weisen die Ergebnisse darauf hin, daß Kinesin-1-Moleküle, welche in vivo Frachten entlang von Mikrotubuli transportieren, auf seitwärts gerichtete Kräfte reagieren können, indem sie von ihrem intrinsisch vorgegebenen Pfad parallel zur Protofilamentachse des Mikrotubulus abweichen. Zwei Motoren, die sich im Gegensatz zu Kinesin-1 in Richtung des Minus-Endes von Mikrotubuli bewegen, sind 22S Dynein und Ncd. Sie sind somit als Gegenspieler von Kinesin-1 in Nanotransportsystemen prädestiniert. Beide Motoren können, ebenso wie Kinesin-1, die Translokation von Mikrotubuli über Oberflächen sowie damit verbundene Rotationen von Mikrotubuli verursachen. Im Gegensatz zu Kinesin-1 tritt die Rotation unabhängig von einer Superhelixstruktur der Mikrotubuli auf. Die Ergebnisse für 22S Dynein lösen Widersprüche zwischen früheren Studien auf, indem sie belegen, daß dieser Motor Rotationen von Mikrotubuli erzeugen kann. Jedoch scheint es unter Verwendung von 22S Dynein nicht möglich zu sein, Bedingungen zu schaffen, unter welchen sich Mikrotubuli in geeigneter Weise als Nanoshuttles homogen und reproduzierbar bewegen. Der Einsatz von Ncd ist hier deutlich erfolgversprechender. Die in diesem Falle erlangten Erkenntnisse bezüglich der Erzeugung von Rotationen von Mikrotubuli decken sich mit früheren Studien. Ein bislang unbekannter, bemerkenswerter Effekt ist dabei ein Rückgang in der Länge der Rotationsperioden mit sinkender ATP-Konzentration. Die mit dem heutigen Wissensstand über den mechanochemischen Zyklus von Ncd konsistente Erklärung ist, daß Ncd-Motoren im nukleotidfrei an Mikrotubuli gebundenen Zustand die Vorwärtskomponente der Bewegung von gleitenden Mikrotubuli stärker hemmen als die Rotationskomponente. Möglicherweise kann die sich hieraus ergebende Möglichkeit der Regulierung der Rotation von Mikrotubuli dazu eingesetzt werden, das Be- und Entladen von Nanoshuttles zu steuern

Studying dynamics of proteins in their biological milieu such as water is interesting because of their strong absorption in the terahertz range that contain information on their global and sub-global collective vibrational modes (conformational dynamics) and global dynamical correlations among solvent water molecules and proteins. In addition, water molecules dynamics within protein solvation layers play a major role in enzyme activity. However, due to the strong absorption of water in the gigahertz-to-terahertz frequencies, it is challenging to study the properties of the solvent dynamics as well as the conformational changes of protein in water. In response, we have developed a highly sensitive megahertz-to-terahertz dielectric spectroscopy system to probe the hydration shells as well as large-scale dynamics of these biomolecules. Thereby, we have deduced the conformation flexibility of proteins and compare the hydration dynamics around proteins to understand the effects of surface-mediated solvent dynamics, relationships among different measures of interfacial solvent dynamics, and protein-mediated solvent dynamics based on the complex dielectric response from 50 MHz up to 2 THz by using the system we developed. Comparing these assets of various proteins in different classes helps us shed light on the macromolecular dynamics in a biologically relevant water environment.
Doctor of Philosophy

Déterminer la structure 3D des protéines expérimentalement est une tâche très lourde et coûteuse, qui peut s'avérer parfois impossible à réaliser. L'arrivée massive de données provenant des programmes de séquençage à grande échelle impose de passer d'une approche biochimique à une approche bioinformatique, et nécessite en particulier de développer des méthodes de prédiction sur des séquences.
Cette thèse propose l'exploration de deux nouvelles pistes pour progresser dans la résolution de prédiction de ponts disulfures dans les protéines. Cette liaison covalente stabilise et contraint fortement la conformation spatiale de la protéine et la connaissance des positions où elle intervient peut réduire considérablement la complexité du problème de la prédiction de la structure 3D. Pour cela, nous utilisons dans un premier temps, l'inférence grammaticale et plus particulièrement les langages de contrôle introduit par Y. Takada, puis dans un deuxième temps, la programmation logique inductive.
Diverses expériences visent à confronter un cadre théorique d'apprentissage et des algorithmes généraux d'inférence grammaticale régulière à une application pratique de prédiction d'appariements spécifiques au sein d'une séquence protéique. D'autres expérimentations montrent que la programmation logique inductive donne de bons résultats sur la prédiction de l'état oxydé des cystéines en inférant des règles interprétables par les biologistes. Nous proposons un algorithme d'induction heuristique dont l'idée est d'effectuer plusieurs phases d'apprentissage en tenant compte des résultats obtenus aux phases précédentes permettant ainsi de diminuer considérablement la combinatoire dans les espaces d'hypothèses logiques en construisant des règles de plus en plus discriminantes.

Durant cette thèse, nous avons travaillé sur l'adaptation des algorithmes d'inférence grammaticale pour la recherche des propriétés communes à un ensemble de protéines. L'inférence grammaticale positive cherche à générer, à partir d'un ensemble de mots appartenant à un langage cible particulier inconnu, une représentation grammaticale qui est "optimale" par rapport à ce langage, c'est-à-dire qui rassemble et organise les particularités des mots du langage. Nous avons utilisé le diagramme de Taylor, qui classe les acides aminés suivant leurs propriétés physico-chimiques, pour construire, sous forme de treillis, un ordre sur les groupes d'acides aminés. Nous avons aussi développé une méthode d'inférence (SDTM) qui calcule les meilleurs alignements locaux entre les paires de protéines suivant un score fondé à la fois sur cet ordre et sur les propriétés statistiques de l'ensemble de protéines donné. Le résultat est une machine séquentielle proche de celle de Mealy avec des sorties réduites à "accepte" et "rejette". L'algorithme commence par construire le plus grand automate reconnaissant exactement les mots du langage et le généralise par fusions successives des paires de transitions correspondant aux acides aminés appariés dans les alignements sélectionnés. Les expérimentations ont montré l'intérêt de cette combinaison de méthodes importées de la découverte de motifs et de l'inférence grammaticale.

The majority of biomolecular motors are powered by nucleoside triphosphate (NTP), especially adenosine triphosphate (ATP). These motors consist of a β-sheet with highly conserved motifs and the nucleotide binding domain around it. The highly conserved protein folds are the engines of these motors, which convert the energy of NTP hydrolysis cycle to mechanical work. Although functions of molecular motors are widely diverse, (including cargo movement, DNA unwinding, protein degradation, ion pumping, etc), the nucleotide binding domains are very similar. In the binding site, NTP undergoes a hydrolysis cycle E+NTP⇄E·NTP⇄E•NTP⇄E•NDP•Pi⇄E•NDP+Pi⇄E+NDP+Pi where E is the enzyme (motor protein), the small dot represents the docking of NTP, and the large dot represents the tightly-bound states. The hydrogen bond network formed in the NTP binding step, as shown in Figure 1 [1], deforms the β-sheet and adjacent structures. The local deformation propagates to conformational changes of functional residues to do mechanical work or to change the affinity to the substrate [2]. For multimeric motor proteins, we must also consider the stress paths among subunits which control the sequence and the activity of the protein. Stress trajectories emanating from a binding site either passes through a circumferential stress loop or a stress loop through the substrate.

Kinematic and workspace analyses are performed to predict the performance of a new nanoscale biomolecular motor: The Viral Protein Linear (VPL) Motor. The motor is based on a conformational change observed in a family of viral envelope proteins when subjected to a changing pH environment. The conformational change produces a motion of about 10 nm, making the VPL a basic linear actuator, which can be further interfaced with other organic/inorganic nanoscale components such as DNA actuators and carbon nanotubes. This paper presents the principle of operation of the VPL motor and the development of direct and inverse kinematic models for workspace analysis. Preliminary results obtained from the developed computational tools are presented.

As technology advances, there is an increasing need to reliably output mechanical work at smaller scales. At the nanoscale, one of the most promising routes is utilizing biomolecular motors such as myosin proteins commonly found in cells. Myosins convert chemical energy into mechanical energy and are strong candidates for use as components of artificial nanodevices and multi-scale systems. Isoforms of the myosin superfamily of proteins are fine-tuned for specific cellular tasks such as intracellular transport, cell division, and muscle contraction. The modular structure that all myosins share makes it possible to genetically engineer them for fine-tuned performance in specific applications. In this study, a parametric analysis is conducted in order to explore the design space of Myosin II isoforms. The crossbridge model for myosin mechanics is used as a basis for a parametric study. The study sweeps commonly manipulated myosin performance variables and explores novel ways of tuning their performance. The analysis demonstrates the extent that myosin designs are alterable. Additionally, the study informs the biological community of gaps in experimentally tabulated myosin design parameters. The study lays the foundation for further progressing the design and optimization of individual myosins, a pivotal step in the eventual utilization of custom-built biomotors for a broad range of innovative nanotechnological devices.

Kinesins, biomolecular motors that move along microtubules (MTs) can potentially be utilized as an actuator in nanoscale transporting systems. Recent studies have reported inverted geometry in vitro, in which MTs randomly moved on kinesins fixed to substrates. To develop the transporting systems, one of key elements includes precise control of the direction of sliding MTs. One possible method is to utilize electric field (EF) to direct the MTs because MTs are negatively charged in neutral solutions [1,2]. For example, MTs have been shown to orient to the direction of uniaxially or biaxially applied EFs [3,4]. However, for a reliable transporting system, further studies are still required to control the direction of sliding MTs dynamically and effectively. In our previous study [5], we applied EF to MTs in random direction and showed that the rate of change in angle (angular velocity) was proportional to the sin of the angle between the directions of MTs and the generated electrophoretic force. The result indicates that it is most efficient to continuously apply EF perpendicular to the direction of MTs. In this study, the direction of sliding MTs was dynamically controlled with EF, particularly demonstrating a circular movement of MTs.

In mechanisms and robotics it is common to describe motions relative to a ground link, or robot base, and the position and orientation of the distal link (or robot hand) is viewed as a rigid-body motion relative to this fixed world frame. Assessing preferred relative rigid-body position and orientation in interacting biomolecules (such as proteins) often uses this approach as well by artificially calling one molecule the ground, and considering the motions of another molecule relative to it. But since both molecules are floating, it is not as natural to take this perspective as it is in the field of mechanisms and robotics. Therefore, this paper introduces a ‘symmetrical’ parameterization of relative biomolecular motions in which the structure of the equations is the same when each molecule views the other. In this way, there is no bias in terms of labeling one molecule as being fixed and the other as moving. The properties of this new parameterization are evaluated and compared with traditional ones known to the kinematics community.