The Atomic Force Microscope (AFM) is an instrument that is capable of measuring intermolecular forces between single molecules. Multi-Parameter Force Spectroscopy (MPFS) is a technique that uses the AFM. MPFS enables the acquisition of force curves and thermal resonance of the system under investigation. This technique can shed light on the mechanical behavior at the molecular level. Improvements described herein have enhanced the sensitivity of MPFS over background noise. This investigation focuses on the mechanical and interfacial properties of three carbon nanostructures: long nanotubes, nanocoils, and nanoloops. Different types of adhesion are encountered, measured and discussed: friction, rupture, and peeling. The elastic modulus of long carbon nanotubes is calculated from frequency shifts when the system is put into tension. An elastica model is applied to the post-buckled carbon nanotubes, which enables the estimation of the static coefficient of friction on chemically modified surfaces. The compression of a nanocoil at large contact angles reveals that changes in oscillation amplitude do not occur from damping, but from adding stiffness into the systems measured herein. This result is counter to the assumptions of dynamic force spectroscopy. Finally, carbon nanoloops are brought into and out of contact with several different surfaces. The force curve and frequency response of the system shows the difference between rupture and peeling. The results presented herein lead to a better understanding of the mechanical and tribological properties of the carbon nanostructures.

We have developed an analytical technique for single molecule force spectroscopy (SMFS) data that avoids filtering prior to analysis and performs pattern recognition to identify distinct SMFS events. The technique characterizes the signal similarity between all curves in a data set and generates a hierarchical clustering tree, from which clusters can be identified, aligned, and examined to identify key patterns. This procedure was applied to alpha-lactalbumin (aLA) on polystyrene substrates with flat and nanoscale curvature, and bacteriorhodopsin (bR) adsorbed on mica substrates. Cluster patterns identified for the aLA data sets were associated with different higher-order protein-protein interactions. Changes in the frequency of the patterns showed an increase in the monomeric signal from flat to curved substrates. Analysis of the bR data showed a high level of multiple protein SMFS events and allowed for the identification of a set of characteristic three-peak unfolding events.

<p>Just as heat, light and electricity do, mechanical forces can also stimulate reactions. Conventionally, these processes - known as mechanochemistry - were viewed as comprising only destructive events, such as bond scission and material failure. Recently, Moore and coworkers demonstrated that the incorporation of mechanophores, i.e., mechanochemically active moieties, can bring new types of chemistry. This demonstration has inspired a series of fruitful works, at both the molecular and material levels, in both theoretical and experimental aspects, for both fundamental research and applications. This dissertation evaluates mechanochemical behavior in all of these contexts. </p><p>At the level of fundamental reactivity, forbidden reactions, such as those that violate orbital symmetry effects as captured in the Woodward-Hoffman rules, remain an ongoing challenge for experimental characterization, because when the competing allowed pathway is available, the reactions are intrinsically difficult to trigger. Recent developments in covalent mechanochemistry have opened the door to activating otherwise inaccessible reactions. This dissertation describes the first real-time observation and quantified measurement of four mechanically activated forbidden reactions. The results provide the experimental benchmarks for mechanically induced forbidden reactions, including those that violate the Woodward-Hoffmann and Woodward-Hoffmann-DePuy rules, and in some cases suggest revisions to prior computational predictions. The single-molecule measurement also captured competing reactions between isomerization and bimolecular reaction, which to the best of our knowledge, is the first time that competing reactions are probed by force spectroscopy. </p><p> Most characterization for mechanochemistry has been focused on the reactivity of mechanophores, and investigations of the force coupling efficiency are much less reported. We discovered that the stereochemistry of a non-reactive alkene pendant to a reacting mechanophore has a dramatic effect on the magnitude of the force required to trigger reactivity on a given timescale (here, a 400 pN difference for reactivity on the timescale of 100 ms). The stereochemical perturbation has essentially no measurable effect on the force-free reactivity, providing an almost perfectly orthogonal handle for tuning mechanochemical reactivity independently of intrinsic reactivity. </p><p>Mechanochemical coupling is also applied here to the study of reaction dynamics. The dynamics of reactions at or in the immediate vicinity of transition states are critical to reaction rates and product distributions, but direct experimental probes of those dynamics are rare. The s-trans, s-trans 1,3-diradicaloid transition states are trapped by tension along the backbone of purely cis-substituted gem-difluorocyclopropanated polybutadiene using the extensional forces generated by pulsed sonication of dilute polymer solutions. Once released, the branching ratio between symmetry-allowed disrotatory ring closing (of which the trapped diradicaloid structure is the transition state) and symmetry-forbidden conrotatory ring closing (whose transition state is nearby) can be inferred. Net conrotatory ring closing occurred in 5.0 ± 0.5% of the released transition states, as compared to 19 out of 400 such events in molecular dynamics simulations.</p><p>On the materials level, the inevitable stress in materials during usage causes bond breakage, materials aging and failure. A strategy for solving this problem is to learn from biological materials, which are capable to remodel and become stronger in response to the otherwise destructive forces. Benzocyclobutene has been demonstrated to mechanically active to ortho-quinodimethide, an intermediate capable for [4+4] dimerization and [4+2] cycloaddition. These features make it an excellent candidate for and synthesis of mechanochemical remodeling. A polymer containing hundreds of benzocyclobutene on the backbone was synthesized. When the polymer was exposed to otherwise destructive shear forces generated by pulsed ultrasound, its molecular weight increased as oppose to other mechanophore-containing polymers. When a solution of the polymer with bismaleimide was subjected to pulsed ultrasonication, crosslink occurred and the modulus increased by two orders of magnitude.</p> / Dissertation

This thesis contains several contributions to the theoretical description and interpretation of biophysical single-molecule measurements: (i) For semiflexible polymers, we derive an efficient formulation of their local transverse dynamics in terms of a Generalized Langevin Equation. The elastic and frictional properties of the polymer are condensed into a memory kernel that is a function of the polymer\'s length and stiffness, the level of backbone tension, the position of the force probe along the polymer backbone and the boundary conditions at the polymer ends. At short times, the memory kernel attains a universal limiting form that depends neither on the polymer length nor on the boundary conditions; we obtain analytical results that accurately describe this regime. We discuss how to quickly and reliably evaluate the memory kernel for arbitrary times using a spectral decomposition method, and use an extensive body of numerical data to obtain analytical approximations to the memory kernel that cover the complementary long-time limit wherein polymer friction can be subsumed under a renormalized drag coefficient. (ii) Based on a systematic nonequilibrium treatment of an overdamped, one-dimensional stochastic escape process driven by external force, we develop a theory of Dynamic Force Spectroscopy (DFS) that generalizes previously available DFS theories to the high loading rates realized in novel experimental assays and in computer simulations. (iii) Extrapolating to future DFS experiments that may operate at far higher time resolution than presently achievable, we discuss the fast nonequilibrium relaxation of a semiflexible linker after bond rupture. Based on a rigorous theory of tension propagation in semiflexible polymers, we predict the relaxation of force within the force actuator, show that this relaxation is dominated by linker contraction, and demonstrate quantitative agreement of our predictions with experimental data obtained by a collaborating experimentalist group.

<p>Proteins are specialized molecules that catalyze most of the reactions that can sustain life, and they become functional by folding into a specific 3D structure. Despite their importance, the question, "how do proteins fold?" - first pondered in in the 1930's - is still listed as one of the top unanswered scientific questions as of 2005, according to the journal Science. Answering this question would provide a foundation for understanding protein function and would enable improved drug targeting, efficient biofuel production, and stronger biomaterials. Much of what we currently know about protein folding comes from studies on small, single-domain proteins, which may be quite different from the folding of large, multidomain proteins that predominate the proteomes of all organisms.</p><p>In this thesis I will discuss my work to fill this gap in understanding by studying the unfolding and refolding of large, multidomain proteins using the powerful combination of single-molecule force-spectroscopy experiments and molecular dynamic simulations.</p><p>The three model proteins studied - Luciferase, Protein S, and Streptavidin - lend insight into the inter-domain dependence for unfolding and the subdomain stabilization of binding ligands, and ultimately provide new insight into atomistic details of the intermediate states along the folding pathway.</p> / Dissertation

<p>The coupling of mechanical stress fields in polymers to covalent chemistry (polymer mechanochemistry) has provided access to previously unattainable chemical reactions and polymer transformations. In the bulk, mechanochemical activation has been used as the basis for new classes of stress-responsive polymers that demonstrate stress/strain sensing, shear-induced intermolecular reactivity for molecular level remodeling and self-strengthening, and the release of acids and other small molecules that are potentially capable of triggering further chemical response. The potential utility of polymer mechanochemistry in functional materials is limited, however, by the fact that to date, all reported covalent activation in the bulk occurs in concert with plastic yield and deformation, so that the structure of the activated object is vastly different from its nascent form. Mechanochemically activated materials have thus been limited to “single use” demonstrations, rather than as multi-functional materials for structural and/or device applications. Here, we report that filled polydimethylsiloxane (PDMS) elastomers provide a robust elastic substrate into which mechanophores can be embedded and activated under conditions from which the sample regains its original shape and properties. Fabrication is straightforward and easily accessible, providing access for the first time to objects and devices that either release or reversibly activate chemical functionality over hundreds of loading cycles. </p><p>While the mechanically accelerated ring-opening reaction of spiropyran to merocyanine and associated color change provides a useful method by which to image the molecular scale stress/strain distribution within a polymer, the magnitude of the forces necessary for activation had yet to be quantified. Here, we report single molecule force spectroscopy studies of two spiropyran isomers. Ring opening on the timescale of tens of milliseconds is found to require forces of ~240 pN, well below that of previously characterized covalent mechanophores. The lower threshold force is a combination of a low force-free activation energy and the fact that the change in rate with force (activation length) of each isomer is greater than that inferred in other systems. Importantly, quantifying the magnitude of forces required to activate individual spiropyran-based force-probes enables the probe behave as a “scout” of molecular forces in materials; the observed behavior of which can be extrapolated to predict the reactivity of potential mechanophores within a given material and deformation.</p><p>We subsequently translated the design platform to existing dynamic soft technologies to fabricate the first mechanochemically responsive devices; first, by remotely inducing dielectric patterning of an elastic substrate to produce assorted fluorescent patterns in concert with topological changes; and second, by adopting a soft robotic platform to produce a color change from the strains inherent to pneumatically actuated robotic motion. Shown herein, covalent polymer mechanochemistry provides a viable mechanism to convert the same mechanical potential energy used for actuation into value-added, constructive covalent chemical responses. The color change associated with actuation suggests opportunities for not only new color changing or camouflaging strategies, but also the possibility for simultaneous activation of latent chemistry (e.g., release of small molecules, change in mechanical properties, activation of catalysts, etc.) in soft robots. In addition, mechanochromic stress mapping in a functional actuating device might provide a useful design and optimization tool, revealing spatial and temporal force evolution within the actuator in a way that might also be coupled to feedback loops that allow autonomous, self-regulation of activity. </p><p>In the future, both the specific material and the general approach should be useful in enriching the responsive functionality of soft elastomeric materials and devices. We anticipate the development of new mechanophores that, like the materials, are reversibly and repeatedly activated, expanding the capabilities of soft, active devices and further permitting dynamic control over chemical reactivity that is otherwise inaccessible, each in response to a single remote signal.</p> / Dissertation

La spectroscopie de champ proche couplée à la possibilité de manipuler des nano-objets uniques permet de sonder des interactions dans des conditions de dilution élevées et de manière contrôlée. Dans ce sujet, deux techniques de spectroscopie à sonde locale ont été mises à profit. D'une part, couplée à la microscopie à effet tunnel (STM), la spectroscopie Raman exaltée par effet de pointe (TERS) a été mise à profit pour l'observation de la signature vibrationnelle de rotaxanes adsorbés sur une surface d'or. Les topographies STM (microscopie à effet tunnel) ont permis de caractériser en conditions ambiantes des rotaxanes individuels. D'autre part, la spectroscopie dynamique de force (AFM-DFS) appliquée à l'étude des interactions entre un oligopeptide CK(AAAAK)2C et une surface d'or a révélé plusieurs signatures s'échelonnant de 100 pN à plus de 300 pN, et plus rarement, quelques signatures supérieures à 1 nN. Les expériences de contrôle garantissent la spécificité de l'interaction. Nous avons également testé une correspondance entre les modèles cinétiques appliqués dans les systèmes ligand-récepteur à notre système par variation du taux de charge : Bell-Evans et Friddle-DeYoreo. Plusieurs méthodes d'exploitation, en fonction de l'expression prise pour le taux de charge tenant compte où non de la dynamique de dépliement de l'agent de couplage ont été testées. Un outil numérique a également été développé pour le traitement automatique des courbes de force. Les biais liés au sous-échantillonnage de données, à une sélection manuelle, ou au choix des critères de classement des variables sont limités par la mise en place d'outils automatisant ce processus. / Beyond simply imaging, near-field techniques allow to probe and control molecular interactions onto a reduced number of molecules, if not a single one. In this subject, we took advantage of two local-probe spectroscopies. On the one hand, dynamic force spectroscopy (AFM-DFS) was applied to measure interactions between the oligopeptide CK(AAAAK)2C and a gold surface. It revealed several signatures ranging from 100 to 300 pN and more rarely some signatures above 1 nN. In order to interpret this complex response, we tested the kinetic models of Bell-Evans and Friddle-De Yoreo generally applied in ligand-receptor systems onto our system when the loading rate, i.e. the variation of the applied force per unit of time, is varied. Several interpretation methods including or not the effect of the unfolding of the coupling agent into the loading rate expression were tested. A numeric tool was also developed for the automatic processing of the force curves. Biases related to under-sampling of data, due to manual selection, or due to variables classification and mis-representation were limited by the implementation of tools automating the whole process. On the other hand, coupled to Scanning Tunneling Microscopy (STM), tip enhanced Raman spectroscopy (TERS) allowed us to observe the vibrational signature of a giant rotaxane on a gold substrate. STM images performed in ambient conditions could moreover highlight domains with individual molecules.

Membrane fusion is essential for survival in eukaryotic cells. Many physiological processes such as endocytosis and exocytosis are mediated by membrane fusion, which is driven by highly specialized and conserved family of proteins. Neuronal soluble Nethylmaleimide- sensitive factor attachment protein receptors (SNAREs) mediate vesicle fusion with the plasma membrane during neurotransmitter release; however, the mechanism for SNARE-mediated membrane fusion remains to be established. In the current work, we aimed at investigating this mechanism using atomic force microscope (AFM) spectroscopy. We established an AFM lipid bilayer system, which proved effective in detecting fusion of bilayers and measuring compression forces required to generate fusion. It also revealed that SNARE-mediated membrane fusion proceeds through an intermediate hemifused state. Using this system, we revealed the energy landscape for membrane fusion using a dynamic force approach. We carried out compression force measurements at different compression rates and a significant reduction in the force was observed when SNAREs were present in the bilayers. The results also indicated that a single energy barrier governed membrane fusion in our experimental system. The energy barrier is characterized by its width and height, which determine the slope of the activation potential. With SNAREs in the opposing (trans) bilayers, the width of the barrier increased > 2 fold, which is interpreted as an increase in the compressibility of the membranes and subsequently a greater ease in their deformation and fusion under compression. Moreover, specific perturbations to the SNARE interaction interfered with the observed facilitation of membrane fusion, which indicated the involvement of SNAREs in the observed fusion facilitation and increase in the fusion rate. Furthermore, dissociation kinetics analysis of the SNARE interaction revealed a strong binding force during trans SNARE-complex formation, and a correlation between the strength of the SNARE interaction and the degree of fusion facilitation was established. In conclusion, the present findings provide support for a mechanism for SNAREmediated membrane fusion, where trans-interaction between SNAREs provides close apposition of the membranes and reduces fusion energy requirements by locally destabilizing the bilayers, in which the SNAREs are anchored, through pulling on or tilting of their transmembrane segments.

The chemokine, Monocyte Chemoattractant Protein (MCP-1), enhances integrin mediated monocyte adhesion to the vascular endothelium during inflammation. In this study, we demonstrate that MCP-1 promotes rapid sub-second adhesion of THP-1 cells to Vascular Cell Adhesion Molecule-1 (VCAM-1), but not to Intercellular Cell Adhesion Molecule-1 (ICAM-1). MCP-1 activates membrane tethered Very Late Antigen 4 (VLA-4, α4β1), but not necessarily cytoskeleton anchored VLA-4. Activated tethered VLA-4 bonds tremendously increased the period of time monocytes remain bound from hundreds of milliseconds to several seconds and also increased the distance over which immunologic surveillance occurs from several microns up to 20 microns along the endothelium. Lastly at the single molecule level, MCP-1 stimulated tethered VLA-4 bonds exhibit increased resistance to pulling force. In conclusion MCP-1 increased tethered VLA-4 bond resistance to force providing a mechanism for monocyte recruitment to the endothelium.

Optische Pinzetten stellen neuartige Werkzeuge in der Biophysik dar, die sich durch eine außerordentliche Präzision auszeichnen. Im Rahmen der vorliegenden Arbeit werden verfeinerte Bildanalysetechniken vorgestellt und neu entwickelt, die es erlauben, die Position eines Mikropartikels mit einer zeitlichen Auflösung von 0,017 s und einer Genauigkeit von ±2nm in lateraler und in axialer Richtung zu bestimmen. Dies ermöglicht eine Kraftauflösung von bis zu ±50 fN. Damit sind die Voraussetzungen für die Untersuchung von Rezeptor/Ligand-Wechselwirkungen auf der Ebene einzelner Bindungsereignisse gegeben. In der vorliegenden Arbeit werden die Wechselwirkungen zwischen den phosphorylierungsspezifischen Antikörpern HPT-101, HPT-104 und HPT-110 und Tau-Peptiden mit verschiedenen Phosphorylierungsmustern sowie zwischen DNA und den Proteinen TmHU und oPrPC untersucht. Die Wechselwirkungen zwischen Tau-Peptiden und Antikörpern werden jeweils anhand ihrer Bindungshäufigkeit sowie der Verteilung der Abrisskräfte charakterisiert. Mit einem aus der Literatur bekannten, theoretischen Modell werden folgende Bindungsparameter bestimmt: Lebenszeit der unbelasteten Bindung, charakteristische Länge und freie Aktivierungsenergie der Dissoziation. Im Einklang mit Ergebnissen einer immunochemischen Messung werden spezifische Wechselwirkungen zwischen HPT-101 und dem biphosphorylierten Tau-Peptid sowie zwischen HPT-104 bzw. HPT-110 und den Peptiden, die eine Phosphorylierung an Thr231 bzw. Ser235 enthalten, beobachtet. Zusätzlich ermöglicht die Einzelmolekülmethode auch eine detaillierte Charakterisierung der unspezifischen Wechselwirkungen mit den Tau-Peptiden, welche das jeweilige spezifisch erkannte Phosphorylierungsmuster nicht beinhalten. Der zweite Teil der Arbeit befasst sich mit dem Einfluss der Proteine TmHU und oPrPC auf einen einzelnen, mit konstanter Kraft gehaltenen DNA-Strang. Der zeitliche Verlauf der TmHU-induzierten Kondensationsreaktion wird bei Kräften zwischen 2 pN und 40 pN sowie in Abhängigkeit von der Proteinkonzentration untersucht. Bei kleinen Kräften ist eine Verkürzung in zwei Phasen auf bis zu 30% der Konturlänge zu beobachten. Unter zusätzlicher Einbeziehung der Ergebnisse einer SMD-Simulation sowie einer rasterkraftmikroskopischen Untersuchung kann die erste Phase der Verkürzung einer primären Anbindung von TmHU zugeordnet werden. Die zweite Reaktionsphase entspricht hingegen vermutlich der Ausbildung einer Überstruktur. Die Wechselwirkung von oPrPC mit DNA wird zusätzlich mit einer kombinierten Anordnung aus Nanokapillare und optischer Pinzette untersucht. Dabei zeigt sich, dass ein Protein/DNA-Komplex ausgebildet wird, der eine negative Oberflächenladung aufweist und sich in seinem Volumen und seiner Ladung von reiner DNA unterscheidet. Allerdings hat oPrPC keinen Einfluss auf den Ende-zu-Ende-Abstand bzw. die Elastizität der DNA.