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Here, we use single molecule force spectroscopy performed with optical tweezers in order to investigate the interaction between Caffeine and the DNA molecule for various different concentrations of the alkaloid and under two distinct ionic strengths of the surrounding buffer. We were able to determine the mechanical changes induced on the double-helix structure due to Caffeine binding, the binding mode and the binding parameters of the interaction. The results obtained show that Caffeine binds to DNA by outside the double-helix with a higher affinity at lower ionic strengths. On the other hand, a considerable cooperativity was found only for sufficient high ionic strengths, suggesting that Caffeine may binding forming dimers and/or trimers along the double-helix under this condition. Finally, it was also shown that Caffeine stabilizes the DNA double-helix upon binding, preventing force-induced DNA melting.

Description of heterogeneous molecular ensembles, such as intrinsically disordered proteins, represents a challenge in structural biology and an urgent question posed by biochemistry to interpret many physiologically important, regulatory mechanisms. Single-molecule techniques can provide a unique contribution to this field. This work applies single molecule force spectroscopy to probe conformational properties of &alpha, synuclein in solution and its conformational changes induced by ligand binding. The goal is to compare data from such an approach with those obtained by native mass spectrometry. These two orthogonal, biophysical methods are found to deliver a complex picture, in which monomeric &alpha, synuclein in solution spontaneously populates compact and partially compacted states, which are differently stabilized by binding to aggregation inhibitors, such as dopamine and epigallocatechin-3-gallate. Analyses by circular dichroism and Fourier-transform infrared spectroscopy show that these transitions do not involve formation of secondary structure. This comparative analysis provides support to structural interpretation of charge-state distributions obtained by native mass spectrometry and helps, in turn, defining the conformational components detected by single molecule force spectroscopy.

Peptide sequences can selectively bind to inorganic substrates in a process similar to the concept of molecular recognition. Here we focused on zinc oxide as an important representative of functional oxides and identified binding peptides with an affinity for different ZnO facets. Five peptides were selected and their conformational ensemble (macrostate) and sensitivity to adsorption were characterised via circular dichroism (CD) spectroscopy. The microstates within their conformational ensembles are accessed by enhanced sampling simulations. To quantify the free energies of adsorption, an optically sectioned indicator displacement assay (O-IDA) was adapted to the peptide/ZnO interface and compared to the results of single molecule force spectroscopy (SMFS), leading to a good agreement between the two approaches.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

Here we report a new study performed at single molecule level on the interaction of the antineoplastic drug Carboplatin and the DNA molecule - the main target of the drug inside cells in cancer chemotherapies. By using optical tweezers, we measure how the mechanical properties of the DNA-Carboplatin complexes changes as a function of the drug concentration in the sample, for two different ionic strengths ([Na] = 150 mM and [Na] = 1 mM). From these measurements, the binding mechanism and the physicochemical (binding) parameters of the interaction were inferred and directly compared to those obtained for the precursor drug Cisplatin under equivalent conditions. As the main conclusion, we show that Carboplatin binds preferentially forming covalent monoadducts in contrast to Cisplatin, which is hydrolyzed easier and presents a higher efficiency in forming covalent diadducts along the double-helix. In addition, we explicitly show that Carboplatin is much less sensitive to ionic strength changes when compared to Cisplatin. These findings provide new insights on the interactions of platinum-based compounds with the DNA molecule, being important to improve the current treatments and in the development of new antineoplastic agents.

International audience; For host cell adhesion and invasion, surface piliation procures benefits for bacteria. A detailed investigation of how pili adhere to host cells is therefore a key aspect inunderstanding their role during infection. Streptococcus pneumoniae TIGR 4, a clinical relevant serotype 4 strain, is capable of expressing pilus-1 with terminal RrgA, an adhesin interacting with host extracellular matrix (ECM) proteins. We used single molecule force spectroscopy to investigate the binding of full-length RrgA and single RrgA domains to fibronectin. Our results show that full-length RrgA and its terminal domains D3 and D4 bind to fibronectin with forces of 51.6 (full length), 52.8 (D3), and 46.2 pN (D4) at force-loading rates of around 1500 pN/s. Selective saturation of D3 and D4 binding sites on fibronectin showed that both domains can interact simultaneously with fibronectin, revealing a two-domain binding mechanism for the pilus-1 tip protein. The high off rates and the corresponding short lifetime of the RrgA Fn bond (τ = 0.26 s) may enable piliated pneumococci to form and maintain a transient contact to fibronectin-containing host surfaces and thus to efficiently scan the surface for specific receptors promoting host cell adhesion and invasion. These molecular properties could be essential for S. pneumoniae pili to mediate initial contact to the host cells andshared with other piliated Gram-positive bacteria_favor host invasion

The mechanical properties of DNA nanostructures are of widespread interest as applications that exploit their stability under constant or intermittent external forces become increasingly common. We explore the force response of DNA origami in comprehensive detail by combining AFM single molecule force spectroscopy experiments with simulations using oxDNA, a coarse-grained model of DNA at the nucleotide level, to study the unravelling of an iconic origami system: the Rothemund tile. We contrast the force-induced melting of the tile with simulations of an origami 10-helix bundle. Finally, we simulate a recently proposed origami biosensor, whose function takes advantage of origami behavior under tension. We observe characteristic stick-slip unfolding dynamics in our force-extension curves for both the Rothemund tile and the helix bundle and reasonable agreement with experimentally observed rupture forces for these systems. Our results highlight the effect of design on force response: we observe regular, modular unfolding for the Rothemund tile that contrasts with strain-softening of the 10-helix bundle which leads to catastropic failure under monotonically increasing force. Further, unravelling occurs straightforwardly from the scaffold ends inward for the Rothemund tile, while the helix bundle unfolds more nonlinearly. The detailed visualization of the yielding events provided by simulation allows preferred pathways through the complex unfolding free-energy landscape to be mapped, as a key factor in determining relative barrier heights is the extensional release per base pair broken. We shed light on two important questions: how stable DNA nanostructures are under external forces and what design principles can be applied to enhance stability.

Protein aggregation is linked with the onset of several neurodegenerative disorders, including Parkinson's disease (PD), which is associated with the aggregation of α‐synuclein (αSyn). The structural mechanistic details of protein aggregation, including the nature of the earliest protein–protein interactions, remain elusive. In this study, we have used single molecule force spectroscopy (SMFS) to probe the first dimerization events of the central aggregation‐prone region of αSyn (residues 71–82) that may initiate aggregation. This region has been shown to be necessary for the aggregation of full length αSyn and is capable of forming amyloid fibrils in isolation. We demonstrate that the interaction of αSyn71‐82 peptides can be studied using SMFS when inserted into a loop of protein L, a mechanically strong and soluble scaffold protein that acts as a display system for SMFS studies. The corresponding fragment of the homolog protein γ‐synuclein (γSyn), which has a lower aggregation propensity, has also been studied here. The results from SMFS, together with native mass spectrometry and aggregation assays, demonstrate that the dimerization propensity of γSyn71‐82 is lower than that of αSyn71‐82, but that a mixed αSyn71‐82: γSyn71‐82 dimer forms with a similar propensity to the αSyn71‐82 homodimer, slowing amyloid formation. This work demonstrates the utility of a novel display method for SMFS studies of aggregation‐prone peptides, which would otherwise be difficult to study.

We have performed a biophysical characterization, at single molecule level, of the interaction between the DNA molecule and the biogenic polyamine putrescine. By using force spectroscopy, we were able to monitor the complexes formation as putrescine is added to the sample, determining the mechanical properties of such complexes and the physicochemical (binding) parameters of the interaction for three different ionic strengths. In particular, it was shown that the behavior of the equilibrium binding constant as a function of the counterion concentration deviates from the prediction of the Record-Lohman model. The measured constants were (1.3 ± 0.2) × 10^5 M^- 1 for [Na] = 150 mM, (2.1 ± 0.2) × 10^5 M^- 1 for [Na] = 10 mM, and (2.2 ± 0.3) × 10^5 M^- 1 for [Na] = 1 mM. The cooperativity degree of the binding reaction, on the other hand, increases with the ionic strength. From these analysis, the DNA-putrescine binding mechanisms are inferred, and a comparison with results reported for ordinary bivalent ions like magnesium is performed. Such study provides new insights on the general behavior of the DNA interactions with biogenic polyamines.

Many biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 kBT per C2 domain. Regulation by bilayer composition or Ca2+recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.

International audience; Mechanical and conformational properties of type 1 fimbriae were evaluated on live bacterial cells by Single Molecule Force Spectroscopy (SMFS) and Dynamic Force Spectroscopy (DFS) in buffered solutions whose pH varied from 3 to 9. We evidenced that both fimbrial extension and fimbrial binding force to mannosylated-surface are modulated with changing the externally applied shear force and the solution pH. In particular, intertwined FimA-FimA and FimH-mannose interactions lead to a 5 to 25-fold decrease of the fimbrial unwinding for pulling rates larger than 10 μm/s and for pH values outside the range 5 to 7. In this pH range, the FimH-mannose binding force is maximal with a magnitude of -150-200 pN and the fimbriae extension reaches 8 μm. The enhancement of the FimH-mannose binding force at neutral pH, as evidenced from molecular AFM analyses, strongly correlates with an optimum in yeast agglutination detected at pH 5 to 7. The results reported in this work suggest that "catch bond effect" was negligible over the range of pulling rates tested, and both FimA-FimA and FimH-mannose interactions under given pH and external shear force conditions modify the ability of the bacteria to efficiently colonize host surfaces.

Folding may be described conceptually in terms of trajectories over a landscape of free energies corresponding to different molecular configurations. In practice, energy landscapes can be difficult to measure. Single-molecule force spectroscopy (SMFS), whereby structural changes are monitored in molecules subjected to controlled forces, has emerged as a powerful tool for probing energy landscapes. We summarize methods for reconstructing landscapes from force spectroscopy measurements under both equilibrium and nonequilibrium conditions. Other complementary, but technically less demanding, methods provide a model-dependent characterization of key features of the landscape. Once reconstructed, energy landscapes can be used to study critical folding parameters, such as the characteristic transition times required for structural changes and the effective diffusion coefficient setting the timescale for motions over the landscape. We also discuss issues that complicate measurement and interpretation, including the possibility of multiple states or pathways and the effects of projecting multiple dimensions onto a single coordinate. Copyright © 2014 by Annual Reviews. All rights reserved.

It is now possible to manipulate individual molecules using a nanopore to read DNA and proteins, or write DNA by inserting mini-genes into cells. Furthermore, development of these methodologies will kick open the door to new biology and chemistry that has been logistically intractable previously. Nanopore technology will place molecular and sub-molecular analysis within the reach of the typical bench-top scientist or clinical lab-no longer limited to genomics or mass spectrometry specialists. Moreover, the prospects for synthetic biology-using nanopores to program or reprogram cells-are promising as well, but have been examined only at the level of a single cell, so far.

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response and have broad activity against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalyzed DNA elongation. Here we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.

The present article offers an overview on the use of atomic force microscopy (AFM) to characterize the nanomechanical properties of polymers. AFM imaging reveals the conformations of polymer molecules at solid– liquid interfaces. In particular, for polyelectrolytes, the effect of ionic strength on the conformations of molecules can be studied. Examination of force versus extension profiles obtained using AFM-based single molecule force spectroscopy gives information on the entropic and enthalpic elasticities in pN to nN force range. In addition, single molecule force spectroscopy can be used to trigger chemical reactions and transitions at the molecular level when force-sensitive chemical units are embedded in a polymer backbone.

Large multi-domain proteins, which are ubiquitous in the proteomes in eukaryotic and prokaryotic organisms, still lack intensive studies on their folding mechanisms due to their complicated interactions between and inside of their domains. My work is broadly aimed at characterize folding behaviors and mechanisms of large, multi-domain proteins.In the first part of my thesis work, I developed a novel mechanical folding polypeptide probe based on the anti-parallel coiled-coil domain of a natural protein (Archeal Box C/D sRNP Core Protein) to enable us to capture the progress of the unfolding front along the host protein structure. In this way, the structural origin of the signal from single molecule force spectroscopy (SMFS) based on atomic force microscopy (AFM) can be directly identified. Beyond this published work, I also characterized and compared the unfolding pathway of two homologous multi-domain proteins – yeast phosphoglycerate kinase (PGK) and its E.coli homolog. I found that yeast PGK has much higher mechanical stability than E.coli PGK although previous literature reported that they had similar thermodynamics stability determined by bulk measurements. In collaboration with Mr. Zackary N. Scholl, we characterized another multi-domain protein, protein S, and its two isolated domains (protein S N terminal domain and C terminal domain). By matching the statistical distributions of the unfolding forces from the truncated domains with the distributions of forces from full protein S, we solved the problem of assigning force peaks in the force-extension (FE) curves to individual domains. However, accurate determination of the structural correspondence to the force peaks still needs insertion of CC probes into the loop regions of these multi-domain proteins. In the second part of my thesis work, I focused on integrating our AFM-SMFS with fluorescence microscopy methods. In particular, I have focused on constructing and mounting a home-made AFM force spectrometer on a high magnification inverted microscope to monitor fluorescence signal changes when stretching a single protein molecule. I am also setting up a total internal reflection fluorescence microscope (TIRF) for combination of Förster resonance energy transfer (FRET) and SMFS measurements in the future.In Chapter 1, I will discuss background of multi-protein unfolding, SMFS method principles and coarse-grained (CG) steered molecular dynamics (SMD) simulation. This chapter will focus on the importance of understanding multi-protein unfolding pathway, the advantage of using SMFS as an experimental way of characterizing multi-domain protein unfolding behavior. As a theoretical way to analyze what event happened during the forced unfolding of multi-domain protein, CG-SMD simulations usually support our SMFS results, and providing details for interpreting FE curves obtained in SMFS measurements.In Chapter 2, I present my recently completed work on developing a mechanical force probe based on an anti-parallel coiled-coil polypeptide chain. This work has just been published by Angewandte Chemie International Edition. The probe I developed provides a new way to determine the structural relation of the peaks shown in protein unfolding FE curves. In Chapter 3, I report my recently started work on characterization of yeast PGK. I will show the preliminary data we obtained, (this work is also in collaboration with Mr. Zackary N. Scholl), and discuss differences in the FE traces of the two homologs and propose future work directions.In Chapter 4, I briefly mention another work, Ca2+dependence of protein S unfolding pathway. The NPS and CPS domains were also individually studied by AFM, and their unfolding data statistics helped exploring comprehensively the structural origin of full protein S unfolding data.In Chapter 5, I will introduce my completed work on combination of AFM and inverted Zeiss microscope. The completion of this work will enable simultaneous recording of the fluorescence signal in an SMFS experiment performed on a single molecule in the future.In Chapter 6, I will describe my recent work on building a TIRF microscope to realize TPM measurement on single molecule multi-domain protein in our lab. We hope that the integrated instrument could enable us to detect FRET signal coming from unzipping of the CC probe when stretching by AFM.

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell–surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB–MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer.

Pseudomonas aeruginosa (PA) is a human opportunistic pathogen responsible for 20% -30% of nosocomial infections in French hospitals. For healthy people, it presents no real danger, but for people with cystic fibrosis disease and immune-compromised patients, it is the leading cause of mortality and lung infections. PA has developed antibiotic multi-resistant strains and new and more effective therapeutic approaches are needed. It binds to the surface of the host cells by an interaction between proteins (lectins) present on the membrane and sugars of the host-cell membrane. The lectin-sugar interaction plays an important role in adherence of the bacteria and in the manufacture of a pathogenic biofilm.A new therapeutic approach is to create synthetic molecules (glycoclusters) of greater affinity than the natural sugars present on the cells. To this aim, more than 150 glycoclusters have been synthetized and screened to find the best candidate to inhibit the bacteria infection process. Some of them have been selected and studied by Atomic Force Microscopy (AFM). In particular, this thesis is devoted to study the lectin-glycocluster and cell-bacteria interactions by AFM. The combination of AFM imaging with molecular dynamic simulations let understanding the role of the geometry of the glycoclusters on the complex formation, while AFM spectroscopy accesses the lectin-glycocluster interaction forces at the molecular and cellular levels. The reduction of bacterial adhesion has been observed upon the addition of the glycocluster. This confirms the anti-adhesive properties of the glycocluster and validates the procedure. The ultimate goal is the identification of the best glycoclusters in order to develop new drugs.; La bactérie Pseudomonas aeruginosa (PA) est un pathogène responsable de 20%-30% des infections nosocomiales en milieu hospitalier. Pour les individus sains, elle ne présente pas de réel danger, mais pour les personnes atteintes par la mucoviscidose et les patients immunodéprimés, elle est la cause principale de mortalité et des infections pulmonaires. PA a développé des souches multi-résistantes aux antibiotiques et des nouvelles approches thérapeutiques plus efficaces sont donc nécessaires. Elle se fixe à la surface des cellules-hôtes par une interaction entre des protéines (lectines) présentes sur sa membrane et des sucres présents sur la membrane cellulaire. L’interaction lectine-sucre joue un rôle important dans l’adhésion de la bactérie puis dans la fabrication d’un biofilm pathogène.Une nouvelle approche thérapeutique consiste à créer des molécules synthétiques (glycomimes) de plus grande affinité que les sucres présents sur les cellules. Pour cela, plus de 150 glycomimes ont été synthétisés et examinés afin de trouver le meilleur candidat pour empêche le processus d'infection de bactéries. Certains d'entre eux ont été choisis et étudiés par la Microscopie à Force Atomique (AFM). Cette thèse est consacrée à l’étude des interactions lectine-glycomime et aussi cellule-bactérie par AFM. L’imagerie combinée avec la modélisation permet de comprendre le rôle du glycomime sur la géométrie des complexes créés et la spectroscopie permet de mesurer les forces d’interaction présentes lors de l’adhésion, au niveau moléculaire et cellulaire. Une réduction de l’adhésion bactérienne a été observée après l’introduction du glycomime, confirmant son rôle d’inhibiteur et la validité de toute la démarche. L’objectif ultime est l’identification des meilleurs glycomimes à introduire afin de développer de nouveaux médicaments.

Proteins fold by the initial, preferential folding of secondarystructures 1, 2, however surprisingly little is known about the basic mechanicalproperties of isolated α-helices and β-sheets from an experimental standpoint.Previous investigations into studying the generic unfolding behaviour of α-heliceshave proved inconclusive 3-5, and to our knowledge the study of an isolated,intramolecular β-sheet is unprecedented.Bioinspired PEG114-b-poly(L-glutamic acid)85-(2-pyridyl disulphide),PEG114-b-poly(L-lysine)134-(2-pyridyl disulphide) and PEG114-b-poly(Llysine)134–b-PEG114 were designed, synthesized and utilized as model systems toprobe the mechanical properties of α-helix and β-sheet secondary motifs. Theobtained results were shown to be in good agreement with theoretical resultsobtained by utilizing a AGAGIR-based statistical mechanical model 6. Thedifference in unravelling force comparing the helices of poly(L-Lysine) ≈30 pNand poly(L-glutamic acid) ≈20 pN diblock copolymers was attributed to thediffering hydrophobicity of the side chains. The greater hydrophobicity of thelysine allowed greater interactions between the side chains and sterically hinderedrandom helix-coil fluctuations, which lead to a superior α-helix stability. Whenexperiments were conducted in conditions promoting the solubility of the lysineside chains, the interactions decreased to a force of ≈20 pN, similar to the force ofinteractions observed for the poly(L-glutamic acid). We infer that a minimum of≈20 pN is needed to rupture the hydrogen bonding maintaining the α-helix as thisforce was obtained in conditions where the side chain interactions wereminimized.The presence of constant force plateaus and corresponding inflectionsdemonstrates a length independent unfolding force, which supports a turn-by-turnunfolding mechanism for the α-helix.In addition, the greater hydrophobicity of the side chains was suggestedto not only stabilize the α-helix structure, but also to inhibit the formation of anintermediate metastable β-hairpin-like structure when entropic forces dominate.Preliminary studies were also conducted on the PEG114-b-poly(LLysine)134-(2-pyridyl disulphide) system after a α-β transition had been inducedby heat in basic conditions, where an inflection at a much higher force of ≈ 70 pNwas obtained suggesting the formation of a β-sheet interaction.A bottom-up, investigative strategy has thus been successfully proposeddemonstrating the potential of utilizing such artificial systems to simplify andexemplify real biological systems. The comprehension of these simpler isolatedmodels will no doubt aid the understanding of more complex systems.; Le repliement des protéines est principalement gouverné par les interactions spécifiques des structures secondaires. 1, 2 Toutefois, il existe expérimentalement peu d’informations sur les propriétés mécaniques fondamentales des hélices α et des feuillets β isolées. Les recherches antérieures sur l'étude du déploiement des hélices sont peu concluantes 3-5 et à notre connaissance l'étude des propriétés mécaniques d'un feuillet β isolé, intramoléculaire est sans précédent. Les copolymères PEG114-b-poly(L-lysine)134-(2-pyridyl disulfure),PEG114-b-poly(L-lysine)-b-PEG114 et poly(L-acide glutamique)85-b-(2-pyridyldisulfure) été synthétisés et utilisés comme systèmes modèles pour tester les propriétés mécaniques des motifs secondaires de type hélice α et feuillet β. Les résultats obtenus se sont révélés être en bon accord avec les résultats théoriques obtenus en utilisant un modèle statistique basé sur AGAGIR 6. La différence de force de déroulement comparant les hélices de poly(L-Lysine) ≈ 30 pN et de poly(L-acide glutamique) ≈ 20 pN des copolymères diblocs a été attribuée à l'hydrophobicité différente des chaînes latérales. La plus grande hydrophobie dumotif lysine conduit à de plus grandes interactions entre les chaînes latérales qui empêchent les fluctuations aléatoires au sein de l’hélice, et conduisent à une stabilité supérieure de l'hélice α. Lorsque les expériences ont été conduites dans des conditions favorisant la solubilité des chaînes latérales de lysine, les interactions ont diminué à une force de ≈ 20 pN, similaire à la force des interactions observées pour le poly(L-acide glutamique). Nous supposons qu'un minimum de ≈ 20 pN est nécessaire pour rompre la liaison hydrogène en maintenant l'hélice α, car cette force a été obtenue dans des conditions où les interactions de la chaîne latérale étaient minimisées. La présence de plateaux de force constants et d'inflexions correspondantes démontre une force de dépliement indépendante de la longueur, qui supporte un mécanisme de déroulement tour-par-tour pour l'hélice. De plus, la plus grande hydrophobie des chaînes latérales a été suggérée non seulement pour stabiliser la structure en hélice, mais également pour inhiber la formation d'une structure de type β-turn métastable intermédiaire lorsque les forces entropiques dominent. Des études préliminaires ont été effectuées sur le système de PEG114-bpoly(L-Lysine)134-(2-pyridyl disulfure) après induction d’une transition - β par un traitement thermique dans des conditions basiques. Une inflexion à une force≈ 70 pN a été obtenue, ce qui suggère la formation d'une interaction de type feuillet β. Une stratégie bottom-up a ainsi été proposée avec succès, démontrant le potentiel d'utilisation de tels systèmes artificiels pour simplifier et modéliser des systèmes biologiques réels. La compréhension de ces modèles isolés plus simples aidera sans doute la compréhension de systèmes plus complexes.

Heart failure is a public health problem with 1 million patients this year in France. This pathology is defined inability to heart pump sufficiently to maintain blood flow to meet the body's needs. This decrease is explicated by the loss of contractile function of the heart, caused by the necrosis of the contractile cells: cardiomyocytes. In this study, I was able to study the topographic and biomechanical modification of the cardiomyocyte membrane upstream of its rupture during necrosis, by technology derived from nanosciences : atomic force microscopy (AFM). My work reveals a highly structured membrane in healthy cardiomyocytes and a loss of this architecture in an early stage of the heart failure installation. In a second study I was interested in the oligomeric organization of a transmembrane receptors family , G protein-coupled receptors. These proteins are a privileged target for the pharmacological treatments on heart failure such as beta- Blockers and vasodilators. This oligomerization mechanism could be the key to the side effects associated with treatments. In order to study the oligomeric conformation, I used single molecule force spectroscopy and I reveal different oligomeric populations of these receptors on the membrane. The results showed a oligomeric populations distribution according the conditions (plasmid density coding for receptors / stimulation with synthetic or natural agonist). It is possible that there is a regulation of the signaling pathways, using the oligomerization for specific activation receptors. The possible difference in activity of each oligomeric population (monomer / dimer / tetramer / hexamer) appears to be a plausible explanation for the side effects of pharmacological agents. My thesis work allowed the discovery of a new track by an innovative technology, atomic force microscopy, in the treatment of heart failure.; L'insuffisance cardiaque est un réel problème de santé publique avec 1 millions de patients souffrant de cette pathologie cette année en France. Elle est définie incapacité de fournir un débit sanguin suffisant à l'organisme. Cette diminution de débit est traduite par la perte de fonction contractile du coeur provoqué par la nécrose des cellules responsable de cette fonction : les cardiomyocytes. Dans cette étude j'ai pu étudier les modifications topographiques et biomécaniques de la membrane du cardiomyocyte vivant en amont de sa rupture lors de la nécrose, par une technologie issue des nanosciences : la microscopie à force atomique (AFM). Mes travaux ont fait apparaitre une membrane très structurée chez le cardiomyocyte sain et une perte de cette architecture dans un temps précoce de l'installation de l'insuffisance cardiaque. L'utilisation de la microscopie électronique à transmission à montrer que les anomalies mises en évidences par AFM ont pour origine un réarrangement mitochondriale. Dans une seconde étude je me suis intéressée à l'organisation oligomérique d'une famille particulière de récepteur transmembranaire, les récepteurs couplés aux protéines G. Ces protéines sont une des cibles privilégiées pour les traitements pharmacologiques de l'insuffisance cardiaque tel que le bêta-bloquants et les vasodilatateurs. Ce mécanisme d'oligomérisation pourrait être la clef des effets secondaires liés à ces traitements. Afin d'étudier la conformation oligomérique, j'ai utilisé la spectroscopie de force à l'échelle de la molécule unique pour mettre en évidence différentes populations oligomérique de ces récepteurs sur la surface membranaire. Les résultats ont montré une distribution des populations oligomériques en fonction des conditions (densité de plasmide codants pour les récepteurs/stimulation avec agoniste synthétique ou naturel). Il est possible qu'il y ait une régulation des voies de signalisations par l'oligomérisation des récepteurs activés. La différence d'activité possible de chaque population oligomérique (monomère/dimère/tétramère/hexamère) semble être une explication plausible aux effets secondaire des agents pharmacologique. Mes travaux de thèse ont permis la mise en évidence de nouvelle piste par une technologie innovante, la microscopie à force atomique, dans le traitement de l'insuffisance cardiaqu

Lamins are mechanosensitive and elastic components of the nuclear lamina that respond to external mechanical cues by altering gene regulation in a feedback mechanism. Numerous mutations in A-type lamins cause a plethora of diverse diseases collectively termed as laminopathies, the majority of which are characterized by irregularly shaped, fragile, and plastic nuclei. These nuclei are challenged to normal mechanotransduction and lead to disease phenotypes. Here, we review our current understanding of the nucleocytoskeleton coupling in mechanotransduction mediated by lamins. We also present an up-to-date understanding of the methods used to determine laminar elasticity both at the bulk and single molecule level.

Atomic force microscopy based single molecule force spectroscopy is an important tool for directly investigating the interactions between synthetically engineered biomolecules and different materials interfaces. Based on this technique, biosensors for the detection of adenosine, mercury ions, and thrombin are developed in the thesis. The biosensors based on specific aptamers for the detection of adenosine and mercury ions show extremely high sensitivity and selectivity. A novel method based on single molecule force mapping method is also developed and applied for the detection of mercury ions. The method is simple, quick, and also shows excellent sensitivity and selectivity. In addition, the interactions of avidin biotin and streptavdin biotin are investigated by single molecule force spectroscopy in combination with a specific oligopeptide sequence, with the aim to detecting thrombin in aqueous solution. This system and the underlying sensing principle are relatively complex, so more efforts are needed to improve this biosensora s performance. In summary, it is believed the AFM based single molecule force spectroscopy sensing technique developed in the present thesis will be useful and promising also for many other analytes. It has the potential to be exploited in commercial devices especially because of the low detection limit, if a sufficient degree of automatization and reproducibility are achieved.

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

Proteins fold by the initial, preferential folding of secondarystructures 1, 2, however surprisingly little is known about the basic mechanicalproperties of isolated α-helices and β-sheets from an experimental standpoint.Previous investigations into studying the generic unfolding behaviour of α-heliceshave proved inconclusive 3-5, and to our knowledge the study of an isolated,intramolecular β-sheet is unprecedented.Bioinspired PEG114-b-poly(L-glutamic acid)85-(2-pyridyl disulphide),PEG114-b-poly(L-lysine)134-(2-pyridyl disulphide) and PEG114-b-poly(Llysine)134–b-PEG114 were designed, synthesized and utilized as model systems toprobe the mechanical properties of α-helix and β-sheet secondary motifs. Theobtained results were shown to be in good agreement with theoretical resultsobtained by utilizing a AGAGIR-based statistical mechanical model 6. Thedifference in unravelling force comparing the helices of poly(L-Lysine) ≈30 pNand poly(L-glutamic acid) ≈20 pN diblock copolymers was attributed to thediffering hydrophobicity of the side chains. The greater hydrophobicity of thelysine allowed greater interactions between the side chains and sterically hinderedrandom helix-coil fluctuations, which lead to a superior α-helix stability. Whenexperiments were conducted in conditions promoting the solubility of the lysineside chains, the interactions decreased to a force of ≈20 pN, similar to the force ofinteractions observed for the poly(L-glutamic acid). We infer that a minimum of≈20 pN is needed to rupture the hydrogen bonding maintaining the α-helix as thisforce was obtained in conditions where the side chain interactions wereminimized.The presence of constant force plateaus and corresponding inflectionsdemonstrates a length independent unfolding force, which supports a turn-by-turnunfolding mechanism for the α-helix.In addition, the greater hydrophobicity of the side chains was suggestedto not only stabilize the α-helix structure, but also to inhibit the formation of anintermediate metastable β-hairpin-like structure when entropic forces dominate.Preliminary studies were also conducted on the PEG114-b-poly(LLysine)134-(2-pyridyl disulphide) system after a α-β transition had been inducedby heat in basic conditions, where an inflection at a much higher force of ≈ 70 pNwas obtained suggesting the formation of a β-sheet interaction.A bottom-up, investigative strategy has thus been successfully proposeddemonstrating the potential of utilizing such artificial systems to simplify andexemplify real biological systems. The comprehension of these simpler isolatedmodels will no doubt aid the understanding of more complex systems., Le repliement des protéines est principalement gouverné par les interactions spécifiques des structures secondaires. 1, 2 Toutefois, il existe expérimentalement peu d’informations sur les propriétés mécaniques fondamentales des hélices α et des feuillets β isolées. Les recherches antérieures sur l'étude du déploiement des hélices sont peu concluantes 3-5 et à notre connaissance l'étude des propriétés mécaniques d'un feuillet β isolé, intramoléculaire est sans précédent. Les copolymères PEG114-b-poly(L-lysine)134-(2-pyridyl disulfure),PEG114-b-poly(L-lysine)-b-PEG114 et poly(L-acide glutamique)85-b-(2-pyridyldisulfure) été synthétisés et utilisés comme systèmes modèles pour tester les propriétés mécaniques des motifs secondaires de type hélice α et feuillet β. Les résultats obtenus se sont révélés être en bon accord avec les résultats théoriques obtenus en utilisant un modèle statistique basé sur AGAGIR 6. La différence de force de déroulement comparant les hélices de poly(L-Lysine) ≈ 30 pN et de poly(L-acide glutamique) ≈ 20 pN des copolymères diblocs a été attribuée à l'hydrophobicité différente des chaînes latérales. La plus grande hydrophobie dumotif lysine conduit à de plus grandes interactions entre les chaînes latérales qui empêchent les fluctuations aléatoires au sein de l’hélice, et conduisent à une stabilité supérieure de l'hélice α. Lorsque les expériences ont été conduites dans des conditions favorisant la solubilité des chaînes latérales de lysine, les interactions ont diminué à une force de ≈ 20 pN, similaire à la force des interactions observées pour le poly(L-acide glutamique). Nous supposons qu'un minimum de ≈ 20 pN est nécessaire pour rompre la liaison hydrogène en maintenant l'hélice α, car cette force a été obtenue dans des conditions où les interactions de la chaîne latérale étaient minimisées. La présence de plateaux de force constants et d'inflexions correspondantes démontre une force de dépliement indépendante de la longueur, qui supporte un mécanisme de déroulement tour-par-tour pour l'hélice. De plus, la plus grande hydrophobie des chaînes latérales a été suggérée non seulement pour stabiliser la structure en hélice, mais également pour inhiber la formation d'une structure de type β-turn métastable intermédiaire lorsque les forces entropiques dominent. Des études préliminaires ont été effectuées sur le système de PEG114-bpoly(L-Lysine)134-(2-pyridyl disulfure) après induction d’une transition - β par un traitement thermique dans des conditions basiques. Une inflexion à une force≈ 70 pN a été obtenue, ce qui suggère la formation d'une interaction de type feuillet β. Une stratégie bottom-up a ainsi été proposée avec succès, démontrant le potentiel d'utilisation de tels systèmes artificiels pour simplifier et modéliser des systèmes biologiques réels. La compréhension de ces modèles isolés plus simples aidera sans doute la compréhension de systèmes plus complexes.

We here give information for a deeper understanding of single molecule force spectroscopy (SMFS) data through the example of the blood protein von Willebrand factor (VWF). It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD) loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK) was used for this demonstration. Further, Brownian dynamics (BD) simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, “Mutual A domain interactions in the force sensing protein von Willebrand Factor” (Posch et al., 2016) [1]. Keywords: Atomic force microscopy, Single molecule force spectroscopy, Molecular dynamics simulation, Brownian dynamics simulation, von Willebrand factor

We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers, while a probe is scanned along the molecule. Interaction forces between the probe and the sample are measured with the optical tweezers. The instrument thus enables us to correlate mechanical and functional properties of bound proteins with the tension within the DNA molecule. The typical friction force between a micropipette used as probe and a naked DNA molecule was found to be

Progress in the development of a redox-driven macromolecular motor and the characterization of its redox-mechanical cycle using electrochemical AFM-based single-molecule force spectroscopy (SMFS) is described. The elasticities of individual neutral and oxidized poly(ferrocenyldimethylsilane) (PFS) macromolecules were reversibly controlled in situ by adjusting the potential in electrochemical SMFS experiments. For the operating cycle of one individual PFS-based molecular motor, an output of 3.4 × 10-19 J and an efficiency of 5% have been estimated.

eingereicht von: Sandra Posch Zusammenfassung in deutscher Sprache Universität Linz, Univ., Dissertation, 2015 OeBB

Folding of proteins and nucleic acids involves diffusion over a multidimensional energy landscape. Optical tweezers provide a powerful method for measuring this landscape using single-molecule force spectroscopy, whereby individual molecules are unfolded and refolded under mechanical load applied by the tweezers and the resulting changes in extension of the molecule are monitored to follow changes in the molecular structure. Methods are discussed for measuring energy landscapes, approximated as a 1D projection of the full multi-dimensional energy surface, via equilibrium fluctuations of the extension or non-equilibrium force-ramps. The correct approach for determining the diffusion coefficient for structural changes from such measurements, wherein the effects of instrumental compliance and time resolution are taken into account, is described. Finally, the approximation of folding as a 1D diffusive process is shown to be good for simple molecules like DNA hairpins, despite the high dimensionality of the full folding problem., Optical Trapping Applications, OTA 2015, 12 April 2015 through 15 April 2015