Physical properties and interactions of biomolecules

The aim of this thesis was to explore the statistical physics and the mechanical properties of individual biomolecules, to study their interactions, and to develop new techniques based on atomic force microscopy (AFM). First, we exploit the high resolution imaging of AFM over long DNA molecules adsorbed on a surface to measure the average end-to-end distance as a function of the DNA length, along with its full distribution function. The latter is almost never determined experimentally, and is not available with ensemble techniques, such as light scattering. We found that the scaling exponents are closer to the values predicted by the polymer theory for a 3D self-avoiding random walk (SAW) than for a 2D SAW. These results suggest that the adsorption process of the DNA molecules is akin to a geometric projection from 3D to 2D, known to preserve the scaling properties of fractal objects of dimension df < 2. Second, taking advantage of the possibility of AFM to also manipulate biomolecules, we investigate the specific interactions between the glycoprotein avidin and the vitamin biotin, the ribonuclease barnase and its inhibitor barstar, and the West Nile Virus surface glycoprotein E and the laminin binding protein. The dependence of the most probable unbinding force on the force loading rate has revealed details of the energy landscapes of the unbinding process of these highly efficient and specific interactions. Beside this standard method, we have measured the lifetime of the avidin-biotin bond, kept stretched at a constant force, as a function of this force, and combined this approach with a small dithering of the AFM tip in order to further explore the avidin-biotin energy landscape. Finally, the viscoelastic properties of biomolecules, such as dextran and titin, have been studied using standard force spectroscopy combined with a small oscillation of the AFM tip. A theoretical description for excitation frequencies near the first and the second resonance of the cantilever has been developed to be able to extract, from the amplitude and phase measurement of the oscillations, the stiffness and friction coefficients of the biomolecule being stretched. We conclude that an excitation frequency of 3 kHz gives reliable results for the stiffness measurement whereas frequencies of 15-25 kHz are more appropriate for the dissipation measurements.

Study of Topoisomerase-DNA Interaction Using Atomic Force Microscopy

Livan Bladimir Alonso Sarduy

The aim of this thesis was to explore the topoisomerase-DNA interactions using atomic force microscopy (AFM) in a liquid environment. Firstly, we present a programmable microcontroller-driven injection system for the exchange of an imaging medium while using AFM. Using this low-noise system, high-resolution imaging can be performed during the process of injection without disturbance. The use of the injection system was put into practice when studying the conformational changes of DNA molecules during the injection of intercalating agents, such as daunorubicin (an anticancer drug) and ethidium bromide, into the fluid chamber. An observation of their mode of interaction with DNA might help elucidating their mechanism of action, thereby facilitating the development of more specific drugs. Part of this thesis deals with the way enzymes interact with DNA molecules. We found that human type II topoisomerases (Topo II) bind preferentially to DNA crossovers. About 50% of the DNA crossings, where the Topo II were bound to, presented an angle of a nearly perpendicular orientation, suggesting a favoured binding geometry in the Topo II recognition. Our studies with AFM also helped us visualize the dynamics of the unknotting action of Topo II in knotted DNA molecules. Additionally we investigated the interactions between Topo II and DNA by applying single molecule force spectroscopy. This study evidenced the inhibitor effect of the aclarubicin (anticancer drug) and evaluated the preferential binding of Topo II to specific DNA supercoiled forms. Finally, we explored the Topo II dynamics using a novel technique that detects motion from nano- to micro-metre sized systems. This method opens new possibilities for the study of conformational changes of single proteins, biochemical reactions, as well as drug-target inhibition.

EPFL2012

Investigation of Biophysical Properties of Biomolecules by Means of Atomic Force Microscopy

Jae Sun Jeong

Biomolecules are the building blocks of living organisms and perform the most important functions in a biological process. The aim of biophysics is to understand the biological functions of biomolecules in terms of their structure: structure and function of biomolecules has been an active research for several decades. Despite the evolution and emergence of new investigation techniques during the last 60 years, biological processes still present enormous challenges to our understanding due to the complex biological system spanning a wide range of spatial and temporal scales. An ideal biophysical method should have the capability to observe atomic level structures and dynamics of biological molecules in their physiological environment. It would also permit the visualization of the structures that form throughout the course of conformational changes or chemical reactions, regardless of the time scale. In this thesis, we have investigated two representative biomolecules using the well-defined biophysical methods in terms of behaviour and biophysical properties of biomolecules: Deoxyribonucleic acid (DNA) and the Amyloid-β (Aβ) peptide. Many different biophysical and biochemical tools were employed, where atomic force microscopy (AFM) was the primary instrument to observe the behaviour of the biomolecules in physiological conditions as well as the dynamic changes of structure of biomolecules. Moreover, AFM was used to measure the mechanical properties of biomolecules via direct and indirect methods. What is the behaviour of DNA molecules in an electric field? The behaviour of DNA molecules in an electric field is crucial for understanding interactions of DNA with electrically charged membrane that serves as a template for DNA based sensors and microarray applications, particularly under an electric field. Taken together, the ability to deposit a single DNA molecule on the electrode is essential to increase its specificity. To achieve this, we optimized the operational conditions in order to control a single DNA molecule adsorbing at +300 mV of potential and desorbing to/from the electrode at -25 mV of potential. Simultaneously dynamic imaging enabled us to analyse its morphological behaviour using real-time Electrochemical AFM (EC-AFM) in aqueous conditions. What is the molecular mechanism underlying Aβ42-fibrillogenesis and its role in pathogenesis? The molecular mechanism underlying Aβ42-fibrillogenesis is essential for defining the key determinant in the pathogenesis of Alzheimer’s disease. In this thesis, we carried out detailed biophysical studies to elucidate the structural changes associated with different stages of amyloid formation and provided unique perspectives on the molecular and structural basis underling the polymorphism of amyloid fibrils in Alzheimer’s disease. For the first time, we provided direct evidence of the existence of secondary-nucleation sites on the surfaces of Aβ42-fibrils and demonstrated that Aβ42-monomers play a crucial role in determining the structural properties and heterogeneity of the formed fibrils. In addition, real-time observation with in situ AFM was performed to define the mechanisms underlying oligomerization and the formation of protofibrils of Aβ42 in physiological conditions. Investigation of the elastic property of Aβ42 fibrils using two techniques based on the AFM. The self-assembly process of Aβ peptides into highly ordered amyloid fibrils is a crucial phenomenon in the cascade of pathological events that culminates in Alzheimer’s disease. Further, the assessment of the elastic properties of Aβ fibrils has recently attracted a lot of attention due to their potential biological applications. We measured the elastic property of Aβ42-fibrils immobilized on two types of substrate (positively charged mica and hydrophobic highly oriented pyrolytic graphite) and compared their properties using two techniques based on the AFM: Statistical analysis of thermal fluctuations and AFM based nanoindentation. The results from our studies show that the values of Young’s modulus of Aβ42 fibrils from the two substrates were similar (1.8 to 2.0 GPa), however they were different in the statistical analysis of thermal fluctuations (1.4 GPa, 2.3 GPa on mica and highly oriented pyrolytic graphite, respectively). These results imply that the method of thermal fluctuations was based on the shape of fibrils affected by the interaction force between the substrate and the fibrils.

EPFL2012

Scaling properties of DNA knots studied by atomic force microscopy

Erika Ercolini

The main subject of the present thesis is the experimental study of the scaling properties of DNA knots of different complexities by tapping mode atomic force microscopy (AFM) in air. Homo- or heterogeneous mixture of DNA knot types were deposited onto mica in regimes of (i) strong adsorption, which induces a kinetic trapping of the molecules, and of (ii) weak adsorption, which permits relaxation on the surface. The contour of each knotted molecule was analyzed by a box counting algorithm, giving the number of boxes containing a part of the molecule, N(L), as a function of the box size, L, allowing to recover the relation N(L) ≈ L-df, where df is the fractal dimension and ν = 1/df is the scaling exponent. This relationship is complicated by the presence of a persistence length of DNA (about 50 nm) which introduces a crossover from a rigid rod behavior to a self-avoiding walk behavior. In (i) the radius of gyration of the adsorbed DNA knot scales with the 3D Flory exponent ν ≈ 0.58 within error. In (ii), the value ν ≈ 0.66, intermediate between the 3D and 2D (ν = 3/4) exponents, was found, indicating an incomplete 2D relaxation or a different polymer universality class. A different analysis, where the fractal dimension was determined by a customized box counting algorithm giving the knot mass as a function of the box size, yielded compatible results. In the case of weak binding conditions, AFM images show evidence of the localization of knot crossings, which is an effect theoretically predicted for knotted polymers confined in two dimensions (flat knots). Part of this thesis is dedicated to a fascinating application of AFM: the imaging of biomolecules in an aqueous buffer. In particular, images of plasmids adsorbed to mica strong enough to be visualized and loosely enough to see them moving in consecutive scans will be shown. The possibility of imaging under buffer molecules loosely anchored to the substrate opens the way to the investigation of biological processes near physiological conditions. The first step of the study of homologous recombination will be presented. We will also show images concerning our study of the activity of topoisomerase I on supercoiled plasmids. Clusters of DNA molecules and proteins were found when imaging in presence of magnesium, in agreement with recent findings by AFM in air. The research on topoisomerases and their interactions with inhibitors or poisons is a hot topic, being these enzymes targets of anti-cancer drugs. Our study paves the way to the investigation of the activity of topoisomerase II, the enzyme which removes knots from DNA. Finally, the principles of static and dynamic light scattering, and the results of dynamic light scattering on latex beads of known size will be presented. Particular emphasis will be given to the discussion of the contribution of light scattering techniques for future experiments on the study of the static and dynamic properties of DNA knotted molecules in solution. These experiments would contribute to shed light on the possible dependence of the gyration radius of knotted polymers on the knot type, and would allow testing theoretical predictions about their relaxation time.

EPFL2008

Investigation of Biophysical Properties of Biomolecules by Means of Atomic Force Microscopy

Jae Sun Jeong

EPFL2012

Scaling properties of DNA knots studied by atomic force microscopy

Erika Ercolini

EPFL2008