Nano-scale mechanics of protein and DNA ... Nano-scale mechanics of protein and DNA assemblies...

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  • Nano-scale mechanics of protein and DNA assemblies

    Single molecule imaging and probing by atomic force microscopy

    Iwan A. T. Schaap


    Nano-scale mechanics of protein and DNA assemblies

    Single molecule imaging and probing by atomic force microscopy


    ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus

    prof.dr. T. Sminia, in het openbaar te verdedigen

    ten overstaan van de promotiecommissie van de faculteit der Exacte Wetenschappen op woensdag 12 april 2006 om 15.45 uur

    in het auditorium van de universiteit, De Boelelaan 1105


    Iwan Alexander Taco Schaap

    geboren te Delft

  • promotor: prof.dr. C.F. Schmidt

  • 3

    Contents 1 Introduction 5 2 Observation of microtubules with scanning force microscopy

    in liquid 15

    3 Resolving the molecular structure of microtubules under

    physiological conditions with scanning force microscopy 23

    4 Small versus large cantilevers for non-invasive imaging at

    single protein resolution in liquids with atomic force microscopy


    5 Displacements of single kinesin motors followed by atomic

    force microscopy 43

    6 Deformation and collapse of microtubules on the nanometer

    scale 53

    7 Elastic response, buckling and instability of microtubules

    under radial indentation 61

    8 Tau protein binding forms a 1 nm thick layer along

    protofilaments without affecting the radial stability of microtubules


    9 Rapid chiral assembly of rigid DNA building blocks for

    molecular nanofabrication 99

    10 Samenvatting 109 11 List of publications 111

  • 4

    On the cover: A microtubule (a protein tube present in cells) one million times magnified, imaged with an atomic force microscope.

  • Chapter 1 5

    Chapter 1 Introduction During my doctoral research I worked on several projects in which I have studied mechanical properties of biological macromolecules. Insight in the mechanics of bio- molecules, proteins and DNA, can teach us more about their function in their very complex natural environment, the cell. On the other hand, we can use the ideas offered by nature to design mechanically stable constructs from biomaterials for nanotechnological applications. Our samples are only a few nanometers in size (1 nanometer is 10-9 meter). To perform accurate mechanical measurements it is crucial to be able to image with molecular resolution in a close-to-native environment (in liquid, at room temperature). Electron microscopy, which provides molecular resolution, requires fixed and stained or frozen samples, precluding the observation of dynamic processes. Optical microscopy, especially combined with contrast enhancing techniques, like differential interference contrast (DIC) is suitable for studying biologically active specimens in liquid, but the ~200 nm resolution limit is not sufficient to resolve single proteins. By labeling the sample with fluorescent markers in single molecule fluorescence assays, nanometer displacements of single molecular motors have been resolved (1). Optical tweezers are also a powerful tool to study single bio-molecules (2). A molecule, that can be a single protein or a DNA strand, is coupled to a bead which is trapped in a focused laser beam. The displacements of the bead and the related force can be monitored with nanometer and pico-Newton accuracy (1 pico-Newton is 10-12 N). For example the step size of the motor protein kinesin has been resolved using this technique (3). Most of my experiments have been performed with an atomic force microscope (AFM). This technique, were a very fine probe is scanned over the sample, combines single molecule resolution, with the ability to work in liquids at room temperature. In addition AFM can be used as a very sensitive force transducer, to measure forces exerted by the sample or to apply forces to the sample. The achievements described in this thesis can be summarized in three points: i) We have developed the experimental procedures to study bio-molecules in a close to natural environment (in liquid at room temperature), such that the samples could be imaged with nanometer resolution without destroying them. For AFM imaging, the molecule of interest needs to be attached to a surface. Adsorption should be strong enough to hold the molecule, but not so strong that it deforms its structure. Critical for all our experiments was the force applied to the sample by the AFM probe. We applied new AFM techniques like tapping mode with small cantilevers and jumping mode and succeeded in limiting the forces to just tens of pico-Newtons. Using these techniques we have published the first high-resolution images of intact microtubules (4). These techniques can now be easily adapted to study other biological systems. ii) We have measured the mechanical response (deformation under load) of various bio- molecular assemblies: natural protein tubes (microtubules), artificial protein tubes ((5), this work was performed in a collaboration and is not expanded in this thesis), and

  • Chapter 1 6

    artificially created DNA pyramids (tetrahedra). We applied forces up to a nano-Newton and monitored the indentation of the objects with sub-nanometer resolution. We have used analytical and finite element modeling to understand and explain this mechanical response in detail. Our experiments demonstrate the effectiveness of various strategies to engineer robust nanometer sized constructs. DNA tetrahedra use a triangulated architecture, which gives them a very high resistance against deformations. Microtubules rely on a tubular design with longitudinal reinforcements to achieve a very high axial rigidity. iii) We studied the interactions between bio-molecules like they occur in the living cell. We have investigated the morphology and mechanical consequences of tau proteins binding to microtubules. Furthermore we have studied the action of kinesin motor proteins that walk over the microtubule and are responsible for transport in the cell. Using AFM we imaged the displacements of individual motors and investigated how they move and what they do at roadblocks. Bio-molecules All the samples we have studied were made of deoxyribonucleic acid (DNA) or proteins (6). The main function of DNA is storage of the genetic code. It is a very stable molecule, which under optimal conditions can persist for millions of years (7). DNA is a helical filament of two paired chains. It is composed of only 4 different building blocks, the nucleotides (figure 1). By varying their sequence an almost infinite number of combinations can be made. In chapter 9 we have used DNA as construction material in self-assembling nanostructures. The advantage of DNA in nanotechnology is that the specific recognition between the bases makes it possible to design well-defined interactions that can result in self-assembling higher ordered structures. Only very recently we found that also in nature DNA is used as construction material to enhance the mechanical rigidity of a Parvo virus (8).

    Figure 1. DNA is made of four types of nucleotides, which are paired into a helical filament. Each nucleotide consist of a sugar- phospate unit to which one of the four bases is attached, adenine (A), cytosine (C), guanine (G) or thymine (T). Adenine pairs only with thymine (A-T), and guanine with cytosine (G-C). The diameter of the helix is ~ 2 nm (image from

  • Chapter 1 7

    Proteins consist of a sequence of twenty different amino acids, encoded by sequences of DNA (genes). The three-dimensional structure of a protein depends on its amino acid composition and can be very complicated. Proteins have many functions in the cell: they are used as building material, they form enzymes that can catalyze all kinds of chemical reactions, and some are specialized to act as little machines, for example to copy DNA or for transporting compounds through the cell. In chapter 2 to 8 we investigated microtubules (MTs), a tubular assembly of tubulin proteins (figure 2). MTs are the most rigid filaments of those forming the eukaryotic cell skeleton (6). This cytoskeleton is a highly dynamic system, enabling the cells to regulate their shape, for example during growth, locomotion or division. MTs interact with a multitude of MT associated proteins (9) in order to adjust the chemical or mechanical properties and thereby their function. Tau is one of the most abundant MT-associated proteins involved in stabilization and bundling of MT, and tau malfunctioning has been found related to many neuro- degenerative diseases (10, 11) including Alzheimer's disease. In chapter 8 we studied the morphology and mechanical consequences of tau proteins binding to MTs. MTs also facilitate intracellular transport by serving as tracks for molecular motors of the kinesin and dynein families (12). They are involved in organelle and vesicle transport, but also in the separation of chromosomes during cell division. Kinesin was found to walk in 8 nm steps (3), each powered by the hydrolysis of one ATP molecule. The exact coordination between the biochemical and the mechanical cycle of kinesin is still under debate. In chapter 5 we studied by AFM imaging the dynamic interaction between MTs and the kinesin motor-pro