Feeling sugar-protein interactions using carbon nanotubes : a molecular
recongition force microscopy study
Klein, D.C.G.
Citation
Klein, D. C. G. (2004, November 11). Feeling sugar-protein interactions using carbon
nanotubes : a molecular recongition force microscopy study. Retrieved from
https://hdl.handle.net/1887/106077
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Klein, D.C.G.
Chapter 1
Introduction
1.1 Introduction and motivation
The development of atomic force microscopy (AFM) for application to biological systems has initiated a new form of molecular biology or biomolecular physics. With the direct observation of single molecules at work, such as the activity of RNA polymerase1, and real-time observation of the complex formation of
single GroEL and GroES molecules2, a new approach to study
biological processes at the level of individual molecules has been introduced. Combining its ability to study single proteins, membranes and whole cells in physiological conditions with its inherent high resolution, AFM is an excellent tool to study biological processes on a (sub-) molecular level.3,4
Currently, many techniques are being developed or refined to investigate a large multitude of biological systems on the molecular and submolecular level, that have so far only been studied by
traditional, indirect biochemical methods. It is now becoming possible to “see” the molecular mechanisms of biology. Both microscopy and cell biology have now arrived at the single molecule level. This new field, which is often referred to with the buzz word “bio-nanotechnology”, is developing very quickly, because of investments by many research groups in the world.
Our contribution to this new and exciting field is the combination of the high lateral resolution of atomic force microscopy with chemical resolution. In the future, this tool can be applied to complex biological systems and, in addition to “simple” topography, to obtain direct quantitative information such as binding constants of proteins and their dependence on the direct surroundings of a receptor. A key element is the high spatial resolution, which will allow us to extract such information of individual molecules.
In order to compare AFM to other techniques, it is useful to distinguish two different criteria: on the one hand the spatial resolution a technique can achieve and on the other hand the degree in which the conditions, while measuring, approximate real life situations.
Introduction 3
Electron microscopy (EM) is a technique that can provide images of biological structures, such as organelles in cells with a remarkably high resolution in all three dimensions.
Single molecule fluorescence microscopy is a powerful technique that allows to extract information about single molecules in their native environment. A very nice example is the assessment of the degree of dimerization of a G-protein-coupled receptor prior to stimulation and after stimulation, which confirmed the connection between dimerization and functionality that had been suggested for a long time.5 The resolution that can be obtained is in the order of 200
nm. In fluorescence resonance energy transfer (FRET), distances of only a few nm between fluorescent labels can be measured.
In terms of resolution, AFM fills a niche between single molecule fluorescence and EM on the one hand and X-ray diffraction and NMR on the other hand. In addition, measuring under physiological conditions, can only be performed by NMR, single molecule fluorescence microscopy and AFM.
The first part of this thesis has an exploratory character. We first acquaint ourselves with the possibilities of bio-AFM by studying various proteins and membrane fragments. In the second part we select one topic from the large multitude of relevant biological questions that can be answered with the AFM techniques described here. We focus on the specific interaction between sugars and sugar-binding proteins, which serves as an example of a more general approach that can be applied to many other biological systems.
The ultimate goal of the research that is described in this thesis is to recognize binding sites on proteins, with (sub-) molecular resolution. In order to reach this goal, very detailed topographical information is necessary, combined with specific chemical binding information. In order to make this possible, standard AFM had to be adapted. Shortly: a small ligand was bound to the sharp needle of the AFM, and the interaction between this ligand and receptors on a surface was probed.
single experiment with all ingredients working simultaneously, we investigated two different kinds of sugar-binding proteins.
1.2 Receptor-ligand interactions
Our present knowledge about receptor-ligand interactions is mainly the result of decades of research on the scale of many cells. This research provided information about the average behavior of many cells. The behavior of single cells or, on an even smaller scale, single receptors, remained unexplored for a long time. New techniques make it possible to study not only single cells, but even single molecules (1.3). This allows us to gain fundamental understanding of the binding process of a ligand to a receptor on a (sub-) molecular level, which provides us with new insight on how single ligands and receptors interact and how this affects the activity of the receptor. For example, coupling between receptors, as in dimerization5 or even between different types of receptors6, seems to
have a large influence on the activity of the receptor. At present, the molecular mechanisms at play are not known. These new developments hold a significant promise for future biology and medicine. Being able to study receptor behavior on a single receptor level may, for instance, ultimately provide drug development researchers with new strategies to design more effective treatment with less side-effects, by targeting only those receptors that are involved in the disease.
Figure 1 Schematic overview of the adaptations necessary to
perform high-resolution molecular recognition force microscopy with an AFM on a receptor-ligand system.
Sugar binding pockets 5 nm
Ligand on AFM tip: mannose Short spacer: carbon nanotube OHO HOHO O HO O H O H O HO O H O
Sugar binding pockets 5 nm
Introduction 5
Historical background of AFM in receptor-ligand research
Discrete interactions between single ligand and receptor molecules in liquid environment have been measured using AFM with chemically functionalized tips. The first papers in this field demonstrated discrete (strept-) avidin-biotin interactions7,8,9. In this
early work, AFM tips were completely coated with ligands. Later, the interaction between e.g. antibodies and intercellular adhesion molecules10, and lectin and red blood cells11 has been studied. Also,
single molecular interactions in cell-cell adhesion12 and the
cooperativity of molecular adhesion13 have been studied. Several
reviews have been published on biomolecular interactions measured with AFM; see for instance Willemsen14 and Allison15. Hinterdorfer et
al. have pioneered the use of a single flexible linker between the AFM
tip and a coupled ligand16.
Although single-molecule interactions were detected, in most of the above studies the lateral resolution was not optimal. For example, in reference 11, the unbinding events were correlated with the position on the surface with a lateral resolution of 400 nm. A breakthrough came when Raab et al. combined molecular recognition with dynamic AFM17, a technique called molecular recognition force
microscopy (MRFM)18. The lateral resolution was improved from 400
nm to 20 nm and the imaging speed was increased from several tens of minutes to a few minutes for a scan of 500 nm x 500 nm. Recently, single-molecule recognition imaging of histones in nucleosomal arrays was realized.19
Sugar-lectin interactions in this thesis
1.3 Atomic force microscopy
The atomic force microscope was invented by Binnig, Quate and Gerber in 1986.20 A sketch of a commonly used AFM design is shown
in Figure 2.
Shortly, in an atomic force microscope, a very sharp needle at the end of a cantilever is scanned over a surface. The deflection of the cantilever is recorded by a laser beam that is reflected off the cantilever and detected by a 4-segment photodiode. In constant force contact mode, the deflection is kept constant by a feedback system that adjusts the height of the sample surface. The vertical movement of the piezo element provides topographical information about the sample surface. Under optimal conditions, a lateral resolution of 1 nm and a vertical resolution of 0.1 nm can be obtained, when scanning in liquid.
Initially, the AFM was mostly used to image surfaces in air, but soon it was modified in order to make it possible to use the AFM in liquid.21 This makes the AFM the ideal tool to study the relationship
between microstructure and function, because it allows imaging biological samples with (sub-) molecular resolution, while imaging can be performed in liquid, which prevents the biological samples from denaturation.
Piezo scan tube
Cantilever Metal disk Mica Molecules Plastic ring Laser source Mirror 4 Segment photo- detector 10 µm Tip
Figure 2 The atomic force microscope. In the inset, an SEM image of a
Introduction 7
Molecular recognition force microscopy
In addition to protein dynamics and (sub-) molecular topographical information, the AFM can also provide chemical contrast. Chemical force microscopy (CFM)22,23,24,25 is realized by
binding a molecule to the AFM tip, scanning it over a surface that contains other molecules, and detecting the interaction between the molecule on the tip and molecules on the surface. CFM can be used to study the specific interaction between ligands and receptors, a technique which is called molecular recognition force microscopy (MRFM).26,27 The first MRFM experiment was performed in the group
of Hinterdorfer in Linz. Lysozyme molecules were probed with an AFM tip containg an antibody against lysozyme. The lateral resolution was approximately 25 nm as is shown in Figure 3. Important here is the
role of the spacer, which couples the ligand to the AFM tip and provides it with rotational freedom. Several polymers have been used as a spacer. A high density of spacers on the tip resulting in a tip containing many ligands was used to probe multiple interactions.28 A
low density of spacers on the tip, resulting in a tip containing only a few ligands, was used to probe single molecular interactions.29
We aimed for a higher lateral resolution (< 5 nm), which implied using a short spacer, although it should remain flexible enough to let the ligand find and bind the binding site. Decreasing the length of the spacer makes the design for a functionalized tip more
Figure 3 Molecular recognition force microscopy on lysozyme. An
difficult, because the ligand should be attached to the very apex of the tip, as is illustrated in Figure 4.
In order to make sure that the ligand can reach the surface, a tip with a very sharp end that has chemical properties different from the rest of the tip should be used, making it possible to bind the ligand to this sharp tip end. We chose to use a carbon nanotube as a spacer, because it has a diameter in the order of 2-10 nm. Furthermore, covalent chemistry can be used to bind a ligand to the nanotube tip end. Using a carbon nanotube as a spacer makes it possible to position a ligand at the very apex of the tip.
1.4 Carbon nanotubes
Carbon nanotubes are wrapped up sheets of graphene with special mechanical and electronic properties.30 Carbon nanotubes
have a Young modulus of the order of 1 TPa, and they buckle elastically under applied load rather than to fracture, which makes carbon nanotubes a uniquely tough and energy-absorbing material.31
Carbon nanotubes are members of the fullerene family, molecules that were discovered by Kroto, Curl and Smalley, who were awarded the Nobel Prize in chemistry in 1996 for this discovery.32,33 Carbon
nanotubes were discovered by Iijima in 1991, and the first TEM images of nanotubes are shown in Figure 5.34
Figure 4 Schematic picture of chemically modified AFM tips, using a
long spacer (a) and a short spacer (b). As can be seen in (b), the short spacer has to be positioned at the very tip end, otherwise the ligand will not be able to reach the receptor on the surface.
Introduction 9
Why are carbon nanotubes interesting for us? The high stiffness of the nanotubes makes these molecules a good candidate to use them as a linker between an AFM tip and a molecule that one wants to bind to the tip. Secondly, an open ended carbon nanotube that reacted with oxygen, has a carboxylic acid end group, and can be chemically functionalized in a covalent way, using amide chemistry.35
Carbon nanotubes can be chemically functionalized by binding molecules to the walls or to the end of the nanotube. In the first case, antibodies against fullerenes, that also bind to nanotubes, can be used to functionalize nanotube walls.36 Or, nanotubes can be
coated with polymers that are receptive to certain proteins.37 For
scanning probe applications, the end of a nanotube can be functionalized covalently, as was developed by Lieber.35,38 For this
covalent functionalization, the nanotube should have an open end.
Figure 5 First TEM images of multi-walled and double-walled carbon
1.5 Outline of this thesis
This thesis consists of eight chapters. In Chapters 2 and 3, the application of AFM to biological systems is explored, both in air and in liquid. Chapter 2 deals with atomic force microscopy in air and, as an example, contains a study of filaments found in dividing yeast cells. Chapter 3 describes atomic force microscopy in liquid, both in contact mode and in tapping mode.
In Chapters 4, 5, and 6 we discuss all steps that have to be taken in order to make the high-resolution MRFM experiment described in Chapter 7 possible. As a first step, Chapter 4 provides a method for immobilizing well-separated single molecules for AFM studies in liquid. Next, in Chapter 5, the use of carbon nanotubes as AFM tips for molecular recognition force microscopy is discussed, and a description is given of the fabrication process of single-walled and multi-walled carbon nanotube AFM tips. In Chapter 6, the last step is discussed: chemical characterization and functionalization of carbon nanotubes. In Chapter 7, all steps come together, and first results of molecular recognition force microscopy experiments on pea lectin and on mannan binding lectin are shown and discussed.
To conclude, Chapter 8 provides a summarizing discussion and an outlook.
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