Scanning tunneling microscopy on large bio-molecular systems on surfaces

The ever growing demand for the development of new technologies and materials has led to extensive studies inspired by biological functional complexes. Peptides with their outstanding ability to efficiently self-assemble can express a broad spectrum of intriguing functionalities. A key question in mimicking their assembly and function is the precise understanding of the specific interactions between the contained amino acids on the level of a sub-molecular length scale. An excellent tool to probe samples at these length scales is available with scanning tunneling microscopy (STM) and its operation under the controlled conditions of ultra-high vacuum (UHV) and low temperature. Within this thesis it is demonstrated how high-resolution studies by STMin combination with a controlled sample preparation by electrospray ion beam deposition (ES-IBD) under UHV conditions allows for the structural determination of peptides on surfaces. Furthermore the results presented here contribute to the development of a method capable of directly identifying individual amino acids within a peptide sequence. The structural and electronic properties of molecules on surfaces crucially depend on their interaction with the underlying substrate. Implementing a thin dielectric layer on the metallic surface, electronically decouples molecules from the substrate and enables an unperturbed observation of the molecular electronic structure. In Chapter 2 the properties of hexagonal boron nitride (h-BN) on Rh(111) as decoupling layer are assessed on behalf of the structural and electronic properties of the molecular model system pentacene adsorbed on it. In a second part of this chapter the discovery and the characterization of a new phase of h-BN/Rh(111) is described. In Chapter 3, we gained insight into the properties of amino acids on metal surfaces by utilizing the capability of the STM to probe the structure and the electronic characteristics in high resolution imaging and scanning tunneling spectroscopy (STS). As a second important aspect of this chapter, the modification of STM tips with amino acids is investigated as a method to enhance the structural and electronic resolution. An experimental protocol allowing to adhere amino acids on the STM tip be could developed. Using functional STM tips in STS experiments on amino acids enabled the observation of specific molecular resonances. An obstacle in investigating large bio-polymers, such as natural peptides, is their high structural complexity and conformational freedom. Therefore the utilization of custom designed synthetic sequences tailored towards a specific property is a good approach. In Chapter 4 studies performed on two synthetic peptide sequences deposited by ES-IBD on an Au(111) surface are discussed. The first sequence assembled in ordered two-dimensional networks. A folded gas-phase conformation could be utilized to rationalize the observed structures in the networks. Subsequently it was shown that the self-assembly behavior of the peptide could be steered towards chain-like assemblies by modifying the sequence at the peptide C-terminal. Using specific amino acid functionalized STM tips a sensitivity towards an amino acid of the same type in the peptide sequence could be observed. Thereby a partial sequencing of the synthetic peptide was enabled.

Construction of a low temperature STM and studies of large molecular systems

Daniel Pablo Rosenblatt

Several aspects in the field of molecular self assembly are addressed in this thesis. Thanks to the scanning tunnelling microscope (STM), structural, electronic and magnetic properties on surfaces can be studied down to the size of a single atom. In this thesis, I designed and built a STM operating in ultra high vacuum at liquid helium temperature. In Chapter 2 I highlight the design and key features of this system. I describe the development of UHF cabling under severe design constraints, intended to be used for a state of the art STM time resolved technique with nanosecond time resolution. The performance of the instrument is demonstrated by a few benchmark measurements. The arrangement of molecules on surfaces is sensitive to intermolecular interactions, but also to molecule-surface interactions. The surfaces used as support for molecules must therefore be well characterized from the structural, chemical and electronic points of view. Using STM, work function (WF) variations at the nanoscale on hexagonal boron nitride on rhodium (h-BN/Rh(111)) are investigated. These variations are the key to understanding the arrangement of molecules on this surface, yet their magnitude is disputed. Local WF is measured in two ways and compared with KPFM maps. The results provide insight about hot electronic states near surfaces, called image potential state, and their lifetimes. A new phase of h-BN/Rh(111) was discovered and a preliminary characterization of its origin is presented. STM can provide valuable information for understanding the arrangement and the electronic properties of molecules on surfaces. These capabilities are harnessed in Chapter 4 to study large molecules on metallic surfaces, and on thin dielectric surfaces. Angiotensin-II is a peptide, comprising 8 amino acids, small enough to fit in the nanometric cavities of h-BN/Rh(111). This peptide can be brought to the surface intact using electrospray ion-beam deposition. On a Cu surface, we observe that the peptides interact strongly with the substrate, and this interaction determines their arrangement and electronic properties. However, on a single layered sheet of h-BN/Rh(111), the peptides are sufficiently decoupled from the metal, and their electronic properties conserved. In a series of preliminary measurements, these electronic properties are studied in order to discriminate between the amino acids the peptide is made of. Additional strategies that may enable peptide sequencing by STM are discussed. Turning to still larger molecules, cytochrome-C is a protein made of 104 amino acids. On plain metal surfaces, its size and flexibility make the number of conformations available to it enormous. In Chapter 5 this protein is deposited on a nanopatterned surface, forcing it to adhere to a square grid. This makes the number of possible conformations manageable. A detailed look at these conformations reveals many details about how the proteins move on the surface and find their place on the grid. For example, it can inferred that short segments of the protein can diffuse on the surface at room temperature, while longer segments are immobilized. The critical length where diffusion begins is used to estimate the diffusion barrier energy. Furthermore, a simplified simulation of the landing process yields a good fit to the experimentally observed conformation distribution. This fit confirms the flexibility of the proteins and the negligible role of the amino acid side chains.

EPFL2016

"Switch peptides"

Lydiane Jeanine Saucède

Despite of considerable progress in the research at the interface of Chemistry, Biology and Medicine, neurodegenerative diseases affect more and more seriously our ageing population and remain a real therapeutic challenge for researcher. Nevertheless, advances made in this field allow us to better understand the molecular mechanisms responsible for these disorders and to stress the involvement and the key-role of conformational changes of some proteins. Thought to be at the origin of Alzheimer's disease, amyloid-β peptide undergoes structural modifications leading to β-sheet structures, which further aggregate into toxic fibrils and plaques. However, intrinsic properties of these structures result in problems such as insolubility and self-aggregation limiting their experimental access. Among the main research lines to find an efficient therapeutic agent to fight against Alzheimer's disease, fibrillar inhibition is considered as promising strategy. It requires the use of a molecule, called β-breaker, which would be able to block the misfolding triggered by conformational transitions. Consequently, the present thesis is focusing on the design and chemical synthesis of potential inhibitors of amyloid-β fibrillogenesis. As a specific common feature, the designed compounds integrate in their chemical structure a short sequence ("nucleation site") derived from the central part of Aβ(1-42), allowing to specifically interact with the pathogenic peptide. We first envisaged to design cyclic peptides as potential inhibitors containing proline residues, known for its destabilizing effect upon secondary structures (way A). In collaboration with C. Soto et al., Univ. Galveston, the biological activity of the prototype cyclo(Lys-Leu-Pro-Phe-Phe-Glu) was tested. Most notably, electron microscopy, in collaboration with J. Dubochet and M. Adrian, UNIL, Congo red and thioflavin T staining, and CD studies pointed to the potential of cyclic peptides as Aβ inhibitors. Subsequently, conjugate peptides, assembling the recognition sequence of amyloid-β and organic molecules with potential to block fibrillogenesis were synthesized specifically. (+/-)-Trans-4-cotininecarboxylic acid, 3-indolebutyric acid and a tripeptide β-strand mimic were chosen. The biological assessment of these molecules allowed us to highlight their potential in this therapeutic strategy as well as the advantage given by the intercalation of a proline between the two components of these conjugates. In an effort to introduce in the structure of β-breakers a dynamization element, comparable to the cis/trans isomerization of proline, we designed a new type of β-breaking molecules according to strategy B (Figure). The elaboration of this innovating concept termed "switch-peptides" allows to controll the function of a polypeptide by using intramolecular acyl migration as switch-element for the in situ induction of structure and function. By combining an conformational induction unit σ, a switch element S (cysteine or serine) and an amyloid recognition sequence, we obtained a new generation of dynamic β-breakers. At the Soff state, the σ part was linked to the switch element S via an ester bond, deactivating the structural influence of the conformational induction unit on the target peptide, resulting in the absence of biological activity. By removing the protecting group Y from the amino function of the switch element, intramolecular acyl migration was triggered, restoring the native amide bond, setting off the impact of σ (Son state). In applying pseudo-prolines as β-breaking σ-elements (resulting in a "kink" conformation), the corresponding switch-peptide can adopt a recognition state (Soff) and a β-sheet disrupting state (Son), triggered by controlled acyl migration. We explored the use of cysteine as switch element, with special attention to the chemoselective synthesis, kinetics of acyl migration and potential for β-breaking. Interestingly, the S→N acyl migration proceeded very fast at physiological pH, in comparison to serine derived O→N migrations. Of utmost importance for further extensions of the switch-concept, the design of non peptidic S-elements such as trifunctionalized aromatic compounds would allow us to generate the β-breaking element in situ. As a first step, the intramolecular acyl migration O→N via an intermediate of 5, 6, 7 or 9 membered rings, as well as the reversibility of this reaction, was successfully demonstrated. In addition, enzymatically cleavable protecting groups Y for triggering acyl migrations were established. In conclusion, the present thesis presents some promising concepts in the design of fibril disrupting compounds of considerable therapeutical potential.

EPFL2006

Growth and characterization of specific self-assembled supramolecular architectures at crystal surfaces

Marta Canas Ventura

In the framework of this thesis self-assembled supramolecular architectures of several molecular species on crystal metal surfaces are characterized. These investigations combine results obtained by means of scanning tunneling microscopy (STM), scanning tunneling spectroscopy (STS), X-Ray Photoelectron Spectroscopy (XPS), and Near-Edge X-Ray Absorption Fine Structure Spectroscopy (NEXAFS). The interest in controlling the formation of specific arrangements of supramolecular ensembles at surfaces is motivated to a great extent by the prospect to design molecular devices and nanomaterials with customized properties. The goal is a "spontaneous" creation of targeted, well-ordered molecular architectures, which shall be achieved by taking advantage of the molecule's propensity to self-assemble. Control means, in this context, predictability. This term address the accuracy of the prediction of the structures resulting from molecular self-assembly for systems exhibiting different characteristics. Here, system stands for a particular combination of a single-crystal surface and adsorbed molecular species. In a first part, the focus is given to the impact of a Pd(111) surface exhibiting high chemical activity. Due to surface induced deprotonation two resulting chemical states are found for the adsorbed terephthalic acid species. Deprotonation of the carboxyl moieties is observed to affect the topology of the molecular assemblies even though hydrogen bonds (H-bonds) are responsible for the stabilization of assemblies of both, intact and deprotonated molecular species. Therefore, the selective fabrication of a single specific molecular structure becomes a challenge when the surface is prone to induce chemical reactions. Secondly, a molecular species exhibiting conformational flexibility is studied on a largely inert Au(111) surface. It is observed that the characteristic conformational flexibility is also active on the surface, and the molecule is found to adopt different conformations depending on the locally most favorable bonding motif. In particular, phenyl groups may undergo a rotation such that one-dimensional chains can be stabilized by means of intermolecular H-bonds. This phenomenon is very interesting from an adsorbate-substrate interaction point of view, but it limits the predictability of supramolecular structures formed upon self-assembly. In a third step, a comparative study of the self-assembly of three closely related molecular species highlights the impact of concurrent competing intermolecular interactions. Molecules exhibiting an increasing number of possible interaction channels are found to assemble into an increasing number of distinct supramolecular architectures. The most flexible molecular species allowing for different hydrogen-bonding patterns as well as dipolar coupling self-assembles into various structurally complex architectures. Structural predictability, however, is low since the formation of one or the other assembly appears to depend on small variations in sample preparation conditions and on poorly controllable local surface properties. Finally, a system where the resulting structures fully agree with predictions evidences the possibility to control the formation of targeted specific supramolecular architectures. The formation of well ordered bimolecular ribbons and wires over extended length scales is accomplished by the rational choice of both, complementary building blocks for the selective formation of three-fold H-bonds and a nanostructured surface to guide the molecular self-assembly. Furthermore, the impact of H-bond formation on the electronic structure of self-assembled molecular building blocks is probed by STS measurements. The experimental data reveal shifts in the electronic levels depending on the H-bond configuration and on the local characteristics of the underlying surface. In order to improve the precision of such a study, single molecules, homo- and heteromolecular dimers as well as trimers are successfully isolated and assembled by molecular manipulation with the STM tip. Further STS studies on such artificially assembled structures are suggested.

EPFL2008