Kinetics of collagen denaturation in tendon

Collagen is constituted of three polypeptides forming a triple helix maintained by numerous weak chemical interactions, such as hydrogen bonds. In connective tissue it further packs into very long cylinders of few nanometers of diameter, called fibrils. Once heated sufficiently, the molecules loose their original structure and take more random conformations (denaturation). The kinetics at which this reaction occurs is strongly dependent upon temperature, because of its co-operative nature. Although the reaction at the molecular level is likely complex and multiple, modelling the kinetics can be simplified considering a single reaction from the native to the denatured state. The rate limiting step of the reaction involves an activated state to which an activation enthalpy and entropy are attributed. The first part of this work is concerned with the characterisation of this activated state. Rates of denaturation are measured under isothermal conditions by monitoring the shrinkage of native bovine tendon. These measurements permit the extraction of the enthalpy and entropy of the molecular state limiting the kinetics. It is shown here that the model considering the activated state as the denatured state of the co-operative fraction of the molecule also permits a modelling of the effect of tension on the rate of reaction. Based upon the theory of rubber elasticity, we derive a quadratic dependence of the kinetics with linear tension applied to denaturing sample. This quadratic behaviour is verified experimentally. The effect of stress is also suspected to be responsible for the heterogeneous form of denatured tissue. By performing isothermal shrinkage measurements on rat tail tendons, the activation parameters of this tissue are also extracted. Additional cross-linking is realised by Glutaraldehyde fixation. It is found experimentally that additional cross-linking leads to a reduction of the entropy of the activated state. In the second part of this work, we are mainly interested in the kinetics of high temperature denaturation as induced by pulsed infrared lasers during surgical procedures. New tools are developed to monitor the reaction during and after CTH:YAG laser pulses on rat tail tendon. For this purpose, an imaging setup is built to generate images of the phase shift between two orthogonal polarisation directions of a probe beam passing through the sample. By careful analysis of the images obtained, the instantaneous local state of birefringence of the sample is derived. This very sensitive method is first used to analyse reversible structural changes of the tissue below the threshold of laser radiant exposure leading to denaturation. Surprisingly, we measured a totally reversible increase of birefringence with temperature. This mechanism is shown to be of different origin than the denaturation process and is suspected to be linked to a variation of the fibril diameter with temperature. The same optical method permits the observation of the state of denaturation of rat tail tendons that are heated by laser pulses of higher energy. Local sites of denaturation are detected by the irreversible modification of birefringence. The method allows one to determine the pulse threshold radiant exposure for rat tail tendon denaturation. Comparison is then made between the threshold radiant exposures determined experimentally with predictions made using the kinetic model whose parameters were obtained previously with isothermal measurements. This is our first successful attempt to validate the model for higher temperatures in a time scale of 10 ms, ruled by the heat diffusion. In order to characterise even higher rates of denaturation, the optical setup is slightly modified to permit a follow up of the reaction during the laser pulse. A streak imaging principle is added to the phase shift detection. The time scales probed by this technique are in the range of 50 μs. By applying higher power laser pulses on the samples, the method reveals a total denaturation in a fraction of a millisecond. The starting point of denaturation during a laser pulse is compared to the computations realised assuming a first order kinetic model. A good agreement between theory and experiments is observed. These experiments realised under high heating rates validate the use of the phenomenological first order kinetic model up to the 50 μs time scale.

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

Peptide ligands stabilized by small molecules

Alessandro Angelini, Davide Bertoldo, Shiyu Chen, Christian Heinis, Florence Pojer

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle-free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity.

Wiley-V C H Verlag Gmbh2014

Scanning tunneling microscopy on large bio-molecular systems on surfaces

Sebastian Koslowski

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.