Nanoscale Characterization of Amyloid Fibrils and Biomacromolecules by Atomic Force Microscopy Approaches

This thesis focuses on the interdisciplinary biophysical research field, in which physical approaches are applied to study the biological phenomena. Specifically, the main object of the research in this thesis is to target biological species,especially the amyloid fibrils by using Atomic Force Microscope (AFM) and several relevant technologies. In the main section, we firstly present the high-resolution AFM and AFM-IR measurement on the LHCII on the chloroplast lipid membrane, and investigation on the regulation of the xanthophyll pigments, including violaxanthin and zeaxanthin, on the supramolecular organization and structural conformation of LHCIIs on the chloroplast lipid. Then, the novel vis-PTIR technique shows it capability for ultra-sensitive nanoscale imaging and chemical analysis.This AFM-based approach is enhanced by both the gap plasmon and the instrumentation resonance that shows a high-spatial resolution and single-molecule layer sensitivity. Then, my following researches are focused on the protein aggregation and amyloid fibrils that are associated with various neurodegenerative diseases. The first research target is to investigate the environmental factors on protein aggregation kinetics and amyloid formation. We designed the experimental setup to highlight the influence of sedimentation, microgravity, hydrodynamic flow,air-water interface on the protein aggregation kinetics and distinct microscopic steps of aggregation.The results show that the interface promotes the primary nucleation and hydrodynamics enhances the secondary nucleation, whereas the sedimentation and microgravity contribute no significant impact on the aggregation kinetics. Similarly, to understand the polymorphism phenomenon encountered in the preceding research, we performed an additional experiment with well-controlled environmental kinetics. This environmental kinetics originated from the different combinations of hydrodynamics and interface. This experiment proves that the amyloid polymorphism is under the control of environmental kinetics, as well as the order-order transition among the polymorphs. This conclusion is expected to expand our understanding on fibrillization mechanism and also to show its significance in the biomaterial applications. Last, but not the least, we propose another possible mechanism of amyloid aggregation, hierarchically assembly model(HAM), that enables the protofilament intertwining to form higher-ordered mature fibrils. In this experiment, this novel intertwining model based on statistical result of AFM images, is verified with the modelling and multiple experimental evidences, that may enrich our current knowledge of fibrillization mechanism and mature fibril formation.In the end of this thesis, we briefly summarize the presented scientific works and their potential in the applications. In addition, we developed the perspective of these research in the future with several examples of the interesting topics.

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

AFM-Based Single Molecule Techniques: Unraveling the Amyloid Pathogenic Species

Andrea Cerreta, Giovanni Dietler, Francesco Simone Ruggeri

Background: A wide class of human diseases and neurodegenerative disorders, such as Alzheimer's disease, is due to the failure of a specific peptide or protein to keep its native functional conformational state and to undergo a conformational change into a misfolded state, triggering the formation of fibrillar cross-beta sheet amyloid aggregates. During the fibrillization, several coexisting species are formed, giving rise to a highly heterogeneous mixture. Despite its fundamental role in biological function and malfunction, the mechanism of protein self-assembly and the fundamental origins of the connection between aggregation, cellular toxicity and the biochemistry of neurodegeneration remains challenging to elucidate in molecular detail. In particular, the nature of the specific state of proteins that is most prone to cause cytotoxicity is not established. Methods: In the present review, we present the latest advances obtained by Atomic Force Microscopy (AFM) based techniques to unravel the biophysical properties of amyloid aggregates at the nanoscale. Unraveling amyloid single species biophysical properties still represents a formidable experimental challenge, mainly because of their nanoscale dimensions and heterogeneous nature. Bulk techniques, such as circular dichroism or infrared spectroscopy, are not able to characterize the heterogeneity and inner properties of amyloid aggregates at the single species level, preventing a profound investigation of the correlation between the biophysical properties and toxicity of the individual species. Conclusion: The information delivered by AFM based techniques could be central to study the aggregation pathway of proteins and to design molecules that could interfere with amyloid aggregation delaying the onset of misfolding diseases.

Bentham Science Publishers2016

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

Jae Sun Jeong

EPFL2012