Discriminative adsorption of amphiphilic monolayer protected gold nanoparticles on amyloid fibers

This thesis presents a systematic study of how different types monolayer-protected AuNPs interact with amyloid fibers. We report a class of amphiphilic gold nanoparticles capable of adsorbing onto specific surface features on these types of protein fibers. A common disease-associated protein fold is the amyloid state: it is characterized by a cross-beta sheet structure that forces proteins and peptides into a fibrillar state, commonly found in illnesses such as Alzheimerâs disease, Parkinsonâs disease among many others. Amyloid diseases are typically chronic, correlated with ageing and have posed several challenges: the exact structure of the fibers is difficult to determine and their etiologic role is often unclear. This thesis shows, for the first time, that amphiphilic monolayer-protected AuNPs can discriminatively adsorb onto surface features of amyloid fibers made of A1-40 and -synuclein in vitro and that hydrophobicity determines adsorption onto Tau fibers. Given an amyloid fiber that adopts a twisted ribbon morphology, AuNPs protected by a mixture of sulfonated and hydrophobic thiolate molecules adsorb onto specific features on the surface of the fiber, leaving other interfaces uncovered. This generates a novel supra-molecular assembly that directly interfaces an engineered nanomaterial with a biological structure, without using antibodies. Experiments and calculations demonstrated the importance of nanoparticle size and ligand-shell composition: a size cut-off around 4 nm was observed and other types of water soluble nanoparticles did not adsorb discriminatively. Small amphiphilic AuNPs act as surfactants and probably adsorb onto solvent-exposed beta sheets and small amyloidogenic oligomers. The results presented in this thesis provide a systematic framework to understand the interaction between nanoparticles and amyloid fibers. The particles can, moreover, become useful markers for amyloid research and possibly a cross-instrumental probe to reconcile spectroscopic and imaging techniques to help molecular structure determination. During this work, the synthesis and purification of large amounts of sulfonated thiolate molecules was systematized to generate libraries of differently coated water soluble gold nanoparticles (AuNPs). This helped elucidate how amphiphilic AuNPs fuse with lipid bilayers.

Self-Assembly of ß-Amyloid Peptides in Cells and on Solid Supports

Enrico Condemi

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease share common mechanisms characterized by protein misfolding and aggregation, including formation of plaques or inclusion bodies. The rapid growing number of patients has motivated intensive research to understand biological phenomena involved in the pathology and hence developing therapeutic strategies. Here, we will focus on β-amyloid peptide (Aβ) involved in Alzheimer's disease. In the first part of this thesis, tissue transglutaminase (tTGase) has been investigated due to its implication in a number of cellular processes and disease states, where the enzymatic actions of tTGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of tTGase in cell survival or cell death mechanisms still remain elusive. tTGase mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that, tTGase induces intramolecular cross-linking of Aβ peptide, resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that, a large population of Aβ monomers contained an increase in mass of 0.984 Da at a glutamine residue, indicating that glutamine Q15 serves as an indispensable substrate in TGase mediated deamidation to glutamate E15. We provide strong analytical evidence on tTGase mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that, tTGase induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation. In the second part of this work, we explore the idea of using synthetic peptides that can switch conformation in a controlled way as a sensor for β-breakers of Aβ. The concept of switch-peptides allows the controlled onset of polypeptides folding and misfolding at physiological pH by in situ O,N-acyl migration. The synthesized sequence consisted in the core sequence responsible for Aβ aggregation, flanked by two β-sheet-inducing template through ester bonds. The triggering of the acyl migration allows the native sequence to be recovered from the ester bonds and structural change from flexible unordered conformation to β-sheets and subsequently fibrils formation were observed in solution. In a more biological environment made of lipid vesicles, the conformation of the peptide has been shown to act comparably to wild type Aβ peptide by circular dichroism (CD), namely increased negative charges over the surface led to increased helical propensity followed by a transition to β-sheets and accelerated fibrils formation. Thereafter, the conformation of the cysteine analogue of the switch-peptide covalently linked to gold surface was characterized by Fourier-transform infrared spectroscopy (FTIR). In monolayer of switch-peptide tethered to a planar gold surface, the peptide already adopts most likely β-turn conformation and no changes in the infrared spectra were detected after the triggering of the switch. On gold nanoparticles, the switch-peptide folds spontaneously into β-sheet conformation. These unwanted foldings limit the efficiency of the sensor and has driven us to an alternative configuration, where gold nanoparticles were coated with a 95:5 mixture of two peptide sequences able to stabilize the nanoparticles and to act as recognition sequence, respectively. In this configuration, the switch-peptide is added in the gold nanoparticles solution and the aggregation triggered. Here, we want to take advantage of a particular feature of gold nanoparticles that consists in a change of solution color from red to blue as the nanoparticles come closer to each other during aggregation. Preliminary results have shown that a discrimination between aggregated and soluble samples is possible. However, further improvements are necessary before a potential biosensor could be expected.

EPFL2011

Protein-nanoparticle interactions: the effects of surface compositional and structural heterogeneity are scale dependent

Randy Patrick Carney, Francesco Stellacci

Nanoparticles (NPs) in the biological environment are exposed to a large variety and concentration of proteins. Proteins are known to adsorb in a 'corona' like structure on the surface of NPs. In this study, we focus on the effects of surface compositional and structural heterogeneity on protein adsorption by examining the interaction of self-assembled monolayer coated gold NPs (AuNPs) with two types of proteins: ubiquitin and fibrinogen. This work was designed to systematically investigate the role of surface heterogeneity in nanoparticle-protein interaction. We have chosen the particles as well as the proteins to provide different types (in distribution and length-scale) of heterogeneity. The goal was to unveil the role of heterogeneity and of its length-scale in the particle-protein interaction. Dynamic light scattering and circular dichroism spectroscopy were used to reveal different interactions at pH above and below the isoelectric points of the proteins, which is related to the charge heterogeneity on the protein surface. At pH 7.4, there was only a monolayer of proteins adsorbed onto the NPs and the secondary structure of proteins remained intact. At pH 4.0, large aggregates of nanoparticle-protein complexes were formed and the secondary structures of the proteins were significantly disrupted. In terms of interaction thermodynamics, results from isothermal titration calorimetry showed that ubiquitin adsorbed differently onto (1) AuNPs with charged and nonpolar terminals organized into nano-scale structure (66-34 OT), (2) AuNPs with randomly distributed terminals (66-34 brOT), and (3) AuNPs with homogeneously charged terminals (MUS). This difference in adsorption behavior was not observed when AuNPs interacted with fibrinogen. The results suggested that the interaction between the proteins and AuNPs was influenced by the surface heterogeneity on the AuNPs, and this influence depends on the scale of surface heterogeneity and the size of the proteins.

Royal Society of Chemistry2013

Gold nanoparticles as a new tool in amyloid studies

Urszula Beata Cendrowska

The misfolding and self-assembly of proteins into fibrils is a hallmark of several neurodegenerative and systemic diseases. These disease-associated proteins have the propensity to form fibrils with a cross-Î² sheet structure, called amyloids. Amyloids can assemble into structures of various morphologies and increasing evidence suggests, that this polymorphism gives rise to distinct toxicity and pathology spreading patterns. This was observed for fibrils formed under different conditions or derived from different diseased tissues. Validation of this hypothesis remains challenging due to the laborious methods that are employed to enable the direct visualization and analysis of amyloid fibrils polymorphism in hydrated and complex biological samples. This thesis presents a novel microscopy method for assessing amyloid fibrils polymorphism using small gold nanoparticles (~3 nm diameter) coated with a mixture of 1-octanethiol and 11-mercapto-1-undecanesulfonate. These nanoparticles efficiently label amyloid fibrils that were produced in vitro as well as fibrils that were derived from tissues of human patients (ex vivo), that suffered from diseases linked to amyloid aggregates. Decoration with gold nanoparticles enabled the rapid visualization of the labelled contours of the amyloid fibrils that were imaged using Cryogenic transmission electron microscopy (cryo TEM). This method rendered the structural analysis of the fibrils relatively easy. Due to the labelling of the fibrils, we were able to observe a wide range of the morphologically polymorphic amyloids for in vitro derived samples. Moreover, comparative analysis of these fibrils with ex vivo derived amyloids, revealed significant differences in the morphological homogeneity. Our results showed an unexpected uniformity of the ex vivo derived fibrils, highlighting the influence of the milieu on the aggregation of amyloid fibrils (Chapter 4 and 5). The nanoparticles were also used as a probe to analyze amyloid fibrils surfaces. For example, we showed that sequence modification influences the efficiency of amyloid fibril decoration with our nanomaterial. In Chapter 3, we present that two types of fibrils and their truncated versions, were differently labelled due to the modification of their charged domains. This shows, that we can assess the changes in the primary structure of the amyloidogenic protein by monitoring the interactions between the nanoparticles and the surface of amyloids. Furthermore, results presented in Chapter 6 show that the decoration of the amyloids is possible in various types of biological milieu, including cerebrospinal fluids (CSF) and cell lysates. This finding suggests that our gold nanomaterial can potentially be used for targeted amyloid decoration in more complex systems, such as cells or tissues. The results presented in this thesis show that gold nanoparticles may represent a novel tool for rapid determination of the polymorphism of in vitro and ex vivo derived amyloid fibrils. We demonstrate and validate the advantages of our technique such as simplicity, quick data acquisition and analysis, stability of the labelling and high efficiency of decoration in the complex samples. This can be of high value for the studies of amyloid related diseases as this method allows for relatively easy screen of fibrils derived from the patientsâ tissue and recognition of polymorphic species.

EPFL2020