Cryogenic electron tomography to determine thermodynamic quantities for nanoparticle dispersions

Here we present a method to extract thermodynamic quantities for nanoparticle dispersions in solvents. The method is based on the study of tomograms obtained from cryogenic electron tomography (cryoET). The approach is demonstrated for gold nanoparticles (diameter < 5 nm). Tomograms are reconstructed from tilt-series 2D images. Once the three-dimensional (3D) coordinates for the centres of mass of all of the particles in the sample are determined, we calculate the pair distribution function g(r) and the potential of mean force U(r) without any assumption. Importantly, we show that further quantitative information from 3D tomograms is readily available as the spatial fluctuation in the particles’ position can be efficiently determined. This in turn allows for the prompt derivation of the Kirkwood-Buff integrals with all their associated quantities such as the second virial coefficient. Finally, the structure factor and the agglomeration states of the particles are evaluated directly. These thermodynamic quantities provide key insights into the dispersion properties of the particles. The method works well both for dispersed systems containing isolated particles and for systems with varying degrees of agglomerations

Water interactions with complex surfaces

The solvent accessible surface area of proteins is very complex, mainly because it is composed of domains with different degrees of affinity for water. The domains nature and arrangement determine both the interactions of the proteins with water (and therefore the water structure around the protein) as well as the interactions with other particles in suspension (other proteins or other macromolecules). Both these interactions are fundamental to determine the functionality of the proteins. To date, a theoretical model able to predict the wetting properties of proteins has yet to be developed. The goal of this thesis is to improve our understanding of the wetting properties of this kind of complex surfaces. In order to simplify the interpretation of the results a model system has been used: gold nanoparticles protected by a shell composed of a binary mixture of hydrophobic/hydrophilic ligands. These nanoparticles resemble proteins because of their overall size as well as the size of the hydrophilic and hydrophobic domains exposed on their surface. The advantages of working with nanoparticles include the fact that they are stable over a larger range of conditions and that the properties of the ligand shell can be designed and tuned depending on the research question. In this thesis we show that the arrangement of the hydrophobic and hydrophilic domains is responsible for the interaction between the particles and the surrounding solvent (especially water). Indeed, we show that both the water structure and interfacial energy vary significantly between identical particles that differ only in the domain arrangement. A new predictive model is proposed. This model considers, for every molecule in the ligand shell, the contributions of its first-nearest neighbors as a descriptor to determine the wetting properties of the surface. The experiments and theoretical model proposed here provide a starting point to develop a comprehensive understanding of complex interfaces as well as for the engineering of synthetic ones. The interparticle interactions in water are also been characterized. It is shown that there exists the possibility to screen hydrophobic attraction by adding small molecules to the suspension. This stabilization technique seems to be relevant to maintain the stability of biological fluids. An example of the application of this stabilization technique to food science is also presented here. In addition to these studies, an innovative technique to measure the surface energy at the nanoscale is presented here, based on the measurement of the adhesion force between an atomic force microscopy tip and a sample

La spettroscopia di forza basata sull'AFM nello studio dello spazio conformazionale e dei processi aggregativi di proteine prioniche

Le malattie neurodegenerative sono caratterizzate da aggregazione proteica, dipendente dalla perdita della usuale struttura fisiologica funzionale delle proteine coinvolte, a favore di conformazioni tossiche (patologiche). Il modello corrente descrive questi cambiamenti conformazionali come eventi rari e ritiene che non esista una sola conformazione patogena, ma che tali possibili conformazioni siano piuttosto eterogenee. La caratterizzazione di queste strutture è, di conseguenza, difficile con le tradizionali tecniche in bulk che permettono di studiare solo la conformazione media e non rendono possibile il riconoscimento delle caratteristiche dei conformeri individuali. Lo sviluppo delle tecniche di singola molecola ha permesso di studiare in modo approfondito le conformazioni possibili. In questo lavoro la spettroscopia di forza di singola molecola basata sull'AFM viene applicata a PrP (proteina responsabile delle encefalopatie spongiformi trasmissibili). Si studiano gli equilibri conformazionali del monomero e quelli di costrutti oligomerici, allo scopo di caratterizzare gli step iniziali dei processi aggregativi. Nel corso di questo lavoro di tesi è stato, in particolare, sviluppato un sistema di analisi dati, al fine di studiare in modo quantitativo le distribuzioni di eventi ottenute. Grazie a tale strumento è stato possibile riconoscere i segnali di unfolding della conformazione nativa del monomero e notare come essa sia presente anche in costrutti oligomerici, ad indicare come questo ripiegamento sia stabile anche in presenza di più monomeri ravvicinati. Si è osservato l'effetto del pH sulla stabilità di tale struttura, notando come pH acidi destabilizzino il ripiegamento nativo. Inoltre si è studiato il ruolo dell'orientazione dei monomeri nella formazione di strutture dimeriche. Monomeri e oligomeri di PrP sono stati descritti come proteine parzialmente strutturate il cui panorama energetico contiene molti minimi locali, dando origine a parecchie conformazioni transienti

High aspect ratio silicon nanowires control fibroblast adhesion and cytoskeleton organization

Cell-cell and cell-matrix interactions are essential to the survival and proliferation of most cells, and are responsible for triggering a wide range of biochemical pathways. More recently, the biomechanical role of those interactions was highlighted, showing, for instance, that adhesion forces are essential for cytoskeleton organization. Silicon nanowires (Si NWs) with their small size, high aspect ratio and anisotropic mechanical response represent a useful model to investigate the forces involved in the adhesion processes and their role in cellular development. In this work we explored and quantified, by single cell force spectroscopy (SCFS), the interaction of mouse embryonic fibroblasts with a flexible forest of Si NWs. We observed that the cell adhesion forces are comparable to those found on collagen and bare glass coverslip, analogously the membrane tether extraction forces are similar to that on collagen but stronger than that on bare flat glass. Cell survival did not depend significantly on the substrate, although a reduced proliferation after 36 h was observed. On the contrary both cell morphology and cytoskeleton organization revealed striking differences. The cell morphology on Si-NW was characterized by a large number of filopodia and a significant decrease of the cell mobility. The cytoskeleton organization was characterized by the absence of actin fibers, which were instead dominant on collagen and flat glass support. Such findings suggest that the mechanical properties of disordered Si NWs, and in particular their strong asymmetry, play a major role in the adhesion, morphology and cytoskeleton organization processes. Indeed, while adhesion measurements by SCFS provide out-of-plane forces values consistent with those measured on conventional substrates, weaker in-plane forces hinder proper cytoskeleton organization and migration processes

Evidence of Orientation-Dependent Early States of Prion Protein Misfolded Structures from Single Molecule Force Spectroscopy

SIMPLE SUMMARY: Prion diseases are neurodegenerative disorders caused by the amyloidal aggregation of the cellular prion protein. We apply single-molecule force spectroscopy approaches to study the unfolding of prion protein monomers and dimers in different orientations. We find heterogeneous behavior in the prion protein unfolding and an interesting difference between the dimer orientations whereby the dimer in which the C-termini are joined unfolds at a higher force, implying a more stable structure owing to interactions between the C-termini. These results may contribute to a better understanding of the initial steps of oligomer assembly during prion diseases. ABSTRACT: Prion diseases are neurodegenerative disorders characterized by the presence of oligomers and amyloid fibrils. These are the result of protein aggregation processes of the cellular prion protein (PrP(C)) into amyloidal forms denoted as prions or PrP(Sc). We employed atomic force microscopy (AFM) for single molecule pulling (single molecule force spectroscopy, SMFS) experiments on the recombinant truncated murine prion protein (PrP) domain to characterize its conformations and potential initial oligomerization processes. Our AFM-SMFS results point to a complex scenario of structural heterogeneity of PrP at the monomeric and dimer level, like other amyloid proteins involved in similar pathologies. By applying this technique, we revealed that the PrP C-terminal domain unfolds in a two-state process. We used two dimeric constructs with different PrP reciprocal orientations: one construct with two sequential PrP in the N- to C-terminal orientation (N-C dimer) and a second one in the C- to C-terminal orientation (C-C dimer). The analysis revealed that the different behavior in terms of unfolding force, whereby the dimer placed C-C dimer unfolds at a higher force compared to the N-C orientation. We propose that the C-C dimer orientation may represent a building block of amyloid fibril formation

Determination and evaluation of the nonadditivity in wetting of molecularly heterogeneous surfaces

The interface between water and folded proteins is very complex. Proteins have “patchy” solvent-accessible areas composed of domains of varying hydrophobicity. The textbook understanding is that these domains contribute additively to interfacial properties (Cassie’s equation, CE). An ever-growing number of modeling papers question the validity of CE at molecular length scales, but there is no conclusive experiment to support this and no proposed new theoretical framework. Here, we study the wetting of model compounds with patchy surfaces differing solely in patchiness but not in composition. Were CE to be correct, these materials would have had the same solid–liquid work of adhesion (WSL) and time-averaged structure of interfacial water. We find considerable differences in WSL, and sum-frequency generation measurements of the interfacial water structure show distinctively different spectral features. Molecular-dynamics simulations of water on patchy surfaces capture the observed behaviors and point toward significant nonadditivity in water density and average orientation. They show that a description of the molecular arrangement on the surface is needed to predict its wetting properties. We propose a predictive model that considers, for every molecule, the contributions of its first-nearest neighbors as a descriptor to determine the wetting properties of the surface. The model is validated by measurements of WSL in multiple solvents, where large differences are observed for solvents whose effective diameter is smaller than ∼6 Å. The experiments and theoretical model proposed here provide a starting point to develop a comprehensive understanding of complex biological interfaces as well as for the engineering of synthetic ones

Nature-Inspired Circular-Economy Recycling for Proteins: Proof of Concept

The billion tons of synthetic-polymer-based materials (i.e. plastics) produced yearly are a great challenge for humanity. Nature produces even more natural polymers, yet they are sustainable. Proteins are sequence-defined natural polymers that are constantly recycled when living systems feed. Digestion is the protein depolymerization into amino acids (the monomers) followed by their re-assembly into new proteins of arbitrarily different sequence and function. This breaks a common recycling paradigm where a material is recycled into itself. Organisms feed off of random protein mixtures that are "recycled" into new proteins whose identity depends on the cell's specific needs. In this study, mixtures of several peptides and/or proteins are depolymerized into their amino acid constituents, and these amino acids are used to synthesize new fluorescent, and bioactive proteins extracellularly by using an amino-acid-free, cell-free transcription-translation (TX-TL) system. Specifically, three peptides (magainin II, glucagon, and somatostatin 28) are digested using thermolysin first and then using leucine aminopeptidase. The amino acids so produced are added to a commercial TX-TL system to produce fluorescent proteins. Furthermore, proteins with high relevance in materials engineering (beta-lactoglobulin films, used for water filtration, or silk fibroin solutions) are successfully recycled into biotechnologically relevant proteins (fluorescent proteins, catechol 2,3-dioxygenase).ISSN:0935-9648ISSN:1521-409