Ivan Usov

Understanding nanocellulose chirality and structure-properties relationship at the single fibril level

Nanocellulose fibrils are ubiquitous in nature and nanotechnologies but their mesoscopic structural assembly is not yet fully understood. Here we study the structural features of rod-like cellulose nanoparticles on a single particle level, by applying statistical polymer physics concepts on electron and atomic force microscopy images, and we assess their physical properties via quantitative nanomechanical mapping. We show evidence of right-handed chirality, observed on both bundles and on single fibrils. Statistical analysis of contours from microscopy images shows a non-Gaussian kink angle distribution. This is inconsistent with a structure consisting of alternating amorphous and crystalline domains along the contour and supports process-induced kink formation. The intrinsic mechanical properties of nanocellulose are extracted from nanoindentation and persistence length method for transversal and longitudinal directions, respectively. The structural analysis is pushed to the level of single cellulose polymer chains, and their smallest associated unit with a proposed 2 × 2 chain-packing arrangement.

Measurement of intrinsic properties of amyloid fibrils by the peak force QNM method.

We report the investigation of the mechanical properties of different types of amyloid fibrils by the peak force quantitative nanomechanical (PF-QNM) technique. We demonstrate that this technique correctly measures the Youngs modulus independent of the polymorphic state and the cross-sectional structural details of the fibrils, and we show that values for amyloid fibrils assembled from heptapeptides, α-synuclein, Aβ(1-42), insulin, β-lactoglobulin, lysozyme, ovalbumin, Tau protein and bovine serum albumin all fall in the range of 2-4 GPa.

Non-equilibrium nature of two-dimensional isotropic and nematic coexistence in amyloid fibrils at liquid interfaces

Two-dimensional alignment of shape-anisotropic colloids is ubiquitous in nature, ranging from interfacial virus assembly to amyloid plaque formation. The principles governing two-dimensional self-assembly have therefore long been studied, both theoretically and experimentally, leading, however, to diverging fundamental interpretations on the nature of the two-dimensional isotropic-nematic phase transition. Here we employ single-molecule atomic force microscopy, cryogenic scanning electron microscopy and passive probe particle tracking to study the adsorption and liquid crystalline ordering of semiflexible β-lactoglobulin fibrils at liquid interfaces. Fibrillar rigidity changes on increasing interfacial density, with a maximum caused by alignment and a subsequent decrease stemming from crowding and domain bending. Coexistence of nematic and isotropic regions is resolved and quantified by a length scale-dependent order parameter S(2D)(d). The nematic surface fraction increases with interfacial fibril density, but depends, for a fixed interfacial density, on the initial bulk concentration, ascribing the observed two-dimensional isotropic-nematic coexistence to non-equilibrium phenomena.

Polymorphism Complexity and Handedness Inversion in Serum Albumin Amyloid Fibrils

Protein-based amyloid fibrils can show a great variety of polymorphic structures within the same protein precursor, although the origins of these structural homologues remain poorly understood. In this work we investigate the fibrillation of bovine serum albumin--a model globular protein--and we follow the polymorphic evolution by a statistical analysis of high-resolution atomic force microscopy images, complemented, at larger length scales, by concepts based on polymer physics formalism. We identify six distinct classes of coexisting amyloid fibrils, including flexible left-handed twisted ribbons, rigid right-handed helical ribbons and nanotubes. We show that the rigid fibrils originate from flexible fibrils through two diverse polymorphic transitions, first, via a single-fibril transformation when the flexible left-handed twisted ribbons turn into the helical left-handed ribbons, to finally evolve into nanotube-like structures, and second, via a double-fibril transformation when two flexible left-handed twisted ribbons wind together resulting in a right-handed twisted ribbon, followed by a rigid right-handed helical ribbon polymorphic conformation. Hence, the change in handedness occurs with an increase in the level of the fibrils structural organization.

Adsorption at liquid interfaces induces amyloid fibril bending and ring formation

Protein fibril accumulation at interfaces is an important step in many physiological processes and neurodegenerative diseases as well as in designing materials. Here we show, using β-lactoglobulin fibrils as a model, that semiflexible fibrils exposed to a surface do not possess the Gaussian distribution of curvatures characteristic for wormlike chains, but instead exhibit a spontaneous curvature, which can even lead to ring-like conformations. The long-lived presence of such rings is confirmed by atomic force microscopy, cryogenic scanning electron microscopy, and passive probe particle tracking at air- and oil-water interfaces. We reason that this spontaneous curvature is governed by structural characteristics on the molecular level and is to be expected when a chiral and polar fibril is placed in an inhomogeneous environment such as an interface. By testing β-lactoglobulin fibrils with varying average thicknesses, we conclude that fibril thickness plays a determining role in the propensity to form rings.

Polymorphism in bovine serum albumin fibrils: morphology and statistical analysis

We investigate the self-assembly process of the globular protein bovine serum albumin (BSA) into fibrillar structures upon incubating the protein solution at high temperature (90 degreeC) and in an acidic environment (pH 2) for several days. The investigation is performed by atomic force microscopy (AFM) on the self-assembled fibrillar structures, adsorbed on mica substrates from a solution at different fibrillation time snapshots. A rigorous study of structural morphology reveals a sophisticated hierarchy of the BSA fibrils, where two major classes can be identified: flexible and rigid fibrils, with an order of magnitude of difference in their stiffness. Furthermore, each main class can be divided into two subclasses of thin and thick fibrils according to their average height. It is also shown that all types of flexible ribbon-like fibrils at some stage can wrap and close into nanotubes, that is into a rigid class of fibril. A precise statistical analysis of all the subclasses identified is developed throughout the manuscript. The determination of height and contour length distributions, persistence lengths, and other topological characteristics is carried out by processing the coordinates of BSA fibrils acquired from AFM imaging using in-house developed software. The resulting statistical analysis allows better understanding the fibrillation process and the structural properties of the BSA fibrils.

Anomalous stiffening and ion-induced coil-helix transition of carrageenans under monovalent salt conditions.

The macromolecular conformations of anionic polysaccharides with decreasing linear charge densities—lambda, iota, and kappa carrageenan—, at varying NaCl concentrations, are studied by single-chain statistical analysis of high-resolution atomic force microscopy (AFM) images. Lambda remains in the random coil conformation, whereas iota and kappa undergo ion-induced coil-helix transitions, with a 2-3-fold increase in chain rigidity. At low ionic strengths, I, the polymer chains sequester Na⁺, leading to a greater flexibility, and beyond a critical I to the formation of an intramolecular single helix. The persistence length exhibits a sublinear dependence on the Debye screening length, κ⁻¹, L(p)(e) ∼ κ(-y) (with 0 < y < 1), deviating from the classical polyelectrolyte behavior expressed by Odijk-Skolnick-Fixman or Barrat-Joanny models. Above a certain I, the L(p) shows an upturn, resulting in polymer stiffening and nonmonotonic behavior. This phenomenon is inferred from specific ion-polymer interactions and/or nonlinear electrostatic physics involving ion-ion correlations.

Nematic field transfer in a two-dimensional protein fibril assembly

We perform Atomic Force Microscopy and numerical simulations of a bimodal solution containing long, semiflexible β-lactoglobulin fibrils and short, flexible β-lactoglobulin linear aggregates at an air-water interface. Short aggregates orient perpendicular to fibrils at very short distances and preferentially parallel at intermediate distances. At even larger distances an isotropic distribution is observed. Parallel ordering coincides with aggregate stretching: by straightening, small aggregates are able to approach the fibrils within a distance smaller than their radius of gyration. These findings contribute to the understanding of how anisotropic interactions are transferred in two-dimensional bimodal nematic fields of biopolymers at liquid interfaces.

Nanocellulose Fragmentation Mechanisms and Inversion of Chirality from the Single Particle to the Cholesteric Phase

Understanding how nanostructure and nanomechanics influence physical material properties on the micro- and macroscale is an essential goal in soft condensed matter research. Mechanisms governing fragmentation and chirality inversion of filamentous colloids are of specific interest because of their critical role in load-bearing and self-organizing functionalities of soft nanomaterials. Here we provide a fundamental insight into the self-organization across several length scales of nanocellulose, an important biocolloid system with wide-ranging applications as structural, insulating, and functional material. Through a combined microscopic and statistical analysis of nanocellulose fibrils at the single particle level, we show how mechanically and chemically induced fragmentations proceed in this system. Moreover, by studying the bottom-up self-assembly of fragmented carboxylated cellulose nanofibrils into cholesteric liquid crystals, we show via direct microscopic observations that the chirality is inverted from right-handed at the nanofibril level to left-handed at the level of the liquid crystal phase. These results improve our fundamental understanding of nanocellulose and provide an important rationale for its application in colloidal systems, liquid crystals, and nanomaterials.