Jozef Adamcik

Understanding amyloid aggregation by statistical analysis of atomic force microscopy images

The aggregation of proteins is central to many aspects of daily life, including food processing, blood coagulation, eye cataract formation disease and prion-related neurodegenerative infections. However, the physical mechanisms responsible for amyloidosis-the irreversible fibril formation of various proteins that is linked to disorders such as Alzheimers, Creutzfeldt-Jakob and Huntingtons diseases-have not yet been fully elucidated. Here, we show that different stages of amyloid aggregation can be examined by performing a statistical polymer physics analysis of single-molecule atomic force microscopy images of heat-denatured beta-lactoglobulin fibrils. The atomic force microscopy analysis, supported by theoretical arguments, reveals that the fibrils have a multistranded helical shape with twisted ribbon-like structures. Our results also indicate a possible general model for amyloid fibril assembly and illustrate the potential of this approach for investigating fibrillar systems.

Biodegradable nanocomposites of amyloid fibrils and graphene with shape-memory and enzyme-sensing properties

Graphene has exceptional mechanical and electronic properties, but its hydrophobic nature is a disadvantage in biologically related applications. Amyloid fibrils are naturally occurring protein aggregates that are stable in solution or under highly hydrated conditions, have well-organized supramolecular structures and outstanding strength. Here, we show that graphene and amyloid fibrils can be combined to create a new class of biodegradable composite materials with adaptable properties. This new composite material is inexpensive, highly conductive and can be degraded by enzymes. Furthermore, it can reversibly change shape in response to variations in humidity, and can be used in the design of biosensors for quantifying the activity of enzymes. The properties of the composite can be fine-tuned by changing the graphene-to-amyloid ratio.

Single-step direct measurement of amyloid fibrils stiffness by peak force quantitative nanomechanical atomic force microscopy

We present an original application of a new atomic force microscopy mode called peak force tapping for the investigation of the mechanical properties of β-lactoglobulin amyloid fibrils. The values of Young’s modulus obtained by this technique are in perfect agreement with the indirect evaluation of fibrils stiffness obtained by combining polymer physics and topological statistical analysis on fibrils’ structural conformations. This technique shows great promise in the estimation of the elastic properties of nanostructured objects relevant in biology, soft matter, and nanotechnology.

General Self-Assembly Mechanism Converting Hydrolyzed Globular Proteins Into Giant Multistranded Amyloid Ribbons

We report a rationale for the formation of amyloid fibrils from globular proteins, and we infer about its possible generality by showing the formation of giant multistranded twisted and helical ribbons from both lysozyme and β-lactoglobulin. We follow the kinetics of the fibrillation under the same conditions of temperature (90 °C) and incubation time (0-30 h) for both proteins, and we assess the structural changes during fibrillation by single-molecule atomic force microscopy (AFM), circular dichroism (CD), and SDS-PAGE. With incubation time, the width of a multistranded fibril increases up to an unprecedented size, with a lateral assembly of as many as 17 protofilaments (173 nm width). In both cases, a progressive unfolding and hydrolysis of the proteins into very short peptide sequences occurs. The molecular weights of peptide fragments, the secondary structure evolution, and the morphology of the final fibrils present striking similarities between lysozyme and β-lactoglobulin. Because of additional analogies to synthetic peptide fibrils, these findings support a universal common fibrillation mechanism in which hydrolyzed fragments play the central role.

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.

Direct Observation of Time‐Resolved Polymorphic States in the Self‐Assembly of End‐Capped Heptapeptides

Amyloid fibrils resulting from uncontrolled peptide aggregation are associated with several neurodegenerative diseases. Their polymorphism depends on a number of factors including pH, ionic strength, electrostatic interactions, hydrophobic interactions, hydrogen bonding, aromatic stacking interactions, and chirality. Understanding the mechanism of amyloid fibril formation can improve strategies towards the prevention of fibrillation processes and enable a wide range of potential applications in nanotemplating and nanotechnology.

Fractal dimension and localization of DNA knots

The scaling properties of DNA knots of different complexities were studied by atomic force microscope. Following two different protocols DNA knots are adsorbed onto a mica surface in regimes of (i) strong binding, that induces a kinetic trapping of the three-dimensional (3D) configuration, and of (ii) weak binding, that permits (partial) relaxation on the surface. In (i) the radius of gyration of the adsorbed DNA knot scales with the 3D Flory exponent nu approximately 0.60 within error. In (ii), we find nu approximately 0.66, a value between the 3D and 2D (nu=3/4) exponents. Evidence is also presented for the localization of knot crossings in 2D under weak adsorption conditions.

Observation of single-stranded DNA on mica and highly oriented pyrolytic graphite by atomic force microscopy

Atomic force microscopy was used to image single‐stranded DNA (ssDNA) adsorbed on mica modified by Mg2+, by 3‐aminopropyltriethoxysilane or on modified highly oriented pyrolytic graphite (HOPG). ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG graphite ssDNA molecules adopt a conformation without secondary structures. We have shown that the immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base‐pairing, thus this method could be important for studying certain processes involving ssDNA in more details.

Snapshots of fibrillation and aggregation kinetics in multistranded amyloid β-lactoglobulin fibrils

We have investigated the structural time-evolution of multistranded β-lactoglobulin protein fibrils at pH 2 and 90 °C by combining small angle neutron scattering (SANS), dynamic (DLS) and depolarized light scattering (DDLS) as well as atomic force microscopy (AFM). Light scattering techniques, combined with SANS clearly demonstrate the different stages of conversion of β-lactoglobulin monomers (2 wt %) into semiflexible protein fibrils upon heating at 90 °C. In addition, atomic force microscopy allows the resolution of some details of the fibrils at the molecular length scale which bulk scattering techniques cannot capture. Thus, we were able to resolve and identify different individual stages of the fibrillation process, including the formation of protofilaments, their alignment and aggregation into mature multistranded fibrils, and the development of a periodic pitch along their contour length. The picture emerging from combination of the scattering and single molecule techniques is consistent with three critical steps: (i) individual protofilaments align upon approaching due to liquid crystalline interactions; (ii) short range attractions among filaments—presumably of Lennard–Jones or hydrophobicity type—lead to irreversible aggregation of nearly perfectly aligned protofilaments into multistranded ribbon-like fibrils; (iii) intramolecular electrostatic repulsion of fibrils leads to twisting of the ribbon along the axis leading to the development of a periodic pitch along the fibrils contour length. The individual stages of the fibrillation and aggregation process are discussed in detail, in terms of the colloidal physics involved.