Trace_y: Software algorithms for structural analysis of individual helical filaments by three-dimensional contact point reconstruction atomic force microscopy.

Atomic force microscopy (AFM) is a powerful and increasingly accessible technology that has a wide range of bio-imaging applications. AFM is capable of producing detailed three-dimensional topographical images with high signal-to-noise ratio, which enables the structural features of individual molecules to be studied without the need for ensemble averaging. Here, a software tool Trace_y, designed to reconstruct the three-dimensional surface envelopes of individual helical filament structures from topographical AFM images, is presented.

Atomic force microscopy 3D structural reconstruction of individual particles in the study of amyloid protein assemblies.

Recent developments in atomic force microscopy (AFM) image analysis have made three-dimensional (3D) structural reconstruction of individual particles observed on 2D AFM height images a reality. Here, we review the emerging contact point reconstruction AFM (CPR-AFM) methodology and its application in 3D reconstruction of individual helical amyloid filaments in the context of the challenges presented by the structural analysis of highly polymorphous and heterogeneous amyloid protein structures. How individual particle-level structural analysis can contribute to resolving the amyloid polymorph structure-function relationships, the environmental triggers leading to protein misfolding and aggregation into amyloid species, the influences by the conditions or minor fluctuations in the initial monomeric protein structure on the speed of amyloid fibril formation, and the extent of the different types of amyloid species that can be formed, are discussed.

Tuning dynamic DNA- and peptide-driven self-assembly in DNA-peptide conjugates.

DNA-peptide conjugates offer an opportunity to marry the benefits of both biomolecular classes, combining the high level of programmability found with DNA, with the chemical diversity of peptides. These hybrid systems offer potential in fields such as therapeutics, nanotechnology, and robotics. Using the first DNA-β-turn peptide conjugate, we present three studies investigating the self-assembly of DNA-peptide conjugates over a period of 28 days.

An Additive-Free Model for Tau Self-Assembly.

Tau is a natively unfolded protein that contributes to the stability of microtubules. Under pathological conditions such as Alzheimer's disease (AD), tau protein misfolds and self-assembles to form paired helical filaments (PHFs) and straight filaments (SFs). Full-length tau protein assembles poorly and its self-assembly is enhanced with polyanions such as heparin and RNA in vitro, but a role for heparin or other polyanions in vivo remains unclear.

Comparative Analysis of the Relative Fragmentation Stabilities of Polymorphic Alpha-Synuclein Amyloid Fibrils.

The division of amyloid fibril particles through fragmentation is implicated in the progression of human neurodegenerative disorders such as Parkinson's disease. Fragmentation of amyloid fibrils plays a crucial role in the propagation of the amyloid state encoded in their three-dimensional structures and may have an important role in the spreading of potentially pathological properties and phenotypes in amyloid-associated diseases. However, despite the mechanistic importance of fibril fragmentation, the relative stabilities of different types or different polymorphs of amyloid fibrils toward fragmentation remain to be quantified.

Structural Identification of Individual Helical Amyloid Filaments by Integration of Cryo-Electron Microscopy-Derived Maps in Comparative Morphometric Atomic Force Microscopy Image Analysis.

The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations.

Heterotypic Amyloid β interactions facilitate amyloid assembly and modify amyloid structure.

It is still unclear why pathological amyloid deposition initiates in specific brain regions or why some cells or tissues are more susceptible than others. Amyloid deposition is determined by the self-assembly of short protein segments called aggregation-prone regions (APRs) that favour cross-β structure. Here, we investigated whether Aβ amyloid assembly can be modified by heterotypic interactions between Aβ APRs and short homologous segments in otherwise unrelated human proteins.

Insights into the dynamic trajectories of protein filament division revealed by numerical investigation into the mathematical model of pure fragmentation.

The dynamics by which polymeric protein filaments divide in the presence of negligible growth, for example due to the depletion of free monomeric precursors, can be described by the universal mathematical equations of 'pure fragmentation'. The rates of fragmentation reactions reflect the stability of the protein filaments towards breakage, which is of importance in biology and biomedicine for instance in governing the creation of amyloid seeds and the propagation of prions. Here, we devised from mathematical theory inversion formulae to recover the division rates and division kernel information from time-dependent experimental measurements of filament size distribution.

Amyloid particles facilitate surface-catalyzed cross-seeding by acting as promiscuous nanoparticles.

Amyloid seeds are nanometer-sized protein particles that accelerate amyloid assembly as well as propagate and transmit the amyloid protein conformation associated with a wide range of protein misfolding diseases. However, seeded amyloid growth through templated elongation at fibril ends cannot explain the full range of molecular behaviors observed during cross-seeded formation of amyloid by heterologous seeds. Here, we demonstrate that amyloid seeds can accelerate amyloid formation via a surface catalysis mechanism without propagating the specific amyloid conformation associated with the seeds.

On the Structural Diversity and Individuality of Polymorphic Amyloid Protein Assemblies.

The prediction of highly ordered three-dimensional structures of amyloid protein fibrils from the amino acid sequences of their monomeric self-assembly precursors constitutes a challenging and unresolved aspect of the classical protein folding problem. Because of the polymorphic nature of amyloid assembly whereby polypeptide chains of identical amino acid sequences under identical conditions are capable of self-assembly into a spectrum of different fibril structures, the prediction of amyloid structures from an amino acid sequence requires a detailed and holistic understanding of its assembly free energy landscape. The full extent of the structure space accessible to the cross-β molecular architecture of amyloid must also be resolved.

Thermodynamics of amyloid fibril formation from non-equilibrium experiments of growth and dissociation.

Amyloid fibrils are ordered, non-covalent polymers of proteins that are linked to a range of diseases, as well as biological functions. Amyloid fibrils are often considered thermodynamically so stable that they appear to be irreversible, explaining why very few quantitative thermodynamic studies have been performed on amyloid fibrils, compared to the very large body of kinetic studies. Here we explore the thermodynamics of amyloid fibril formation by the protein PI3K-SH3, which forms amyloid fibrils under acidic conditions.

The Division of Amyloid Fibrils: Systematic Comparison of Fibril Fragmentation Stability by Linking Theory with Experiments.

The division of amyloid protein fibrils is required for the propagation of the amyloid state and is an important contributor to their stability, pathogenicity, and normal function. Here, we combine kinetic nanoscale imaging experiments with analysis of a mathematical model to resolve and compare the division stability of amyloid fibrils. Our theoretical results show that the division of any type of filament results in self-similar length distributions distinct to each fibril type and the conditions applied.

Quantification of amyloid fibril polymorphism by nano-morphometry reveals the individuality of filament assembly.

Amyloid fibrils are highly polymorphic structures formed by many different proteins. They provide biological function but also abnormally accumulate in numerous human diseases. The physicochemical principles of amyloid polymorphism are not understood due to lack of structural insights at the single-fibril level.

Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs.

Atomic force microscopy, AFM, is a powerful tool that can produce detailed topographical images of individual nano-structures with a high signal-to-noise ratio without the need for ensemble averaging. However, the application of AFM in structural biology has been hampered by the tip-sample convolution effect, which distorts images of nano-structures, particularly those that are of similar dimensions to the cantilever probe tips used in AFM. Here we show that the tip-sample convolution results in a feature-dependent and non-uniform distribution of image resolution on AFM topographs.

Tau (297-391) forms filaments that structurally mimic the core of paired helical filaments in Alzheimer's disease brain.

The constituent paired helical filaments (PHFs) in neurofibrillary tangles are insoluble intracellular deposits central to the development of Alzheimer's disease (AD) and other tauopathies. Full-length tau requires the addition of anionic cofactors such as heparin to enhance assembly. We have shown that a fragment from the proteolytically stable core of the PHF, tau 297-391 known as 'dGAE', spontaneously forms cross-β-containing PHFs and straight filaments under physiological conditions.

The molecular lifecycle of amyloid - Mechanism of assembly, mesoscopic organisation, polymorphism, suprastructures, and biological consequences.

The formation of a diverse range of amyloid structures from normally soluble proteins and peptides is a hallmark of devastating human disorders as well as biological functions. The current molecular understanding of the amyloid lifecycle reveals four processes central to their growth and propagation: primary nucleation, elongation, secondary nucleation and division. However, these processes result in a wide range of cross-β packing and filament arrangements, including diverse assemblies formed from identical monomeric precursors with the same amino acid sequences.

The Molecular Basis for Apolipoprotein E4 as the Major Risk Factor for Late-Onset Alzheimer's Disease.

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD.

Bioinspired Silicification Reveals Structural Detail in Self-Assembled Peptide Cages.

Understanding how molecules in self-assembled soft-matter nanostructures are organized is essential for improving the design of next-generation nanomaterials. Imaging these assemblies can be challenging and usually requires processing, e.g.

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm.

We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement.

A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology.

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models.

The physical dimensions of amyloid aggregates control their infective potential as prion particles.

Transmissible amyloid particles called prions are associated with infectious prion diseases in mammals and inherited phenotypes in yeast. All amyloid aggregates can give rise to potentially infectious seeds that accelerate their growth. Why some amyloid seeds are highly infectious prion particles while others are less infectious or even inert, is currently not understood.

The involvement of dityrosine crosslinking in α-synuclein assembly and deposition in Lewy Bodies in Parkinson's disease.

Parkinson's disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable α-synuclein (α-syn) amyloid fibrils. α-synuclein is an intrinsically disordered protein that has the capacity to assemble to form β-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly.

Effect of bio-engineering on size, shape, composition and rigidity of bacterial microcompartments.

Bacterial microcompartments (BMCs) are proteinaceous organelles that are found in a broad range of bacteria and are composed of an outer shell that encases an enzyme cargo representing a specific metabolic process. The outer shell is made from a number of different proteins that form hexameric and pentameric tiles, which interact to allow the formation of a polyhedral edifice. We have previously shown that the Citrobacter freundii BMC associated with 1,2-propanediol utilization can be transferred into Escherichia coli to generate a recombinant BMC and that empty BMCs can be formed from just the shell proteins alone.

Insights into the variability of nucleated amyloid polymerization by a minimalistic model of stochastic protein assembly.

Self-assembly of proteins into amyloid aggregates is an important biological phenomenon associated with human diseases such as Alzheimer's disease. Amyloid fibrils also have potential applications in nano-engineering of biomaterials. The kinetics of amyloid assembly show an exponential growth phase preceded by a lag phase, variable in duration as seen in bulk experiments and experiments that mimic the small volumes of cells.