Oligomer Diversity during the Aggregation of the Repeat Region of Tau.

The molecular mechanism of protein aggregation is of both fundamental and clinical importance as amyloid aggregates are linked to a number of neurodegenerative disorders. Such protein aggregates include macroscopic insoluble fibrils as well as small soluble oligomeric species. Time-dependent resolution of these species is prerequisite for a detailed quantitative understanding of protein aggregation; this remains challenging due to the lack of methods for detecting and characterizing transient and heterogeneous protein oligomers.

Kinetic barriers to α-synuclein protofilament formation and conversion into mature fibrils.

Oligomeric and protofibrillar aggregates that are populated along the pathway of amyloid fibril formation appear generally to be more toxic than the mature fibrillar state. In particular, α-synuclein, the protein associated with Parkinson's disease, forms kinetically trapped protofibrils in the presence of lipid vesicles. Here, we show that lipid-induced α-synuclein protofibrils can convert rapidly to mature fibrils at higher temperatures.

Identification and nanomechanical characterization of the fundamental single-strand protofilaments of amyloid α-synuclein fibrils.

The formation and spreading of amyloid aggregates from the presynaptic protein α-synuclein in the brain play central roles in the pathogenesis of Parkinson's disease. Here, we use high-resolution atomic force microscopy to investigate the early oligomerization events of α-synuclein with single monomer angstrom resolution. We identify, visualize, and characterize directly the smallest elementary unit in the hierarchical assembly of amyloid fibrils, termed here single-strand protofilaments.

Enhancing the Resolution of Micro Free Flow Electrophoresis through Spatially Controlled Sample Injection.

Free flow electrophoresis is a versatile technique for the continuous separation of mixtures with both preparative and analytical applications. Microscale versions of free flow electrophoresis are particularly attractive strategies because of their fast separation times, ability to work with small sample volumes, and large surface area to volume ratios facilitating rapid heat transfer, thus minimizing the detrimental effects of Joule heating even at high voltages. The resolution of microscale free flow electrophoresis, however, is limited by the broadening of the analyte beam in the microfluidic channel, an effect that becomes especially pronounced when the analyte is deflected significantly away from its original position.

Determination of Polypeptide Conformation with Nanoscale Resolution in Water.

The folding and acquisition of proteins native structure is central to all biological processes of life. By contrast, protein misfolding can lead to toxic amyloid aggregates formation, linked to the onset of neurodegenerative disorders. To shed light on the molecular basis of protein function and malfunction, it is crucial to access structural information on single protein assemblies and aggregates under native conditions.

On the role of sidechain size and charge in the aggregation of A42 with familial mutations.

The aggregation of the amyloid- (A) peptide is linked to the pathogenesis of Alzheimer's disease (AD). In particular, some point mutations within A are associated with early-onset familial Alzheimer's disease. Here we set out to explore how the physical properties of the altered side chains, including their sizes and charges, affect the molecular mechanisms of aggregation.

Kinetic Analysis of Amyloid Formation.

The formation of amyloid fibrils is a central phenomenon in the progressive pathology of many neurodegenerative diseases, as well as in the fabrication of functional materials. Several different molecular processes acting in concert are responsible for the formation of amyloid fibrils from monomeric protein in solution. Here, we describe a method to determine which microscopic processes drive the overall formation of fibrils by using chemical kinetics in combination with systematic experimental datasets analysed in a global manner.

Budding-like division of all-aqueous emulsion droplets modulated by networks of protein nanofibrils.

Networks of natural protein nanofibrils, such as cytoskeletal filaments, control the shape and the division of cells, yet mimicking this functionality in a synthetic setting has proved challenging. Here, we demonstrate that artificial networks of protein nanofibrils can induce controlled deformation and division of all-aqueous emulsion droplets with budding-like morphologies. We show that this process is driven by the difference in the immersional wetting energy of the nanofibril network, and that both the size and the number of the daughter droplets formed during division can be controlled by modulating the fibril concentration and the chemical properties of the fibril network.

On-chip measurements of protein unfolding from direct observations of micron-scale diffusion.

Investigations of protein folding, unfolding and stability are critical for the understanding of the molecular basis of biological structure and function. We describe here a microfluidic approach to probe the unfolding of unlabelled protein molecules in microliter volumes. We achieve this objective through the use of a microfluidic platform, which allows the changes in molecular diffusivity upon folding and unfolding to be detected directly.

Rapid Growth of Acetylated Aβ(16-20) into Macroscopic Crystals.

Aberrant assembly of the amyloid-β (Aβ) is responsible for the development of Alzheimer's disease, but can also be exploited to obtain highly functional biomaterials. The short Aβ fragment, KLVFF (Aβ), is crucial for Aβ assembly and considered to be an Aβ aggregation inhibitor. Here, we show that acetylation of KLVFF turns it into an extremely fast self-assembling molecule, reaching macroscopic ( i.

Cooperative Assembly of Hsp70 Subdomain Clusters.

Many molecular chaperones exist as oligomeric complexes in their functional states, yet the physical determinants underlying such self-assembly behavior, as well as the role of oligomerization in the activity of molecular chaperones in inhibiting protein aggregation, have proven to be difficult to define. Here, we demonstrate direct measurements under native conditions of the changes in the average oligomer populations of a chaperone system as a function of concentration and time and thus determine the thermodynamic and kinetic parameters governing the self-assembly process. We access this self-assembly behavior in real time under native-like conditions by monitoring the changes in the micrometer-scale diffusion of the different complexes in time and space using a microfluidic platform.

Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes.

Alzheimer's disease is a neurodegenerative disorder associated with the aberrant aggregation of the amyloid-β peptide. Although increasing evidence implicates cholesterol in the pathogenesis of Alzheimer's disease, the detailed mechanistic link between this lipid molecule and the disease process remains to be fully established. To address this problem, we adopt a kinetics-based strategy that reveals a specific catalytic role of cholesterol in the aggregation of Aβ42 (the 42-residue form of the amyloid-β peptide).

FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation.

Water-Dispersible Polydopamine-Coated Nanofibers for Stimulation of Neuronal Growth and Adhesion.

Hybrid nanomaterials have shown great potential in regenerative medicine due to the unique opportunities to customize materials properties for effectively controlling cellular growth. The peptide nanofiber-mediated auto-oxidative polymerization of dopamine, resulting in stable aqueous dispersions of polydopamine-coated peptide hybrid nanofibers, is demonstrated. The catechol residues of the polydopamine coating on the hybrid nanofibers are accessible and provide a platform for introducing functionalities in a pH-responsive polymer analogous reaction, which is demonstrated using a boronic acid modified fluorophore.

Measurement of Tau Filament Fragmentation Provides Insights into Prion-like Spreading.

The ordered assembly of amyloidogenic proteins causes a wide spectrum of common neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. These diseases share common features with prion diseases, in which misfolded proteins can self-replicate and transmit disease across different hosts. Deciphering the molecular mechanisms that underlie the amplification of aggregates is fundamental for understanding how pathological deposits can spread through the brain and drive disease.

Distinct thermodynamic signatures of oligomer generation in the aggregation of the amyloid-β peptide.

Mapping free-energy landscapes has proved to be a powerful tool for studying reaction mechanisms. Many complex biomolecular assembly processes, however, have remained challenging to access using this approach, including the aggregation of peptides and proteins into amyloid fibrils implicated in a range of disorders. Here, we generalize the strategy used to probe free-energy landscapes in protein folding to determine the activation energies and entropies that characterize each of the molecular steps in the aggregation of the amyloid-β peptide (Aβ42), which is associated with Alzheimer's disease.

Microfluidic approaches for probing amyloid assembly and behaviour.

The self-assembly of proteins into supramolecular structures and machinery underpins biological activity in living systems. Misassembled, misfolded and aggregated protein structures can, by contrast, have deleterious activity and such species are at the origin of a number of disease states ranging from cancer to neurodegenerative disorders. In particular, the formation of highly ordered protein aggregates, amyloid fibrils, from normally soluble peptides and proteins, is the common pathological hallmark of a range a group of over fifty protein misfolding disorders.

Chemical Kinetics for Bridging Molecular Mechanisms and Macroscopic Measurements of Amyloid Fibril Formation.

Understanding how normally soluble peptides and proteins aggregate to form amyloid fibrils is central to many areas of modern biomolecular science, ranging from the development of functional biomaterials to the design of rational therapeutic strategies against increasingly prevalent medical conditions such as Alzheimer's and Parkinson's diseases. As such, there is a great need to develop models to mechanistically describe how amyloid fibrils are formed from precursor peptides and proteins. Here we review and discuss how ideas and concepts from chemical reaction kinetics can help to achieve this objective.

Massively parallel C. elegans tracking provides multi-dimensional fingerprints for phenotypic discovery.

Background: The nematode worm C. elegans is a model organism widely used for studies of genetics and of human disease. The health and fitness of the worms can be quantified in different ways, such as by measuring their bending frequency, speed or lifespan.

Real-Time Intrinsic Fluorescence Visualization and Sizing of Proteins and Protein Complexes in Microfluidic Devices.

Optical detection has become a convenient and scalable approach to read out information from microfluidic systems. For the study of many key biomolecules, however, including peptides and proteins, which have low fluorescence emission efficiencies at visible wavelengths, this approach typically requires labeling of the species of interest with extrinsic fluorophores to enhance the optical signal obtained - a process which can be time-consuming, requires purification steps, and has the propensity to perturb the behavior of the systems under study due to interactions between the labels and the analyte molecules. As such, the exploitation of the intrinsic fluorescence of protein molecules in the UV range of the electromagnetic spectrum is an attractive path to allow the study of unlabeled proteins.

Biophotonics of Native Silk Fibrils.

Native silk fibroin (NSF) is a unique biomaterial with extraordinary mechanical and biochemical properties. These key characteristics are directly associated with the physical transformation of unstructured, soluble NSF into highly organized nano- and microscale fibrils rich in β-sheet content. Here, it is shown that this NSF fibrillation process is accompanied by the development of intrinsic fluorescence in the visible range, upon near-UV excitation, a phenomenon that has not been investigated in detail to date.

Direct Observation of Oligomerization by Single Molecule Fluorescence Reveals a Multistep Aggregation Mechanism for the Yeast Prion Protein Ure2.

The self-assembly of polypeptides into amyloid structures is associated with a range of increasingly prevalent neurodegenerative diseases as well as with a select set of functional processes in biology. The phenomenon of self-assembly results in species with dramatically different sizes, from small oligomers to large fibrils; however, the kinetic relationship between these species is challenging to characterize. In the case of prion aggregates, these structures can self-replicate and act as infectious agents.

Microfluidic Diffusion Platform for Characterizing the Sizes of Lipid Vesicles and the Thermodynamics of Protein-Lipid Interactions.

Elucidation of the fundamental interactions of proteins with biological membranes under native conditions is crucial for understanding the molecular basis of their biological function and malfunction. Notably, the large surface to volume ratio of living cells provides a molecular landscape for significant interactions of cellular components with membranes, thereby potentially modulating their function. However, such interactions can be challenging to probe using conventional biophysical methods due to the heterogeneity of the species and processes involved.

Microfluidics for Protein Biophysics.

Microfluidics has the potential to transform experimental approaches across the life sciences. In this review, we discuss recent advances enabled by the development and application of microfluidic approaches to protein biophysics. We focus on areas where key fundamental features of microfluidics open up new possibilities and present advantages beyond low volumes and short time-scale analysis, conventionally provided by microfluidics.

Secondary nucleation of monomers on fibril surface dominates α-synuclein aggregation and provides autocatalytic amyloid amplification.

Parkinson's disease (PD) is characterized by proteinaceous aggregates named Lewy Bodies and Lewy Neurites containing α-synuclein fibrils. The underlying aggregation mechanism of this protein is dominated by a secondary process at mildly acidic pH, as in endosomes and other organelles. This effect manifests as a strong acceleration of the aggregation in the presence of seeds and a weak dependence of the aggregation rate on monomer concentration.