On the lag phase in amyloid fibril formation.

The formation of nanoscale amyloid fibrils from normally soluble peptides and proteins is a common form of self-assembly phenomenon that has fundamental connections with biological functions and human diseases. The kinetics of this process has been widely studied and exhibits on a macroscopic level three characteristic stages: a lag phase, a growth phase and a final plateau regime. The question of which molecular events take place during each one of these phases has been a central element in the quest for a mechanism of amyloid formation. In this review, we discuss the nature and molecular origin of the lag-phase in amyloid formation by making use of tools and concepts from physical chemistry, in particular from chemical reaction kinetics. We discuss how, in macroscopic samples, it has become apparent that the lag-phase is not a waiting time for nuclei to form. Rather, multiple parallel processes exist and typically millions of primary nuclei form during the lag phase from monomers in solution. Thus, the lag-time represents a time that is required for the nuclei that are formed early on in the reaction to grow and proliferate in order to reach an aggregate concentration that is readily detected in bulk assays. In many cases, this proliferation takes place through secondary nucleation, where fibrils may present a catalytic surface for the formation of new aggregates. Fibrils may also break (fragmentation) and thereby provide new ends for elongation. Thus, at least two - primary nucleation and elongation - and in many systems at least four - primary nucleation, elongation, secondary nucleation and fragmentation - microscopic processes occur during the lag phase. Moreover, these same processes occur during all three phases of the macroscopic aggregation process, albeit at different rates as governed by rate constants and by the concentration of reacting species at each point in time.This work was supported by the Swedish Research Council (SL) and its Linneus Centre Organizing Molecular Matter for CD spectrometer, plate readers (SL), the Alzheimer Foundation Sweden (SL), the Frances and Augustus Newman Foundation (TPJK), the BBSRC (TPJK), and the Marie Curie fellowship scheme for career development (PA).This is the final version of the article. It first appeared from RSC via http://dx.doi.org/10.1039/C4CP05563

Universality of filamentous aggregation phenomena.

We use perturbative renormalization group theory to study the kinetics of protein aggregation phenomena in a unified manner across multiple timescales. Using this approach, we find that, irrespective of the specific molecular details or experimental conditions, filamentous assembly systems display universal behavior in time. Moreover, we show that the universality classes for protein aggregation correspond to simple autocatalytic processes and that the diversity of behavior in these systems is determined solely by the reaction order for secondary nucleation with respect to the protein concentration, which labels all possible universality classes. We validate these predictions on experimental data for the aggregation of several different proteins at several different initial concentrations, which by appropriate coordinate transformations we are able to collapse onto universal kinetic growth curves. These results establish the power of the perturbative renormalization group in distilling the ultimately simple temporal behavior of complex protein aggregation systems, creating the possibility to study the kinetics of general self-assembly phenomena in a unified fashion

Nanoscale spatially resolved infrared spectra from single microdroplets

Droplet microfluidics has emerged as a powerful platform allowing a large number of individual reactions to be carried out in spatially distinct microcompartments. Due to their small size, however, the spectroscopic characterisation of species encapsulated in such systems remains challenging. In this paper, we demonstrate the acquisition of infrared spectra from single microdroplets containing aggregation-prone proteins. To this effect, droplets are generated in a microfluidic flow-focussing device and subsequently deposited in a square array onto a ZnSe prism using a micro stamp. After drying, the solutes present in the droplets are illuminated locally by an infrared laser through the prism, and their thermal expansion upon absorption of infrared radiation is measured with an atomic force microscopy tip, granting nanoscale resolution. Using this approach, we resolve structural differences in the amide bands of the spectra of monomeric and aggregated lysozyme from single microdroplets with picolitre volume.Comment: 5 pages, 3 Figure

Crucial role of nonspecific interactions in amyloid nucleation.

Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiologically high peptide concentrations. At low peptide concentrations, which mimic the physiologically relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β-sheet-rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only observed in rare fluctuations, which is why such aggregates might be hard to capture experimentally. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the critical nucleus size increases with increases in both concentration and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory.This is the accepted manuscript. The final published version is available from PNAS at http://www.pnas.org/content/111/50/17869.abstract

Attoliter protein nanogels from droplet nanofluidics for intracellular delivery.

Microscale hydrogels consisting of macromolecular networks in aqueous continuous phases have received increasing attention because of their potential use in tissue engineering, cell encapsulation and for the storage and release of cargo molecules. However, for applications targeting intracellular delivery, their micrometer-scale size is unsuitable for effective cellular uptake. Nanoscale analogs of such materials are thus required for this key area. Here, we describe a microfluidics/nanofluidics-based strategy for generating monodisperse nanosized water-in-oil emulsions with controllable sizes ranging from 2500 ± 110 nm down to 51 ± 6 nm. We demonstrate that these nanoemulsions can act as templates to form protein nanogels stabilized by supramolecular fibrils from three different proteins. We further show that these nanoparticles have the ability to penetrate mammalian cell membranes and deliver intracellular cargo. Due to their biocompatibility and lack of toxicity, natural protein-based nanoparticles present advantageous characteristics as vehicles for cargo molecules in the context of pharmaceutical and biomedical applications

Atomic force microscopy for single molecule characterisation of protein aggregation.

The development of atomic force microscopy (AFM) has opened up a wide range of novel opportunities in nanoscience and new modalities of observation in complex biological systems. AFM imaging has been widely employed to resolve the complex and heterogeneous conformational states involved in protein aggregation at the single molecule scale and shed light onto the molecular basis of a variety of human pathologies, including neurodegenerative disorders. The study of individual macromolecules at nanoscale, however, remains challenging, especially when fully quantitative information is required. In this review, we first discuss the principles of AFM with a special emphasis on the fundamental factors defining its sensitivity and accuracy. We then review the fundamental parameters and approaches to work at the limit of AFM resolution in order to perform single molecule statistical analysis of biomolecules and nanoscale protein aggregates. This single molecule statistical approach has proved to be powerful to unravel the molecular and hierarchical assembly of the misfolded species present transiently during protein aggregation, to visualise their dynamics at the nanoscale, as well to study the structural properties of amyloid-inspired functional nanomaterials

Consistent treatment of hydrophobicity in protein lattice models accounts for cold denaturation

The hydrophobic effect stabilizes the native structure of proteins by minimizing the unfavourable interactions between hydrophobic residues and water through the formation of a hydrophobic core. Here we include the entropic and enthalpic contributions of the hydrophobic effect explicitly in an implicit solvent model. This allows us to capture two important effects: a length-scale dependence and a temperature dependence for the solvation of a hydrophobic particle. This consistent treatment of the hydrophobic effect explains cold denaturation and heat capacity measurements of solvated proteins.Comment: Added and corrected references for design procedure in main text (p. 2) and in Supplemental Information (p. 8

DNA-coated Functional Oil Droplets

Many industrial soft materials often include oil-in-water (O/W) emulsions at the core of their formulations. By using tuneable interface stabilizing agents, such emulsions can self-assemble into complex structures. DNA has been used for decades as a thermoresponsive highly specific binding agent between hard and, recently, soft colloids. Up until now, emulsion droplets functionalized with DNA had relatively low coating densities and were expensive to scale up. Here a general O/W DNA-coating method using functional non-ionic amphiphilic block copolymers, both diblock and triblock, is presented. The hydrophilic polyethylene glycol ends of the surfactants are functionalized with azides, allowing for efficient, dense and controlled coupling of dibenzocyclooctane functionalized DNA to the polymers through a strain-promoted alkyne-azide click reaction. The protocol is readily scalable due to the triblock's commercial availability. Different production methods (ultrasonication, microfluidics and membrane emulsification) are used with different oils (hexadecane and silicone oil) to produce functional droplets in various size ranges (sub-micron, $\sim 20\,\mathrm{\mu m}$ and $> 50\,\mathrm{\mu m}$), showcasing the generality of the protocol. Thermoreversible sub-micron emulsion gels, hierarchical "raspberry" droplets and controlled droplet release from a flat DNA-coated surface are demonstrated. The emulsion stability and polydispersity is evaluated using dynamic light scattering and optical microscopy. The generality and simplicity of the method opens up new applications in soft matter and biotechnological research and industrial advances.Comment: 7 pages, 2 figures, 1 tabl