Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

Liquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP(3). Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS

The mechanism of amyloid fibril growth from Φ-value analysis

Amyloid fibrils are highly stable misfolded protein assemblies playing an important role in several neurodegenerative and systemic diseases. While structural information of the amyloid state is now abundant, mechanistic details about the misfolding process remain elusive. Here we present a Φ-value inspired approach and apply it to PI3K-SH3 amyloid fibrils to examine the rate-limiting step for fibril elongation. We use experimental Φvalues as constraints in biased MD-simulations to provide the first view of the transition state of a protein misfolding reaction. The resulting framework is generally applicaple and provides mechanistic insight into the misfolding reaction comparable to the breakthroughs previously achieved for protein folding. While protein folding proceeds on funnel-shaped landscapes, we find that the misfolding reaction energy landscape consists of a large ’golf course’ region, defined by a single energy barrier and transition state, accessing a sharply funneled region. Thus, misfolding occurs by numerous unsuccesful binding attempts and rare successful monomer-fibril end collisions which rapidly anneals to the final state. Taken together, these insights enable, the first quantitative and highly resolved description of a protein misfolding reaction

Thermodynamic characterization of amyloid polymorphism by microfluidic transient incomplete separation

Amyloid fibrils of proteins such as α-synuclein are a hallmark of neurodegenerative diseases and much research has focused on their kinetics and mechanisms of formation. The question as to the thermodynamic stability of such structures has received much less attention. Here, we newly utilize the principle of transient incomplete separation of species in laminar flow in combination with chemical depolymerization for the quantification of amyloid fibril stability. The relative concentrations of fibrils and monomer at equilibrium are determined through an in situ separation of these species based on their different diffusivity inside a microfluidic capillary. The method is highly sample economical, using much less than a microliter of sample per data point and its only requirement is the presence of aromatic residues (W, Y) because of its label-free nature, which makes it widely applicable. Using this method, we investigate the differences in thermodynamic stability between different fibril polymorphs of α-synuclein and quantify these differences for the first time. Importantly, we show that fibril formation can be under kinetic or thermodynamic control and that a change in solution conditions can both stabilise and destabilise amyloid fibrils. Taken together, our results establish the thermodynamic stability as a well-defined and key parameter that can contribute towards a better understanding of the physiological roles of amyloid fibril polymorphism