Walking Along a Protein Phase Diagram to Determine Coexistence Points by Static Light Scattering.

The physical process of liquid-liquid phase separation (LLPS), where the drive to minimize global free energy causes a solution to demix into dense and light phases, plays many important roles in biology. It is implicated in the formation of so-called "membraneless organelles" such as nucleoli, nuclear speckles, promyelocytic leukemia protein bodies, P bodies, and stress granules along with the formation of biomolecular condensates involved in transcription, signaling, and transport. Quantitative studies of LLPS in vivo are complicated by the out-of-equilibrium, multicomponent cellular environment. While in vitro experiments with purified biomolecules are inherently an oversimplification of the cellular milieu, they allow probing of the rich physical chemistry underlying phase separation. Critically, with the application of suitable models, the thermodynamics of equilibrium LLPS can inform on the nature of the intermolecular interactions that mediate it. These same interactions are likely to exist in out-of-equilibrium condensates within living cells. Phase diagrams map the coexistence points between dense and light phases and quantitatively describe LLPS by mapping the local minima of free energy versus biomolecule concentration. Here, we describe a light scattering method that allows one to measure coexistence points around a high-temperature critical region using sample volumes as low as 10 µl.

A surface plasmon resonance biosensor in conjunction with a DNA aptamer-antibody bioreceptor pair for heterogeneous nuclear ribonucleoprotein A1 concentrations in colorectal cancer plasma solutions.

A new DNA aptamer and antibody pair was incorporated into surface plasmon resonance (SPR) sensing platform to detect heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in plasma at clinically relevant native concentrations for the diagnosis of colorectal cancer (CRC). SPR detection of hnRNP A1 was realized via formation of the surface sandwich complex of aptamer/hnRNP A1/anti-hnRNP A; the specific adsorption of hnRNP A1 onto a gold chip surface modified with a DNA aptamer followed by the adsorption of anti-hnRNP A1. Changes in the refractive unit (RU) with respect to the hnRNP A1 concentration in buffer solutions were monitored at a fixed anti-hnRNP A1 concentration of 90 nM, resulting in a dynamic range of 0.1-10 nM of hnRNP A1. The surface sandwich SPR biosensor was further applied to the direct analysis of undiluted human normal and pooled CRC patient plasma solutions. Our plasma analysis results were compared to those obtained with a commercial enzyme-linked immunosorbent assay kit.

Structural basis for reversible amyloids of hnRNPA1 elucidates their role in stress granule assembly.

Subcellular membrane-less organelles consist of proteins with low complexity domains. Many of them, such as hnRNPA1, can assemble into both a polydisperse liquid phase and an ordered solid phase of amyloid fibril. The former mirrors biological granule assembly, while the latter is usually associated with neurodegenerative disease. Here, we observe a reversible amyloid formation of hnRNPA1 that synchronizes with liquid-liquid phase separation, regulates the fluidity and mobility of the liquid-like droplets, and facilitates the recruitment of hnRNPA1 into stress granules. We identify the reversible amyloid-forming cores of hnRNPA1 (named hnRACs). The atomic structures of hnRACs reveal a distinct feature of stacking Asp residues, which contributes to fibril reversibility and explains the irreversible pathological fibril formation caused by the Asp mutations identified in familial ALS. Our work characterizes the structural diversity and heterogeneity of reversible amyloid fibrils and illuminates the biological function of reversible amyloid formation in protein phase separation.