Formation of β-Lactoglobulin Self-Assemblies via Liquid–Liquid Phase Separation for Applications beyond the Biological Functions

Abstract

Proteins are like miracle machines, playing important roles in living organisms. They perform vital biofunctions by further combining together and/or with other biomacromolecules to form assemblies or condensates such as membraneless organelles. Therefore, studying the self-assembly of biomacromolecules is of fundamental importance. In addition to their biological activities, protein assemblies also exhibit extra properties that enable them to achieve applications beyond their original functions. Herein, this study showed that in the presence of monosaccharides, ethylene glycols, and amino acids, β-lactoglobulin (β-LG) can form assemblies with specific structures, which were highly reproducible. The mechanism of the assembly process was studied through multi-scale observations and theoretical analysis, and it was found that the assembling all started from the formation of solute-rich liquid droplets via liquid–liquid phase separation (LLPS). These droplets then combined together to form condensates with elaborate structures, and the condensates finally evolved to form assemblies with various morphologies. Such a mechanism of the assembly is valuable for studying the assembly processes that frequently occur in living organisms. Detailed studies concerning the properties and applications of the obtained β-LG assemblies showed that the assemblies exhibited significantly better performances than the protein itself in terms of autofluorescence, antioxidant activity, and metal ion absorption, which indicates broad applications of these assemblies in bioimaging, biodetection, biodiagnosis, health maintenance, and pollution treatment. This study revealed that biomacromolecules, especially proteins, can be assembled via LLPS, and some unexpected application potentials could be found beyond their original biological functions.

Amyloid Protein Cross-Seeding Provides a New Perspective on Multiple Diseases In Vivo

“…With assistance from infrared spectroscopy, AFM-IR can be used for studying the oligomers’ structure existing in different stages of aggregation, building up the relationship between the morphology and structure of oligomers, and exploring the structural transformation (Figure F) . High-speed AFM (HS-AFM) enables single-molecule-level visualization of biomolecules with high resolution and rapid visualization (Figure F), which can be used to study the overall structural transition between assembly states such as the structural dynamics of Aβ 42 polymerization, the structural dynamics of α-nucleoprotein fiber extension during cross-seeding, etc. , Transmission electron microscopy (TEM) is also extensively employed to study the morphology of amyloid fibrils, while high-resolution TEM (HR-TEM) can observe information at the atomic level (Figure I). , For example, Zhang et al used HR-TEM to observe the assembly order pattern formed during the early lactoglobulin self-assembly, which also provides a reference for observing the early self-assembly process of amyloid fibers in the future …”

“…The droplets could be fused and coalesced. This characteristic is determined by their surface tension, and then through liquid–solid phase inversion, small complexes are further formed and finally assembled into large polymers. − Liquid–liquid phase separation is a reversible molecular dynamic self-assembly process. The two kinds of macromolecular solutions are reversibly separated, with one part condensing into a dense phase that frequently forms a droplet, and the other part forming a dilute liquid phase …”

“…193,194 For example, Zhang et al used HR-TEM to observe the assembly order pattern formed during the early lactoglobulin selfassembly, which also provides a reference for observing the early self-assembly process of amyloid fibers in the future. 133 5.5. Methods to Detect Protein Interactions.…”

Investigating Chaperone like Activity of Green Silver Nanoparticles: Possible Implications in Drug Development

“…8-Anilino-1-Naphthalene-Sulphonic Acid (ANS) Fluorescence Measurements ANS (molecular formula C 16 H 13 NO 3 S) is a dye that binds to the hydrophobic regions of a polypeptide [24,25], is employed to study the change in the conformation of the protein. The details methodology have been described in the SI File.…”

Compartmentalized Aqueous-in-Aqueous Droplets for Flow Biocatalysis

“…Aqueous-in-aqueous droplets are naturally suitable for compartmentalized efficient bioreactions. − They can be formed by aqueous droplets enclosed by a lipid bilayer, a layer of nanoparticles, or coacervated droplets. − Inspired by living cells, vesicles and coacervated droplets are utilized as bioreactors for cascade enzymatic reactions, ATP synthesis, and nuclei acid replications. − However, in vesicles, water-soluble reagents, catalysts, and products are typically trapped inside the droplets, rendering a decrease in the reaction rate. , Coacervated droplets are easily affected by salt concentrations. , More importantly, efforts have been focused on individual droplet bioreactors, and the effect of droplet clusters as bioreactors in flow chemistry have not been adequately investigated, let alone optimized.…”