Understanding the Impacts of Molecular and Macromolecular Crowding Agents on Protein–Polymer Complex Coacervates

Abstract

Complex coacervation refers to the liquid–liquid phase separation (LLPS) process occurring between charged macromolecules. The study of complex coacervation is of great interest due to its implications in the formation of membraneless organelles (MLOs) in living cells. However, the impacts of the crowded intracellular environment on the behavior and interactions of biomolecules involved in MLO formation are not fully understood. To address this knowledge gap, we investigated the effects of crowding on a model protein–polymer complex coacervate system. Specifically, we examined the influence of sucrose as a molecular crowder and polyethylene glycol (PEG) as a macromolecular crowder. Our results reveal that the presence of crowders led to the formation of larger coacervate droplets that remained stable over a 25-day period. While sucrose had a minimal effect on the physical properties of the coacervates, PEG led to the formation of coacervates with distinct characteristics, including higher density, increased protein and polymer content, and a more compact internal structure. These differences in coacervate properties can be attributed to the effects of crowders on individual macromolecules, such as the conformation of model polymers, and nonspecific interactions among model protein molecules. Moreover, our results show that sucrose and PEG have different partition behaviors: sucrose was present in both the coacervate and dilute phases, while PEG was observed to be excluded from the coacervate phase. Collectively, our findings provide insights into the understanding of crowding effects on complex coacervation, shedding light on the formation and properties of coacervates in the context of MLOs.

Active droplets through enzyme-free, dynamic phosphorylation

“…To produce su cient droplet material for a reproducible increase in optical density, we added 2.5% PEG 8000 as a crowding agent. 44 We added various amounts of MAP as fuel. Above 10 mM of MAP, we found evidence of the rst droplets.…”

Kinetic Study of the Esterase-like Activity of Albumin following Condensation by Macromolecular Crowding

“…Liquid–liquid phase separation (LLPS) is a widely used process, for instance, in the food or pharmaceutics industry . Complex coacervation is the main phenomena driving LLPS, which typically results from the interactions between oppositely charged polyelectrolytes, although other types of interactions including cation-π or hydrophobic forces are also involved .…”

“…Liquid–liquid phase separation (LLPS) is a widely used process, for instance, in the food or pharmaceutics industry . Complex coacervation is the main phenomena driving LLPS, which typically results from the interactions between oppositely charged polyelectrolytes, although other types of interactions including cation-π or hydrophobic forces are also involved . In biology, LLPS is at the center of several physiological processes ranging from stabilization and transport of RNA to regulating their activity by either inhibition or acceleration of enzyme reactions .…”

“…Hence, the term “condensates” will be employed hereafter. Crowding agents are highly soluble and inert cosolutes such as polyethylene glycol (PEG), Ficoll, or smaller molecules (trehalose and sucrose), suitable to create in vitro similar conditions to the intracellular environment. Interestingly, less efforts have been made toward extracellular proteins due to their lower concentration, but recent studies demonstrated that globular proteins could also undergo LLPS when combined with crowding agents, following weak and reversible interactions.…”

Minimal Peptide Sequences That Undergo Liquid–Liquid Phase Separation via Self-Coacervation or Complex Coacervation with ATP

“…Coacervates also have applications in the formulation of pharmaceutical, personal care, food and agricultural products. 17 LLPS is a phase separation process that may result from simple coacervation of a single species in solution (selfcoacervation) or complex coacervation between different species, generally oppositely charged polyelectrolytes. LLPS may result from associative or segregative phase separation.…”