Acceleration of an Enzymatic Reaction in Liquid Phase Separated Compartments Based on Intrinsically Disordered Protein Domains

Abstract

Spontaneous liquid demixing of biomolecules appears to be an efficient strategy developed by cells to organize reactions in space and time. This process allows cells to modulate biochemical reactions by locally changing the concentration and the environment of specific components. Here, we develop a strategy to couple the formation of biomolecular liquid compartments with reactions occuring within them. In particular, we conjugate a kinase enzyme with biologically derived low complexity domains and develop synthetic micro-reactors that locally increase the enzyme concentration up to 140-fold. We show that these micro-reactors are characterized by a polarity comparable to methanol which promotes recruitment of small molecules. Despite exhibiting higher viscosity with respect to the surrounding solution, the reactors are liquid-like and allow molecular diffusion within their interior. We demonstrate that the local increase in enzyme concentration accelerates the corresponding enzymatic rate up to 5-fold. This flexible strategy enables the generation of biomolecular microreactors with enhanced reactivity, with potential applications in heterogeneous biocatalysis.

Observation of liquid–liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy

“… 53–55 Very recently, the Raman band of phenylalanine was used to estimate protein concentration in a droplet. 55 We have shown here that Raman microscopy can be applied to quantitatively analyse the concentration of the protein responsible for LLPS formation using the water Raman band as an intensity standard. Quantification inside liquid droplets was discussed using NMR; however, it is difficult to measure each droplet separately because of insufficient spatial resolution.…”

A framework for understanding the functions of biomolecular condensates across scales

“…This is because the free energy of the system is minimized when oligonucleotides adopt conformations that mini mally distort the meshlike structure of the condensate interior 74 . The chemical environment within conden sates may also play a role, as measured solvent polarities within condensates are lower than that of water, reduc ing both the hydrophobic effect and charge screening by solvent molecules 13,75 . Nuclear magnetic resonance stud ies may allow these mechanisms to be distinguished by interrogating conformations, interactions and dynamics of misfolded macromolecules within condensate dense phases.…”

Liquid–Liquid Phase Separation by Intrinsically Disordered Protein Regions of Viruses: Roles in Viral Life Cycle and Control of Virus–Host Interactions

“…For instance, signaling complexes trapped inside condensates can better coordinate cell response to environmental stimuli [154]. Overall, sequestration inside LLPS droplets, because of the high viscosity, can exclude some interactions (with "external" molecules) and increase some others, by enhancing the probability and kinetics of molecular interactions and enzymatic reactions among "internal" molecules [155][156][157].…”