Sustained enzymatic activity and flow in crowded protein droplets

Area of Science:

• Biochemistry
• Cell Biology
• Chemical Engineering

Background:

• Living cells utilize environmental energy for essential chemical processes.
• Existing artificial systems often lack the scale and density of cellular environments.
• Understanding cellular energy harvesting requires models that replicate in vivo conditions.

Purpose of the Study:

• To develop a minimal, cell-like system for studying biochemical reactions.
• To achieve high metabolic densities and functional enzyme partitioning within microreactors.
• To explore the creation of non-equilibrium conditions, such as pH gradients, in artificial systems.

Main Methods:

• Utilizing phase-separated protein droplets as microreactors.
• Dispersing enzyme-loaded droplets in a substrate-rich buffer.
• Measuring metabolic densities and observing pH gradient formation.
• Characterizing small molecule transport across droplet interfaces.

Main Results:

• The minimal system operated at protein concentrations and metabolic densities comparable to living cells.
• Protein droplets effectively partitioned enzymes while allowing small molecule transport.
• Steady states were achieved at metabolic densities rivaling microorganisms.
• Stable pH gradients were generated, capable of inducing microscopic flows.

Conclusions:

• This protein droplet system serves as a functional analog for cellular microreactors.
• The platform facilitates the study of enzyme function in cytoplasm-mimicking environments.
• It offers a versatile tool for investigating non-equilibrium matter dynamics.