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Abstract
Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium.
Highlights
Living cells harvest energy from their environments to drive the chemical processes that enable life
This means macromolecules cannot diffuse their own diameter without colliding with others. On top of these tight spatial constraints, a large fraction of these macromolecules are enzymes[6,7,8,9], which catalyze chemical reactions that release energy, creating transient mechanical stresses and chemical gradients. While this crowded and active milieu is an essential feature of the cytoplasm[10,11,12,13,14], we usually study the function of its molecular components, and even its collective behavior in dilute conditions, not very far from equilibrium
While the kinetics of urease are unaffected by compartmentalization, we observe a significant increase in the catalytic efficiency of lactate dehydrogenase (LDH)
Summary
Fluorescent imaging of tagged LDH (Fig. 1a) qualitatively shows that it partitions well to the droplet phase. LDH activity is strongly partitioned to the droplet phase With these results, we can compare the kinetics of LDH in the BSA-rich phase and in buffer (Fig. 1d). The LDH reaction velocity, V, is higher in the droplets than in the buffer. Note that the reaction velocities decrease above 1.3 mM pyruvate concentration (Fig. 1d). This substrate inhibition effect is well documented for LDH in buffer[40,41] and is characterized by the inhibition constant, Ki, which we find to be unchanged by compartmentalization. This enzyme hydrolyzes urea to produce carbon dioxide a BF b 300
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