Highly active enzymes in protein-crowded droplets

Living cells can harvest environmental energy to drive chemical processes. Here we design a minimal artificial system that mimics cytosolic protein crowding and metabolic densities similar to microorganisms.

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Enzymes have fascinated many scientists endlessly (1). Since enzymes accelerate all the biochemical reactions, in cells metabolic processes need their presence to occur at a rate fast enough to sustain life. Understanding how this highly-evolved protein machinery catalyzes many different chemical reactions is a key to appreciating biological processes and regulation.

Nature manipulates and modulates enzyme activities in different ways. A decade ago, the pioneering work of Brangwynne C. P. et al. (2) showed that liquid-liquid phase separation (LLPS) provides an excellent tool for the cells to regulate their homeostasis. These membrane-less molecular condensates concentrate biomolecules (e.g., proteins or RNA) from the cellular environment. LLPS can control biochemical reactions in space and time by sequestering enzymes from the cellular environment. In the lab, reproducing these biological compartments allow researchers to bring new insights into several complex biological processes.

Inspired by nature, our phase-separated membrane-less droplets implement for the first time two essential features of the living system, protein crowding and the highly sustained metabolic activity. This powerful, quick and “easy to handle” minimal system can partition different enzymes and allow us to study several exciting phenomena in a controlled and reproducible way. The enzymatic reactions can be compartmentalized into crowded protein-rich droplets, reaching steady metabolic densities as high as the hungriest microorganism on earth! At the same time, both the studied enzymes (L-lactate dehydrogenase and urease) show a significant increase in the turnover number (namely Kcat), suggesting that the high protein crowding present inside the cell might have an essential role in tuning enzymes catalysis. This finding is still unexplored inside biomolecular condensates. If diluted in a substrate-loaded reservoir, these protein-rich droplets can achieve highly sustained metabolic activity lasting several hours. In other words, these droplets allow us to evaluate the effect that the high metabolic density and protein crowding have on specific biological processes of interest (e.g. protein-protein interaction, protein-ligand binding, signalling). In addition, they allow the study of matter’s behaviour driven far from the equilibrium by the enzymatic activity, that is “impossible” to achieve with a concentrated enzyme in a buffer.

At this point, we were pretty satisfied with the results, and since our Institute was going in lockdown because of the COVID-19 situation, we said to ourselves: “let us draft a manuscript”. Fortunately, our curiosity to explore further this system prevails over the pandemic. During the lockdown, we ordered urease (the other enzyme of the study), and as soon as the Institute opened again, we tested it inside the droplets.

Patience pays. Urease activity and the consequent pH gradient generated by ammonia drives a steady flow within the protein-rich droplets, reminiscent of the cytoplasmic streaming. Also, the nearby droplets modify the flow, and it seems like the droplets are literally “communicating” each other using the stable pH gradient!

These results are the fruits of multidisciplinary collaborations and are driven by curiosity. It is fascinating how biochemical features influence the physical ones and vice-versa. We are happy to share this straightforward droplet system with the scientific community, and we hope that this work could inspire the research community. However, there are still many open questions. Stay tuned!

by Mirco Dindo and Paola Laurino

Link: https://www.nature.com/articles/s41467-021-26532-0

References:

(1) For the love of enzymes: The odyssey of a biochemist: By Arthur Kornberg. pp 336. Harvard University Press, Cambridge, MA, USA, 1989.

(2) Brangwynne CP, Eckmann CR, Courson DS, Rybarska A, Hoege C, Gharakhani J, Jülicher F, Hyman AA. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science. 2009;324(5935):1729-32.

Paola Laurino

Assistant professor , Paola Laurino