Biomedical engineers at Duke University have shown a way of controlling the phase separation of an emerging class of proteins to create artificial membrane-less organelles in human cells. The progress, similar to controlling how vinegar forms droplets within oil, creates chances for engineering artificial structures to modulate present cell functions or produce entirely new behaviours within cells.

The results appear online on August 3 at the journal Nature Chemistry.

Proteins function by folding into specific 3-D shapes which interact with different biomolecular structures. Researchers previously believed that proteins needed these mended shapes to function. But in the last two decades, a new class of intrinsically disordered proteins (IDPs) have been found that have large areas that are"floppy"--which is, they don't fold into a defined 3-D shape. It's currently understood these areas play an important, previously unrecognized role in controlling different cellular functions.

IDPs can also be helpful for biomedical applications because they can undergo phase transitions--changing from a liquid into a gel, for example, or by a soluble to an insoluble state, and again--in response to environmental triggers, such as fluctuations in temperature. These attributes also dictate their phase behaviour in cellular environments and are commanded by adjusting attributes of the IDPs for example their molecular weight or the sequence where the amino acids have been connected together.

"Others in the field have taken a top-level strategy where they'll make a change to a natural IDP and determine how its behavior changes inside a cell," explained Michael Dzuricky, a research scientist working in the Chilkoti laboratory and first author of the study. "We are taking the opposite approach and constructing our own artificial IDPs from straightforward thermodynamic principles. This empowers us and others to precisely tune one property--the form of the phase diagram--to better understand how this parameter affects biological behavior"

<em>Although there are many natural IDPs that show phase behavior in cells, they come in many different flavors, and it has been difficult to discern the rules that govern this behavior. This paper provides very simple engineering principles to program this behavior within a cell."-</em>Ashutosh Chilkoti, the Alan L. Kaganov Distinguished Professor of Biomedical Engineering at Duke

These weakly-held-together constructions allow cells to make compartments without also constructing a membrane to encapsulate it.

This simpler version enabled the researchers to make precise adjustments to the molecular weight of their IDP and amino acids of the IDPs. The researchers show that, based on just how these two factors are tweaked, the IDPs come together to form these compartments at different temperatures in a test tube. And by always trying a variety of temperatures and tweaks, the researchers obtained a good understanding of which layout parameters are most important to control the IDP's behaviour.

As predicted, their artificial IDPs grouped together to make a very small droplet within the cell's cytoplasm. And because the IDP's behavior was so well known, the researchers revealed they could predictably control how they coalesced using their evaluation tube principles as a guide.
"We were able to change temperatures in cells to develop a complete description of their phase behavior, which mirrored our evaluation tube predictions," explained Dzuricky. "At this point, we were able to design distinct artificial IDP systems where the droplets that are formed have different material properties."

Put the other way, since the researchers understood how to manipulate the size and composition of their IDPs to react to temperature, they can program the IDPs to form droplets or bubbles of varying densities within cells. To show this ability might be useful to biomedical engineers, the investigators subsequently used their newfound expertise, as nature frequently does, to create an organelle that performs a particular function within a cell.

The researchers showed that they might utilize the IDPs to create an enzyme to control its action level. By varying the molecular weight of the IDPs, the IDPs hold about the receptor either increased or diminished, which in turn affected how much it may interact with the remainder of the cell.

To demonstrate this ability, the researchers chose an enzyme used by E. coli to convert lactose into usable sugars. Nonetheless, in this circumstance, the investigators tracked this enzyme's activity with a fluorescent reporter at real time to determine the way the engineered IDP organelle was affecting enzyme action.

Later on, the investigators believe they could use their new IDP organelles to restrain the activity levels of biomolecules important to illness conditions. Or to learn how normal IDPs fill similar cellular roles and understand exactly how and why they sometimes malfunction.

"That is the first time anyone was able to precisely specify the method by which the protein sequence controls phase separation behavior inside cells," said Dzuricky. "We used an artificial system, but we believe the same rules apply to natural IDPs and are excited to begin testing this concept."

"We can also now start to program this type of phase behavior with almost any protein in a cell by alerting them to those artificial IDPs," said Chilkoti. "We expect that these synthetic IDPs will offer a new tool for synthetic biology to control cell behavior."
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<span style="color: #ff6600;"><a style="color: #ff6600;" href="http://www.duke.edu/" target="_blank" rel="noopener noreferrer">Duke University</a></span>

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<div class="content-src-label"><strong>Journal reference:</strong></div>
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Dzuricky, M., <em>et al.</em> (2020) De novo engineering of intracellular condensates using artificial disordered proteins. Nature Chemistry<em>.</em><span style="color: #ff6600;"> <a style="color: #ff6600;" href="https://doi.org/10.1038/s41557-020-0511-7" target="_blank" rel="noopener noreferrer">doi.org/10.1038/s41557-020-0511-7</a>.</span>

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