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Biological Chemistry

Wiggly Proteins Prevent Aggregation

Biochemistry: Researchers enhance proteins’ solubility by attaching a disordered sequence

by Erika Gebel
September 6, 2012

Proteins Come Unglued
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Credit: Biochemistry
Aggregation-prone proteins stick together (colorful blobs, left) unless they are tagged with disordered protein sequences called entropic bristles (red coils, right).
Cartoon demonstrating how entropic bristle works to keep proteins from aggregating.
Credit: Biochemistry
Aggregation-prone proteins stick together (colorful blobs, left) unless they are tagged with disordered protein sequences called entropic bristles (red coils, right).

Some proteins just love to stick together when biochemists pluck them out of cells. Their aggregation is bad news for researchers trying to study the molecules, however, because most protein analysis methods require soluble, well-behaved samples. Now researchers can improve the solubility of problematic proteins by tagging them with a disordered protein sequence that wiggles enough to disrupt protein clumping (Biochemistry, DOI: 10.1021/bi300653m).

Insoluble proteins create widespread problems for researchers, contributing to a research group’s observation that half or more of human proteins resist standard methods to produce them in a state suitable for further study (Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.042684199). Currently, scientists can tag aggregation-prone proteins with a protein that is highly soluble, a process that increases the solubility of the complex as a whole. A. Keith Dunker of Indiana University, Indianapolis, wondered whether disordered proteins, which lack a well-defined three-dimensional structure, might work better as a solubilizer than those structured, soluble proteins.

He got the idea from previous research on unstructured protein regions called entropic bristles. Unlike ordered protein sections, these amino acid sequences move around frequently, taking up a large volume of space near the rest of the protein. As a result, these bristle sequences may keep other molecules from coming into contact with the protein, says Dunker. He thought that attaching one of these floppy sequences to a sticky protein could keep it from aggregating. “If you can’t touch each other, how can you aggregate?” wondered Dunker.

He and his colleagues looked for protein-solubilizing bristle sequences by studying dehydrins, which are naturally disordered proteins that scientist think protect plant cells from protein aggregation during drought and cold. They compared the amino acid sequence of dehydrins to those of structured and other unstructured proteins, looking for sequence elements that may have caused disorder. This comparison led the team to create a set of rules that leads to maximum disorder, says Dunker. For example, clusters of negatively charged or of polar amino acids enhance disorder, while sequences with many hydrophobic amino acids create order. The researchers fed these rules into a computer program to generate sequences of potential entropic bristles.

The researchers studied four sequences the program had predicted would form entropic bristles. They varied in length from about 60 to 250 amino acids. The researchers added DNA sequences coding for the bristles to the genes of 20 aggregation-prone proteins, all of which other researchers had struggled to solubilize with conventional tags.

The scientists expressed the genes for the bristle-tagged proteins in bacteria. After allowing the microbes to synthesize the proteins, the team cracked the cells open and centrifuged the lysate. Insoluble proteins ended up in a pellet of cell material, while soluble proteins remained in solution.

The entropic bristles increased the solubility for all 20 proteins, enhancing the amount of protein staying in solution by 65 to 100% compared to the proteins without a solubility-enhancing tag. In general, the bristles also performed better than four commonly used solubility tags.

Richard Kriwacki of St. Jude Children’s Research Hospital calls the results with the stubborn proteins exciting. He hopes to try the bristles with a protein he studies that he calls “a brick” because of its stubborn insolubility.

However, Kriwacki points out that scientists may need to remove the bristle tag for some experiments. “The open question is,” he says, “once you cleave the bristle off, does the protein crash out of solution?” Dunker plans to address this in future research.

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