U.S. study charts new approach for designing proteins

SAN FRANCISCO, July 18 (Xinhua) -- As advances in deoxyribonucleic acid (DNA) synthesis technology merge with improvements in computational design of new proteins, the stage is set for a new era of data-driven protein molecular engineering, according to the researchers in a new study.

Gabriel Rocklin, a postdoctoral fellow in biochemistry at the University of Washington School of Medicine, is the lead author of a paper published in the latest issue of the journal Science on the testing of folding stability for computationally designed proteins, which was made possible by a new high-throughput approach.

Made of amino acid chains with specific sequences, proteins are biological workhorses. As natural protein sequences are encoded in cellular DNA, these chains fold into three-dimensional conformations. The sequence of the amino acids in the chain guides where it will bend and twist, and how parts will interact to hold the structure together.

Researchers have studied these interactions for decades by examining the structures of naturally occurring proteins. However, natural protein structures are typically large and complex, with thousands of interactions that collectively hold the protein in its folded shape. Measuring the contribution of each interaction becomes very difficult.

The problem could be addressed by computationally designing simpler proteins, which would make it easier for researchers to analyze the different types of interactions that hold all proteins in their folded structures.

In addition, researchers want to build new molecules, not found naturally, that can perform tasks in preventing or treating disease, in industrial applications, in energy production, and in environmental cleanups. "However, computationally designed proteins often fail to form the folded structures that they were designed to have when they are actually tested in the lab," Rocklin noted.

"Still, even simple proteins are so complicated that it was important to study thousands of them to learn why they fold," Rocklin said. "This had been impossible until recently, due to the cost of DNA. Each designed protein requires its own customized piece of DNA so that it can be made inside a cell. This has limited previous studies to testing only tens of designs."

In the new study, the researchers tested more than 15,000 newly designed mini-proteins that do not exist in nature to see whether they form folded structures. And their testing led to the design of 2,788 stable protein structures and could have many bioengineering and synthetic biology applications. Their small size may be advantageous for treating diseases when the drug needs to reach the inside of a cell.

In comparison, major protein design studies in the past few years have generally examined only 50 to 100 designs.

To encode their designs of short proteins in this project, the researchers used what is called DNA oligo library synthesis technology, which was originally developed for other laboratory protocols, such as large gene assembly. By repeating the cycle of computation and experimental testing over several iterations, they learned from their design failures and progressively improved their modeling. Their design success rate rose from 6 percent to 47 percent. They also produced stable proteins in shape where all of their first designs failed.

The most stable natural protein the researchers identified was a much-studied protein from the bacteria Bacillus stearothermophilus. It basks in high temperatures, like those in hot springs and ocean thermal vents. Most proteins lose their folded structures under such high temperature conditions. Organisms thriving there have evolved into highly stable proteins that stay folded even when hot.

"A total of 774 designed proteins had higher stability scores than this most protease-resistant monomeric protein," the researchers noted in their paper. Proteases are enzymes that break down proteins, and are essential tools the researchers used to measure stability for their thousands of proteins.

The authors predict that, as DNA synthesis technology continues to improve, high-throughput protein design will become possible for larger, more complex protein structures. "Protein designers were like master craftsmen who used their experience to hand-sculpt each piece in their workshop. Sometimes things worked, but when they failed it was hard to say why," Rocklin was quoted as saying in a news release. "Our new approach lets us collect an enormous amount of data on what makes proteins stable. This data can now drive the design process."