U-M experiments take the express route to evolution
ANN ARBOR— A University of Michigan molecular biologist and his colleagues bypassed half a billion years of evolution by coaxing proteins to subtly alter their shape and perform new tasks in laboratory Petri dishes.
Using a technique called directed evolution, James Bardwell’s team modified a protein called DsbG so that it more closely resembled a distant relative, DsbC, that has the natural ability to protect cells from the toxic effects of exposure to the element copper.
The laboratory-altered version of DsbG gained the capacity to protect against copper toxicity, a function that DsbC acquired through several hundred million years of evolution. The power to reshape protein structure in this manner could help researchers make better pharmaceuticals, said Bardwell, a U-M professor of molecular, cellular and developmental biology and a Howard Hughes Medical Institute investigator.
“If you can understand something, you can build it,” Bardwell said. “We took one protein that was related to another and tried to see if we could convert the first protein into one having the function of the second protein.”
The team’s findings are expected to be published online in late June by the Proceedings of the National Academy of Sciences.
Bardwell’s research focuses on the molecular machinery that helps proteins fold into their proper shapes. The work could eventually lead to a better understanding of disorders such as cystic fibrosis and Alzheimer’s disease, which are thought to involve protein-folding errors.
His team is particularly interested in disulfide bonds, which act like bolts or stiffening struts to keep proteins correctly configured.
“The correct arrangement of disulfide bonds is often so important to a protein that without the right arrangement, the protein does not fold correctly and is destroyed,” said Annie Hiniker, a medical degree and doctoral student at U-M and first author of the new paper.
DsbC counteracts the toxic effects of copper inside cells by ensuring that the sulfur bonds are properly positioned in proteins. The goal of the latest study was to see if directed evolution could reproduce those protective effects by reconfiguring a related protein.
To do that, the researchers tinkered with the gene that makes DsbG. They created 110,000 DsbG mutants that differed from the original protein by one to four amino acids. Genes carry the DNA code that guides the synthesis of proteins; all proteins are assembled from various combinations of 20 amino acids.
Modified versions of the DsbG gene were spliced into E. coli bacteria, which were then grown in Petri dishes containing copper. Of the 110,000 bioengineered DsbG mutants, four displayed some copper-protecting activity.
Surprisingly, each of the four differed from the original DsbG by just one amino acid.
“You could change one protein into one having the function of another just by single mutational changes,” Bardwell said.
Besides offering valuable insights into how evolution alters the structure and function of proteins, the experiment has important practical implications, he said.
Most proteins produced as pharmaceutical agents have sulfur bonds that are critical to their function. But when bioengineered bacteria are used to make pharmaceuticals, they are less efficient than human cells at making those bonds.
Bardwell and his colleagues would like to make E. coli a better “alchemist” by improving its ability to form sulfur bonds in the correct places, he said.
“If we can understand how E. coli creates these bonds, we can build the E. coli we want, and this might allow us to create medically important proteins more quickly and easily.”
The research was supported by grants and awards from the U.S. National Institutes of Health, the Australian Research Council, and the University of Queensland, Australia.