In the past, it was extremely difficult to solve the structure of hIMPs, due to the difficulty of harvesting them from cells and the difficulty of labeling the amino acids that compose the proteins, a key step in determining their three-dimensional configuration.
"One problem was that hIMPs serve many functions in a cell, so if you tried to engineer cells with many copies of the proteins on their membrane, they would die before you could harvest the hIMPs," says Christian Klammt, a postdoctoral researcher in Choe's lab and a first author on the paper.
To get around this, the scientists created an outside-the-cell environment, called cell-free expression system, to synthesize the proteins. They used a plexiglass chamber that contained all the biochemical elements necessary to manufacture hIMPs as if they were inside the cell. This system provided the researchers with enough of the proteins to conduct structural analysis.
The cell-free method also allowed them to easily add labeled amino acids into the biochemical stew, which were then incorporated into the proteins. These amino acids gave off telltale structural clues when analyzed with nuclear magnetic resonance spectroscopy, a method for using the magnetic properties of atoms to determine a molecule's physical and chemical properties.
"It was very difficult and inefficient to introduce labeled amino acids selectively into the protein produced in live cells," says Innokentiy Maslennikov, a Salk staff scientist and co-first author on the paper. "With a cell-free system, we can precisely control what amino acids are available for protein production, giving us isotope-labeled hIMPs in large quantities. Using a proprietary labeling str
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