Unwinding an individual single-molecule composed of a helical string of amino acids stitched through the boundary of a cell—while measuring the force and time that takes—seems to be a fairly tall order in itself. But that wasn’t enough for University of Colorado Boulder researcher Tom Perkins, who spent the last seven years improving the techniques needed to exactly understand the steps needed to unfold these proteins.
“What we achieved is a 10-fold increase in force precision, and a 100-fold increase in time resolution,” said Perkins, a professor of molecular cellular and developmental biology and also a fellow at JILA, CU Boulder’s partnership with the National Institute of Standards and Technology.
“Now we can see 14 intermediate steps in the unfolding process, whereas previously measurements only saw two; in short, we were missing about 85 percent of the intermediate steps.”
Of course, creating that precision took a lot of effort to improve the atomic-force microscope used in the research. Specifically, the JILA research team developed modified cantilevers—a microscopic diving-board-like structure—that is used to pull out the helix structure of the protein.
However, Perkins’ JILA team, led by co-first authors Hao Yu and Matthew Siewny, apparently pulled out a plum, landing the research – “Hidden dynamics in the unfolding of individual bacteriorhodopsin proteins”—in the March 3 edition of Science.
“We made a set of three improvements to the AFM cantilevers” documented in three previous papers, Perkins said. “What you see in this paper led by Hao and Matt is really a capstone of everything we’ve done.”
These improvements included modifying a commercial AFM cantilever with a tool of nanoscience, the focused-ion-beam mill, to essentially sandblast the cantilever with atoms to modify its shape. Of course, underlying all of it was the team’s firm belief that the research of the last 17 years simply didn’t have the time resolution to document all of the protein dynamics that occur over times much shorter than a millisecond and, hence, were hidden in previous studies.
Essentially, biophysicists look at the forces needed to unwind these proteins to better understand the process of folding. In particular, the researchers studied a membrane protein bacteriorhodopsin, which lives at the boundary between the inside and outside of the cell. When expressed by mRNA, these strings of amino acids are essentially one-dimensional; it’s not until they start winding up into coils, or helices, that they assume their functional three-dimensional form.
Proteins that fail to fold correctly will probably be inactive or potentially toxic to the cell. But Perkins said understanding the complexity of protein folding will also create more useful computer models of membrane proteins and potentially create more efficient drug discovery.
“These types of experiments provide more details on the energetics of the membrane proteins,” he said. “If we do that well, then perhaps our colleagues can better predict how drugs binding on the outside of cells lead to a signal on the inside.” The complexity for membrane proteins is that they fold in an environment consisting of water and oil, a so-called lipid bilayer, similar in structure to a soap bubble.
Despite their efforts to improve the AFM instrument, it remains very difficult to create a strong-enough bond between the cantilever and the protein to make the measurement.
“We’re working on that right now,” Perkins said. “The success rate is about 1 percent of the time, but when it works, it has incredible results.”