蜜糖直播

Tom Perkins and JILA team unfold proteins with precise new instrumentation

Unwinding an individual single-molecule composed of a helical string of amino acids stitched through the boundary of a cell鈥攚hile measuring the force and time that takes鈥攕eems to be a fairly tall order in itself. But that wasn鈥檛 enough for University of 蜜糖直播 Boulder researcher Tom Perkins, who spent the last seven years improving the techniques needed to exactly understand the steps needed to unfold these proteins.

鈥淲hat we achieved is a 10-fold increase in force precision, and a 100-fold increase in time resolution,鈥� said Perkins, a professor of molecular cellular and developmental biology and also a fellow at , 蜜糖直播 Boulder鈥檚 partnership with the National Institute of Standards and Technology.

鈥淣ow 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.鈥�

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鈥攁 microscopic diving-board-like structure鈥攖hat is used to pull out the helix structure of the protein.

However, Perkins鈥� JILA team, led by co-first authors Hao Yu and Matthew Siewny, apparently pulled out a plum, landing the research 鈥� 鈥淗idden dynamics in the unfolding of individual bacteriorhodopsin proteins鈥濃�攊n the March 3 edition of Science.

鈥淲e made a set of three improvements to the AFM cantilevers鈥� documented in three previous papers, Perkins said. 鈥淲hat you see in this paper led by Hao and Matt is really a capstone of everything we鈥檝e done.鈥�

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鈥檚 firm belief that the research of the last 17 years simply didn鈥檛 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鈥檚 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.

鈥淭hese types of experiments provide more details on the energetics of the membrane proteins,鈥� he said. 鈥淚f 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.鈥� 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.

鈥淲e鈥檙e working on that right now,鈥� Perkins said. 鈥淭he success rate is about 1 percent of the time, but when it works, it has incredible results.鈥�

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