Technique reveals never-before-seen states as protein unfolds | March 2, 2017 Issue - Vol. 95 Issue 10 | Chemical & Engineering News
Volume 95 Issue 10 | p. 7 | News of The Week
Issue Date: March 6, 2017 | Web Date: March 2, 2017

Technique reveals never-before-seen states as protein unfolds

Modified force spectroscopy method captures protein unraveling with improved time resolution
Department: Science & Technology
News Channels: Biological SCENE, Analytical SCENE
Keywords: protein folding, atomic force microscopy (AFM), single-molecule force spectroscopy (SMFS)
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A laser beam detects forces while an AFM cantilever pulls on two bacteriorhodopsin helices (light blue). The SMFS system plots force (vertical axis) versus time as three amino acids reversibly unfold and refold between two intermediate states (dashed lines).
Credit: JILA
Schematic shows how a laser beam detects bends in a cantilever as it pulls on two bR helices. An accompanying detector plot of force versus time shows three amino acids reversibly unfolding and refolding between two intermediate states on a millisecond time scale.
 
A laser beam detects forces while an AFM cantilever pulls on two bacteriorhodopsin helices (light blue). The SMFS system plots force (vertical axis) versus time as three amino acids reversibly unfold and refold between two intermediate states (dashed lines).
Credit: JILA

An improved version of single-molecule force spectroscopy (SMFS) has enabled researchers to study the unfolding of a membrane protein with much higher time resolution and force precision than ever before, revealing a multitude of new details.

Proteins must adopt specific three-dimensional shapes to work properly. If they don’t, they can malfunction or cause protein-misfolding conditions such as Alzheimer’s disease. Scientists would therefore like to better understand how proteins fold and unfold, including all the intermediate states they adopt along the way.

One type of SMFS technique probes protein-folding intermediates by using an atomic force microscopy (AFM) cantilever to pull on a single protein molecule, unraveling it a little at a time. Most unfolding steps are reversible, so studying unfolding reveals a protein’s folding behavior as well.

In the past, SMFS studies found two intermediate states in the unfolding of two helices in the membrane protein bacteriorhodopsin. But molecular dynamics simulations predicted that this process is much more complex, with 10 unfolding intermediates.

Thomas T. Perkins of JILA in Boulder, Colo., and coworkers shortened, reshaped, and surface-modified AFM cantilevers to develop an SMFS system that analyzes proteins 100 times as fast as conventional AFM-based SMFS and with 10 times better force precision. The researchers have now used it to reveal experimentally that two of bacteriorhodopsin’s helices unfold with 14 intermediates (Science 2017, DOI: 10.1126/science.aah7124).

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Perkins uses a modified AFM instrument to study protein unfolding.
Credit: NIST
Photo shows Thomas T. Perkins using his group’s modified AFM instrument to study protein unfolding.
 
Perkins uses a modified AFM instrument to study protein unfolding.
Credit: NIST

The findings show that the molecular dynamics results were not far off. “Without sufficiently high time resolution, many intermediate states are masked by instrumental limitations,” Perkins says.

The modified system can observe the unfolding of as few as two amino acids over microseconds, whereas conventional AFM-based SMFS typically observes the unfolding of six to 60 amino acids over milliseconds. Another technique called optical trapping can analyze protein unfolding with microsecond time resolution and higher stability than AFM-based SMFS, but it isn’t well suited for studying membrane proteins.

The new system’s time-resolution and force-precision improvements “represent a tour de force, and I use the pun intentionally,” comments Steven Block of Stanford University, an expert on nanoscale biomolecular motions. “The study shows that, with the right instrumentation, you can begin to resolve all the intermediates in protein unfolding,” bringing experiment and theory closer together. “The work is a true breakthrough that will in the future lead to all kinds of new insights,” Block says.

Perkins notes that the modifications have not been patented in hopes that AFM instrument manufacturers will adopt them. “The improvements in data quality are so compelling that we essentially do not take any AFM-based data with a standard commercial cantilever anymore,” he says.

 
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