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Au naturel protein: No staples required

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Scrutinizing a single molecule for more than a few milliseconds used to require effectively “stapling” it down, inhibiting its normal behavior. Now,  Stanford chemist W. E. Moerner and colleagues have for the first time confined a protein, observed its behavior for more than a second, and learned things about it that could influence solar energy technology and biofuels. (Credit: L.A. Cicero)

STANFORD (US)—For the first time, researchers have been able to confine and study an individual protein without having to pin it down so tightly as to alter its fundamental behavior.

Observing molecules one at a time is valuable because it allows for a clear picture of changing behavior over time, without the picture being confused by the presence of other molecules.

Up until now, a molecule needed to be removed from its normal environment—typically a solution such as the bloodstream or the fluids inside a cell—and then researchers had to “basically staple it to some surface such as a glass slide or a large plastic bead, or imbed it in a synthetic polymer to observe it,” says Randall Goldsmith, a postdoctoral researcher in chemistry at Stanford University.

The result, he explains, is like trying to discern how a tiger behaves in the wild by watching it pace back and forth in a cage at the zoo. “You have every reason to be suspicious that you might profoundly alter the behavior of the molecule by binding it to a surface.”

In the first practical application to proteins of a recently developed technique, Goldsmith and W.E. Moerner, professor of chemistry, were able to make detailed observations of the dynamic behavior of the molecule for more than one second, a 50- to 100-fold increase in viewing time compared to other methods, and thereby “set a new standard in single-molecule spectroscopy.

Details of the work appear in the March issue of Nature Chemistry.

Goldsmith and Moerner “trapped” in solution a molecule of a fluorescent photosynthetic protein called allophycocyanin, which is found in red algae and cyanobacteria (formerly known as blue-green algae). Both algae hold promise as key components in next-generation solar technologies and biofuels.

The researchers used a device developed in Moerner’s lab several years ago by former graduate student and postdoctoral scholar Adam Cohen, called an Anti-Brownian Electrokinetic (ABEL) trap.

Brownian motion is the random movement of small particles in a gas or liquid. The movement arises from the particles being bumped by molecules of the fluid; the trap works by cancelling out a molecule’s Brownian motion.

“If the molecule moves east, we give it a kick west. If it moves west, we give it a kick back east. And we have that process going about 40,000 times a second,” explains Goldsmith.

The “kicks” are produced by controlled flows of the solution in which the molecule is placed for observation. The flows are driven by four electrodes evenly spaced around the perimeter of the trap.

Although the molecule is actually tumbling around slightly in the solution in response to the many little kicks it receives, it tumbles in such a confined area that for practical purposes, it is being held in suspension and is stable enough for extended viewing by the researchers.

In the case of the protein in this study, Goldsmith says, they were often able to hold onto a molecule and view it for more than an entire second.

“That may not sound like very much, but if you don’t have the trap and you don’t want to staple your molecule to a surface, you are basically limited to 10 or 20 milliseconds,” he explains.

All the current single-molecule techniques—whether the older, more confining ones or Moerner and Goldsmith’s comparatively free-range method—involve fluorescence microscopy, which employs a laser to excite the molecule of interest into emitting photons.

Not all parts of a protein fluoresce, only certain subgroups within the structure, but the brightness levels of the fluorescence tell the researchers something about how the fluorescent subgroups are interacting with each other.

Goldsmith says one of the previous studies had observed three brightness levels, whereas he saw four or more distinct levels in their experiments.

“That doesn’t sound like it makes a big difference, but for these particular molecules it speaks to a fundamentally different type of behavior.

“We saw that these proteins were undergoing dynamics that would have been more or less impossible to see, had you had them confined,” Goldsmith explains. “What we think is happening is that the protein that encompasses these fluorescent groups is actually changing shape.”

The study was funded by the U.S. Department of Energy and the National Center for Research Resources of the National Institutes of Health.

Stanford University news: http://news.stanford.edu/

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