Stanford, CA, USA. 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. Scrutinizing a single molecule for more than a few milliseconds used to require effectively "stapling" it down, inhibiting its normal behavior.

This is the first practical application to proteins of a technique recently developed in the Moerner lab at Stanford University. They confined a protein and made detailed observations of the dynamic behavior of the molecule for more than one second.

Observing molecules one at a time is valuable because it lets researchers get a clear picture of that molecule's changing behavior over time, without the picture being confused by the presence of other molecules. Protein molecules are among the most important of all, playing a central role in living organisms. They are the chief actors within a cell, carrying out the duties specified by information encoded in genes.

Structural biology is an important branch of molecular biology that focuses on the architecture and shape of biological macromolecules. Macromolecules coil into specific three-dimensional shapes to carry out most cell functions. This architecture (the tertiary structure of molecules) depends on the basic composition (primary structures).

Of special concern to biologists is how proteins and nucleic acids acquire their structures.

Even the most advanced light microscopes cannot examine tiny biomolecules in sufficient detail. Structural biologists usually measure vast numbers of identical molecules at the same time. They use a variety of advanced techiques: crystallography, electron microscopy, electron cryomicroscopy (cryo-EM), circular dichroism, Dual Polarisation Interferometry, Nuclear magnetic resonance spectroscopy (NMR), and ultra fast laser spectroscopy.

Structural biologists also look to bioinformatics in their search for an understanding of complex structure, searching for patterns among the diverse sequences that give rise to particular shapes. Researchers can predict protein structures by basing deductions on the membrane topology.

The past few years have seen advances that support the development of highly accurate physical molecular models and clues from model organisms when studying biological structures.

Up until now, researchers have had to remove a molecule from its normal environment — typically a solution such as the bloodstream or the fluids inside a cell — and "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," said Goldsmith, a postdoctoral researcher in chemistry.

The result 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," Goldsmith said.

That perspective is buttressed by the example results of their study, in which they "trapped" in solution a molecule of a fluorescent photosynthetic protein called allophycocyanin. The protein is found in red algae and cyanobacteria (formerly known as blue-green algae).

Moerner, a professor of chemistry, and Goldsmith 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. In his thesis work Cohen used the trap to precisely measure shape fluctuations in single DNA molecules.

Brownian motion is seemingly random and highly unpredictable movement of particles suspended in a gas or fluid. The movement arises when particles are bumped by molecules of the fluid, preventing extended observation of a single molecule over long periods.

The ABEL trap works by cancelling out a molecule's Brownian motion. Goldsmith described the method this way: "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." 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 said 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 said.

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 said 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," he said.

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

Currently thereare only two ABEL traps in the world besides the one in Moerner's lab. "It is a complex scientific instrument. It is not something trivial to build," Goldsmith said. "But whenever we take this story on the road and talk to other researchers about what the ABEL trap can do, we always see everyone's eyes get really wide, when they see the sort of unperturbed behavior you can get."

"It is not perfect, but we have moved a lot closer to the ideal," Goldsmith said. "And the ABEL trap is being used for other exciting experiments on single molecules," Moerner said.

Citation[C1] Watching conformational- and photodynamics of single fluorescent proteins in solution. Randall H. Goldsmith and W. E. Moerner. Nature Chemistry 2010; 2(3): 179-186. doi:10.1038/nchem.545

Abstract

Observing the dynamics of single biomolecules over prolonged time periods is difficult to achieve without significantly altering the molecule through immobilization. It can, however, be accomplished using the anti-Brownian electrokinetic trap, which allows extended investigation of solution-phase biomolecules—without immobilization — through real-time electrokinetic feedback. Here we apply the trap to study an important photosynthetic antenna protein, allophycocyanin. The technique allows the observation of single molecules of solution-phase allophycocyanin for more than one second. We observe a complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models. Light-induced conformational changes are shown to occur and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant. Our methods provide a new window into the dynamics of fluorescent proteins and the observations are relevant for the interpretation of in vivo single-molecule imaging experiments, bacterial photosynthetic regulation and biomaterials for solar energy harvesting.

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[C2] Single-molecule spectroscopy: Caught in a trap. Peter Dedecker and Johan Hofkens. Nature Chemistry 2010; 2(3): 157-159. doi:10.1038/nchem.562

Abstract

Monitoring the dynamics of a single molecule is impeded by their motion in solution, and immobilizing them without changing their properties is problematic. By using a trapping method that counteracts a molecule's Brownian motion, the complex dynamics of a fluorescent protein, allophycocyanin, have been investigated.

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