Researchers at the National Institute of Standards and
Technology (NIST) have demonstrated a new
device that creates nanodroplet “test tubes” for
studying individual proteins under conditions that mimic the crowded
confines of a living cell. “By confining individual proteins
in nanodroplets of water, researchers can directly observe the dynamics
and structural changes of these biomolecules,” says physicist
Lori Goldner, a coauthor of the paper published in Langmuir.
Researchers have demonstrated a new device that creates nanodroplet test tubes for studying individual proteins under conditions that mimic the crowded confines of a living cell.
Researchers recently have turned their attention to the role
that crowding plays in the behavior of proteins and other
biomolecules—there is not much extra space in a cell.
NIST’s nanodroplets can mimic the crowded environment in
cells where the proteins live while providing advantages over other
techniques to confine or immobilize proteins for study that may
interfere with or damage the protein. This more realistic setting can
help researchers study the molecular basis of disease and supply
information for developing new pharmaceuticals. For example, misfolded
proteins play a role in many illnesses including Type 2 diabetes,
Alzheimer’s and Parkinson’s diseases. By seeing how
proteins fold in these nanodroplets, researchers may gain new insight
into these ailments and may find new therapies.
The NIST nanodroplet delivery system uses tiny glass
micropipettes to create tiny water droplets suspended in an oily fluid
for study under a microscope. An applied pressure forces the water
solution containing protein test subjects to the tip of the
micropipette as it sits immersed in a small drop of oil on the
microscope stage. Then, like a magician whipping a tablecloth off a
table while leaving the dinnerware behind, an electronic switch causes
the pipette to jerk back, leaving behind a small droplet typically less
than a micrometer in diameter.
The droplet is held in place with a laser “optical
tweezer,” and another laser is used to excite fluorescence
from the molecule or molecules in the droplet. In one set of
fluorescence experiments, explains Goldner, “The molecules
seem unperturbed by their confinement - they do not stick to the walls
or leave the container - important facts to know for doing
nanochemistry or single-molecule biophysics.” Similar to a
previous work (see “‘Micro-boxes’ of
Water Used to Study Single Molecules”, Tech Beat July 20,
2006), researchers also demonstrated that single fluorescent protein
molecules could be detected inside the droplets.
Fluorescence can reveal the number of molecules within the
nanodroplet and can show the motion or structural changes of the
confined molecule or molecules, allowing researchers to study how two
or more proteins interact. By using only a few molecules and tiny
amounts of reagents, the technique also minimizes the need for
expensive or toxic chemicals.