Single Amino Acid Mutation
Dramatically Alters Direction Of A
DURHAM, N.C. -- Researchers from Duke University Medical
Center and Tohoku University, Japan, report finding two single
amino acid mutations that disrupt the sense of direction in a
molecular "motor," and creating one for the first time that is
equally likely to travel either up or down its track.
Molecular motors are proteins made up of amino acids like any
other protein in a cell. They move along tiny filaments, called
microtubules, as they transport vesicles around the cell or herd
chromosomes during cell division. Most motors move toward the
fast-growing end of these microtubules, but some move toward
the opposite, more stable end of the microtubules. Until now, it
was thought that these motors could only do one or the other.
Scientists believe that malfunctioning molecular motors might be
responsible for some diseases caused by incorrect distribution of
chromosomes during cell division, such as Down syndrome. By
understanding how motors work, how they organize
chromosomes and how they lead the cell through the division
process, researchers hope to be able to understand what causes
these diseases and how to prevent them.
Changing a certain amino acid in a molecular motor called Ncd,
which was discovered at Duke, caused an "astonishing" result in
the motor's behavior, said Sharyn Endow, lead author of the
research report, which appears in the Aug. 24 issue of the journal
"We were able to create the very first bi-directional molecular
motor by changing only a single amino acid," said Endow,
professor of microbiology at Duke. "We didn't even know it
could be done, and it's very surprising that a single mutation can
do it. This solves the question of the mechanism of directionality
for Ncd and probably applies to other motor proteins as well."
From the time Endow discovered Ncd in fruit flies about 10 years
ago, the little motor has been an enigma. At the time, it was the
first molecular motor of its kind that moved toward the more
stable, or "minus" end of microtubules. The motor protein that it
was closely related to, called kinesin, moved toward the
fast-growing, or "plus" end.
"We thought that all motor proteins closely related to kinesin
would move similarly, but Ncd did not," Endow said. "How the
directionality of a motor is determined has remained an
outstanding question, and it has grabbed all of the researchers in
the motors field."
The Ncd motor consists of a "neck" region that connects the
"stalk" -- a long rod -- to the bundled motor core. Like all
proteins, the amino acid sequence on paper doesn't hint at how
the protein is actually folded -- sometimes amino acids separated
by hundreds of intervening amino acids actually are quite close to
each other in the functional structure of the molecule. Because of
this folding, one amino acid in the neck of Ncd touches an amino
acid in the motor core. The researchers initially found that
changing this particular neck amino acid caused Ncd to move in
either direction. To their surprise, changing the motor core amino
acid that touches the neck amino acid caused bi-directionality as
"These Ncd mutants are motors with no apparent preference in
direction," Endow explained. "Based on where the amino acids
lie in the motor's structure, this motor core and neck interaction is
crucial for proper directionality." Endow created the mutant
protein motors, and the function of single motors was tested and
analyzed by her collaborator, biophysicist Hideo Higuchi of
Tohoku University in Sendai, Japan. Higuchi has perfected the
use of laser microscopy to trap tiny plastic beads that are attached
to these motors. When the motor moves -- that is, when the
protein changes its shape -- the bead moves, amplifying the tiny
motion of the motor.
Through the careful use of this technique and the painstaking
analysis of the resulting data, Higuchi was able to detect the
direction of the motor's movement in two directions down the
length of the microtubule and also around the circumference of
The analysis showed that the normal Ncd motor moves only
toward the minus end of the microtubule and that it also rotates to
the right around the tubule. The mutant Ncd, on the other hand, is
equally likely to move to the plus end as to the minus end, and it
rotates either to the right or left.
The international collaboration wasn't too difficult, Endow said.
Thanks to an unrestricted grant from the Human Frontiers
Science Program, Endow and Higuchi were able to travel to the
other's institution for direct interaction, and other exchanges were
done via e-mail or telephone.
Now Endow plans to study what effect the bi-directional mutant
Ncd has in a real situation. Ncd is needed to assemble structures
called spindles that are critical for segregating chromosomes
during cell division, in particular for making egg cells. Endow
will place the neck mutant form of Ncd back into fruit flies, the
species it was discovered in, to see what effect bi-directionality
has on motor function.
sources of funding were from the U.S. National Institutes of
Health and the Japanese Ministry of Education.
Note: This story has been adapted from a news release issued by Duke
University Medical Center for journalists and other members of the public.
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