Single Amino Acid Mutation Dramatically Alters Direction of Molecular "Motor"
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 Nature.
"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 well.
"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 tubule.
The analysis showed that the normal Ncd motor moves only toward the minus end of the microtubule and that it also rotates to theright 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.
Other sources of funding were from the U.S. National Institutes of Health and the Japanese Ministry of Education.