Reverse-direction movement of a molecular motor

02/06/04



Molecular models of the artifical backwards moving myosin motor attached to F-actin. The recombinant protein consists of the myosin I motor domain (grey), the hGBP four-helix bundle (red) and the lever arm (orange). The motors are modelled in the ‘pre-power-stroke’ state attached to an actin protofilament consisting of five actin monomers (green and blue).

In a new study, which appears in the Feb. 5 issue of Nature, researchers based at Hannover Medical School and the Max Planck Institute for Medical Research in Germany describe the engineering of an artificial backwards-moving myosin from three pre-existing molecular building blocks. These blocks are: a forward-moving class-I myosin motor domain, a directional inverter formed by a four-helix bundle segment of human guanylate-binding protein-1 and an artificial lever arm formed by two alpha-actinin repeats. Drs Tsiavaliaris, Fujita-Becker and Manstein's results demonstrate that reverse-direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180°.

Myosins are mechano-enzymes that contain a common motor domain, by which they convert the energy from the hydrolysis of adenosine triphosphate into movement exerted against polar actin filaments. On the basis of sequence comparisons the myosin superfamily can be divided into at least 18 classes. Most myosins move towards the "barbed" end of actin filaments, but recent studies have established that at least one member of the family, myosin VI, moves towards the "pointed" end.

"The results lend support to a model that suggests that myosins and microtubule-based molecular motors of the kinesin family, which share a common fold consisting of seven beta-strands and six alpha-helices, are intrinsically plus-end directed motors. Conformational changes in the core motor domain are either amplified or amplified and redirected by the neck region in both protein classes," note the researchers.



The translational movement of the tip of a lever (black arrow) depends on the angle of rotation and the direction in which the lever projects away from the axis of rotation. Simply by attaching the lever to the opposite site of the axis of rotation, the same rotation leads to a reversal of the translational movement of the lever.

"The work is based on a very simple idea namely that the translational movement of the tip of a lever depends on the angle of rotation and the direction in which the lever projects away from the axis of rotation. The difficult part was to rotate the direction of the lever arm in precisely the right orientation without creating sterical clashes between domains and compromising the stiffness of the domains and the joints between them," explains Dr. Manstein, who moved his laboratory from the MPI in Heidelberg to the Institute for Biophysical Chemistry in Hannover while the work was in progress.

"Our work shows beautifully how far protein design and engineering approaches have evolved. The generation of an artificial backwards moving myosin was not the result of an extensive trial and error approach but rather we had to reach only once into the toolbox of molecular building blocks to produce a protein with the predicted activity," Manstein notes.

The researchers suggest that the engineering of proteins with new and well-defined properties from known building blocks derived from biologically unrelated proteins has a wide range of applications. "Currently we are combining fluorescent probe techniques with the engineered attachment of long amplifier elements to study the conformational dynamics of enzymes with high spatial and temporal resolution using near-field microscopy techniques," says Tsiavaliaris, who started working on the project during the final phase of his PhD project and is now one of the youngest professors in Germany.

###

The research was supported by a "Molecular Motors" project grant by the Deutsche Forschungsgemeinschaft (DFG).

Original work:
Tsiavaliaris, G., Fujita-Becker, S. & Manstein, D.J.
Molecular engineering of a backwards moving myosin motor
Nature, 5 February 2004

Tunas, sharks and seals And now, in addition to tagging bluefin, Block and her TOPP colleagues from NMFS and ICCAT are tagging all three tuna species that gather off the California and Mexican coasts: bluefin, yellowfin and albacore. TOPP researchers also are tagging numerous shark species – including white, mako, blue and salmon sharks – that range from Alaska to Baja California. These fishes cross the largest ocean on Earth to move from one feeding area back to a breeding region. How and when such migrations occur has been a mystery, Block said: "We are watching salmon sharks do things we never expected them to do. We also have brought together a dedicated group of leatherback sea turtle biologists to tag animals in Costa Rica, and we're going to figure out how to keep these animals from going extinct. It's a very exciting time for the TOPP program, and we're just getting started." One realization TOPP scientists had as they began shaping their research collaboration was that the animals themselves could provide valuable information about their ocean habitats. Because many of the electronic tags collect oceanographic data, such as water temperature, tagged animals can act as "autonomous ocean sensors," going places and collecting data that would be costly and difficult for humans to obtain. "Northern elephant seals swim from northern California to Alaska's Aleutian Islands in about six months," Costa explained. "And every day they'll make 50 to 60 dives to depths of 500 to 600 meters [1,500-1,800 feet]. Since the tags they wear record the water temperature every 10 seconds, every one of those dives represents a detailed thermal profile of the ocean. In many cases, these are the only data we have about the subsurface ocean conditions at that particular place and time." TOPP scientists also are beginning to recognize that the data they gather, similar to those collected on Atlantic bluefin tunas, may provide useful information to resource managers working in the North Pacific. "Right now the Pacific open ocean is one of the last frontiers on our planet," Block said. "We don't have the basic information that would be needed to ensure the long-term health of this ecosystem. Our objective is to garner the knowledge that will lay the foundation for future management." By better understanding how large oceanic animals use the ocean and how they interact with their environment as well as the humans that rely on it, TOPP scientists hope to create new tools to help shape ocean policy. "If we can predict how large predators use the ocean and what are the common points of interaction between sea turtles, tunas, whales and seabirds – where, for example, they all might come together to feed – we can better understand how humans can develop sustainable fisheries without as much turtle and seabird bycatch," Block concluded. Randall E. Kochevar is the science communication manager at the Monterey Bay Aquarium in Monterey, Calif. -30- -By Randall E. Kochevar- Stanford News Service website: http://www.stanford.edu/news/ Stanford Report (university newspaper): http://news.stanford.edu Most recent news releases from Stanford: http://www.stanford.edu/dept/news/html/releases.html To change contact information for these news releases: news-service@lists.stanford.edu Phone: (650) 723-2558

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