Graduate School of Frontier Biosciences
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Professor NAMBA, Keiichi, Ph.D. TEL +81-774-98-2543
Associate Prof. IMADA, Katsumi, Ph.D. TEL +81-774-98-2543
Assistant Prof. YONEKURA, Koji, Ph.D. TEL +81-774-98-2543
FAX +81-774-98-2575
Postal Mail Address Protonic NanoMachine Project, ERATO, JST 3-4 Hikaridai, Seika, Kyoto 619-0237 Japan

Protonic NanoMachine Group aims at the ultimate understanding of the mechanisms of self-assembly and its regulation, conformational switching, force generation, and energy transduction by biological macromolecular complexes. By convergence of complementary techniques, such as X-ray diffraction and electron cryomicroscopy for high-resolution analysis of three-dimensional structures, and optical and electronic measurements on individual molecular complexes for analyzing their dynamic behaviors, we try to reveal the basic principles behind their functional mechanisms, in the hope that they will become a basis for artificial nanomachine design and nanotechnology.

1 Mechanism of self-assembly and its regulation

The bacterial flagellum, a motile organelle, for example, is a huge protein complex made of about 25 proteins, each of which forms either a ring or rod-like structure with a range of subunit copy numbers from a few to few tens of thousands. The whole structure spans from the cytoplasmic face of the cell membrane to the extra-cellular space, where the helical filament grows up to around 15 micrometer. The assembly proceeds one part after another from the base to the tip in an efficient and well-regulated manner. We try to reveal the regulatory mechanisms based on the structure and folding dynamics of individual component proteins and sub-complexes.

2 Mechanism of conformational switching

Quick reversal of the flagellar motor rotation and polymorphic switching of the flagellar filament between left- and right-handed helical forms switch the direction of cell swimming. The universal joint function of the flagellar hook is essential in transmitting the torque to the long helical filament that could be oriented in various directions. All these processes involve highly precise and cooperative switching in the subunit protein conformation coupled with switching in the interactions with other protein subunits. We try to visualize these conformational switching and understand the mechanisms of mechanical signal transduction based on the structure and dynamics.

3 Torque generation mechanism

The flagellar motors are only 30 to 40 nm in diameter, and yet, they rotate as fast as 20,000 to 100,000 rpm. We try to understand the mechanism of torque generation by studying the three-dimensional structure and dynamic behavior of its rotation at high spatial and temporal resolution.

4 Energy transduction mechanism
The high-speed rotation of the flagellar motor is powered by the flow of protons or Na ions through a membrane channel protein complex that performs the stator function of the flagellar motor. The proton or ion flow is driven by the proton or ion motive force across the cytoplasmic membrane, and the current amounts only to several tens femtoampere. We try to measure this extremely small electric current precisely to obtain clues to the energy transduction mechanism.

5 Protein export mechanism
The assembly of the flagellar axial structure with tens of thousands of protein subunits always occurs at the distal end of the growing flagellum. The component proteins are selectively exported from the cytoplasm into the narrow central channel of the flagellum by a protein complex attached on the cytoplasmic face of the motor. This flagellar protein export apparatus uses the energy of ATP hydrolysis and is highly homologous to the type III protein secretion system of pathogenic bacteria, by which pathogenic effecter proteins are secreted into host cells. We try to visualize the export process by single molecule technique and understand the mechanism of selective export based on the structure.

6 Design principles for protein nanomachine
Protein nanomachines have a unique and special ability to form three-dimensional structures and large complexes so that individual atoms take well-defined three-dimensional positions to perform specific and yet various functions in a highly precise manner. This ability of self-organization is a great advantage in nanotechnology development, because, without this feature, mass production of nanomachines is impossible and therefore practical applications cannot be expected no matter how useful individually made nanomachines could be. The outcome of our studies on protein nanomachines, which work flexibly and precisely at the same time, is expected to produce much useful knowledge to eventually form a basis for design principles for artificial nanomachines of practical use.
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