Potential applications of Biomolecular Mechanisms in Nanotechnology

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Potential applications of Biomolecular Mechanisms
in Nanotechnology

• Keiichi Namba
• Protonic NanoMachine Project, ERATO, JST
• Graduate School of Frontier Biosciences, Osaka
  University

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Electron micrograph of Salmonella
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Bacteria Swimming
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Architecture of the bacterial flagellum
(FliE,FlgB,FlgC,FlgF,FlgG)
(FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)
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Flagellar bundle rotation
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Questions
Being constructed as a protein polymer tube with
chemically identical subunits of flagellin, how
can the filament form a helical propeller? how
can the helical propeller switch its helical form?
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A model of polymorphic supercoiling
Based on the proposal by Asakura (1970) and
Calladine (1975)
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Two straight filaments for structure analysis
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Electron CryoMicroscope JEM3000SFF (300 kV FEG
Specimen holder at 1.5 K)
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Three dimensional structure of the flagellar
filament
(Mimori et al., 1995)
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Comparison of the two structures at 10 Å
resolution
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X-ray fiber diffraction from well-oriented liquid
crystallinefor accurate measurement of the
structural parameters
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SPring-8 at Harima, Hyogo
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BL40B2 at SPring-8
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X-ray fiber diffraction from bacterial flagellar
filaments
L-type
R-type
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3D map obtained by X-ray amplitudes and EM phases
(Yamashita et al., 1998)
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X-ray fiber diffraction from the L- and R-type
filaments (comparison at 20 Å
resolution)
L-type
R-type
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Repeat distance along the protofilament
R-type
L-type
(Yamashita et al., 1998)
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L- and R-type lattices of the subunit packing
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Models of 10 supercoils and 2 straight filaments
Connecting two planes with short and long elastic
rods for simple mechanical simulation
of flagellar supercoils as formulated by
Calladine (1978)
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Models of 10 supercoils with 2 straight filaments
L
R

the number of the R-type protofilaments
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Comparison of observed and simulated supercoils
(Kamiya Asakura, 1975)
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Twist-shear conversion by the protofilament
structure
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Sliding switch from the L- to R-type lattice by
shearing
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Switching from a left- to right-handed supercoil
the number of the R-type protofilaments
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Assembly regulation by unfolded terminal regions
?

?
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Crystal of the F41 fragment of flagellin
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SPring-8 at Harima, Hyogo
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Electron density map of F41 crystal at 2.0 Å
resolution
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Ca backbone trace of F41
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Crystal packing of F41 (a-c plane)
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Fit of a protofilament to an EM map (20 Å)
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A long protofilament on an EM map
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Model of the flagellar filament with F41 subunits
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Axial interaction of flagellin along the
protofilament
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Crystal packing of F41 (a-c plane)
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Simulated extension of the protofilament
(animation)
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Distribution of hydrophobic side chains
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Architecture of the bacterial flagellum
hook
(FliE,FlgB,FlgC,FlgF,FlgG)
(FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)
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Questions
Being constructed as a protein polymer tube with
a helical symmetry very similar to that of the
filament, how can the hook be so flexible and
highly curved compared to the filament? why are
the two junction proteins, HAP1 and HAP3, are
necessary to connect the hook and the filament?
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Architecture of the axial structure of the
flagellum
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Flagellar protein export at the base and
self-assembly at the distal end
(FliE,FlgB,FlgC,FlgF,FlgG)
(FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)
Cytoplasmic chaperones
Cytoplasmic chaperones (FlgN,FliJ,FliS,FliT)
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Questions
While flagellin has an intrinsic ability to
self-assemble into the helical filament
structure, why in vivo assembly of flagellin at
the growing end of the bacterial flagellum
requires the cap? how the cap is stably
attached during the whole process of the
flagellar growth while allowing flagellin
subunits to insert between the cap and the
filament end?
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3D structure of the reconstituted cap-filament
complex
Solid representation in end-on and side views
Central section of cylindrical average
Half cut structure
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HAP2 binding to the filament with symmetry
mismatch
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Five views of the cap-filament complex
Inverted L-shaped gap is the site of next
flagellin assembly
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Model of HAP2 cap binding
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HAP2 cap rotation (animation)
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Architecture of the bacterial flagellum
(FliE,FlgB,FlgC,FlgF,FlgG)
Rotor
(FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)
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The basal body structure- central section of a
cylindrically averaged map -
DeRosier (1998) Cell
Francis et al. (1996) J. Mol. Biol.
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The assembly process of the bacterial flagellum
(update 2002)
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Proton flow network in a bacterial cell
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Questions
How do protons drive the motor rotation? How
does the motor rotates? Are there steps? - step
speed, step rate, step distance -
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Old methods for the motor rotation measurements
Laser Dark Field Photometry
Tethered cell
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Torque-Speed measurement
Ref Ryu, Berry Berg (2000) Nature 403,
444-447 Chen Berg (2000) Biophys. J. 78,
1036-1041
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Single motor rotation measurementthrough a 40 nm
fluorescent bead attached to the hook
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The method of single motor rotation measurement
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Single motor rotation measurement system
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Pyramid mirror with four photomultipliersfor the
position sensitive sensor
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Optical path in the optics
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Data from single motor measurements
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Member of the Protonic NanoMachine Project,
ERATO, JST(0ctober 2001)
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Protonic NanoMachine Project, ERATO,
JST(1997-2002)