Potential applications of Biomolecular Mechanisms in
Nanotechnology
Keiichi Namba
Protonic NanoMachine Project, ERATO, JST
& Graduate School of Frontier Biosciences, Osaka University
Electron micrograph of Salmonella
Protonic NanoMachine Project, ERATO
Bacteria Swimming
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Protonic NanoMachine Project, ERATO
Architecture of the bacterial flagellum
HAP2
Filament
HAP3
HAP1
Hook
Rod (FliE,FlgB,FlgC,FlgF,FlgG)
L ring
FliF
P ring
Outer membrane
MotB
MotA
FliG
FliM
FliN
Peptidoglycan layer
Inner membrane
S ring
M ring
Export apparatus (FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)
C ring
Protonic NanoMachine Project, ERATO
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?
Protonic NanoMachine Project, ERATO
A model of polymorphic supercoiling
L-type
Normal
Semicoil
R-type
Based on the proposal by Asakura (1970) and Calladine (1975)
Protonic NanoMachine Project, ERATO
Two straight filaments for structure analysis
L-type
SJW1660
R-type
SJW1655
Protonic NanoMachine Project, ERATO
Electron CryoMicroscope JEM3000SFF
(300 kV FEG & Specimen holder at 1.5 K)
Protonic NanoMachine Project, ERATO
Three dimensional structure of the flagellar filament
(Mimori et al., 1995)
Protonic NanoMachine Project, ERATO
Comparison of the two structures at 10 Å resolution
100 Å
Protonic NanoMachine Project, ERATO
X-ray fiber diffraction from well-oriented liquid crystalline
for accurate measurement of the structural parameters
Protonic NanoMachine Project, ERATO
SPring-8 at Harima, Hyogo
Protonic NanoMachine Project, ERATO
BL40B2 at SPring-8
Protonic NanoMachine Project, ERATO
X-ray fiber diffraction from bacterial flagellar filaments
L-type
R-type
3.3 Å
Protonic NanoMachine Project, ERATO
3D map obtained by X-ray amplitudes and EM phases
Inner tube
Outer tube
(Yamashita et al., 1998)
Protonic NanoMachine Project, ERATO
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)
Protonic NanoMachine Project, ERATO
L- and R-type lattices of the subunit packing
12
11
0.8 Å
6
1.8 Å
1
0
2.6 Å
-5
0.8 Å
-11
-10
Local lattice of the subunit packing
L-type
R-type
Protonic NanoMachine Project, ERATO
Models of 10 supercoils and 2 straight filaments
L
0
R
1
2
3
4
5
6
7
8
9
The number of the R-type protofilaments
10
11
Connecting two planes with
short and long elastic rods
for simple mechanical simulation of
flagellar supercoils
[as formulated by Calladine (1978)]
Protonic NanoMachine Project, ERATO
Models of 10 supercoils with 2 straight filaments
L
# 0
R
1
2
3
4
5
6
#: the number of the R-type protofilaments
7
8
9
10 11
Protonic NanoMachine Project, ERATO
Comparison of observed and simulated supercoils
4
3
2
5
7
8
9
10
1
0
11
L
R
Curvature-twist plot of supercoils
open circle: model simulation
all other symbols: observed data
(Kamiya & Asakura, 1975)
Protonic NanoMachine Project, ERATO
Twist-shear conversion by the protofilament structure
Conversion of the twisting force by quick reversal of the
motor rotation into shearing between the protofilaments
Protonic NanoMachine Project, ERATO
Sliding switch from the L- to
R-type
lattice
bydomain
shearing
Switching
of the
outer-tube
interactions
The 0-11 distance: L-type, 52.7 Å; R-type, 51.9 Å
The red-blue distance: AR-AL, 1.8 Å; BR-BL, 2.6 Å
11
11
6
5
6
5
0
0
AR
AL
-5
-6
BR
BL
AR
AL
A'
B'
-11
L-type
A'
-5
-6
BR
BL
B'
-11
R-type
: Forced deformation by quick reversal of the motor rotation
: Sliding switching resulting from the forced deformation
Protonic NanoMachine Project, ERATO
Switching from a left- to right-handed supercoil
# 0
1
2
3
4
5
6
#: the number of the R-type protofilaments
7
8
9
10 11
Protonic NanoMachine Project, ERATO
Assembly regulation by unfolded terminal regions
D3
D3
○
D2
D2
D3
D3
D1
D1
D0
D0
D2
×
D0
D1
D2
D1
○
D0
(66)
(450)
F40
(178)
(422)
F27
(494)
(1)
S JW1103
D0
D1
D3
D2
D1
D0
Protonic NanoMachine Project, ERATO
Crystal of the F41 fragment of flagellin
100 mm
Protonic NanoMachine Project, ERATO
SPring-8 at Harima, Hyogo
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Electron density map of F41 crystal at 2.0 Å resolution
Protonic NanoMachine Project, ERATO
Ca backbone trace of F41
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Crystal packing of F41 (a-c plane)
51.9 Å
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Fit of a protofilament to an EM map (20 Å)
51.9 Å
Protonic NanoMachine Project, ERATO
A long protofilament on an EM map
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Model of the flagellar filament with F41 subunits
Protonic NanoMachine Project, ERATO
Axial interaction of flagellin along the protofilament
D3
D2
D1
N
N
C
C
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Crystal packing of F41 (a-c plane)
51.9 Å
52.7 Å
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Simulated extension of the protofilament (animation)
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Distribution of hydrophobic side chains
Protonic NanoMachine Project, ERATO
Architecture of the bacterial flagellum
HAP2
Filament
hook
HAP3
HAP1
Hook
Rod (FliE,FlgB,FlgC,FlgF,FlgG)
L ring
FliF
P ring
Outer membrane
MotB
MotA
FliG
FliM
FliN
Peptidoglycan layer
Inner membrane
S ring
M ring
Export apparatus (FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)
C ring
Protonic NanoMachine Project, ERATO
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?
Protonic NanoMachine Project, ERATO
Architecture of the axial structure of the flagellum
HAP3
HAP3
HAP1
HAP1
Export
Protonic NanoMachine Project, ERATO
HAP2
Flagellar protein export at the base
and self-assembly at the distal end
Filament
HAP3
HAP1
Hook
Rod (FliE,FlgB,FlgC,FlgF,FlgG)
L ring
FliF
P ring
Outer membrane
MotB
MotA
FliG
FliM
FliN
Peptidoglycan layer
Inner membrane
S ring
M ring
Export apparatus (FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)
C ring
Cytoplasmic chaperones (FlgN,FliJ,FliS,FliT)
Protonic NanoMachine Project, ERATO
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?
Protonic NanoMachine Project, ERATO
3D structure of the reconstituted cap-filament complex
Solid representation in end-on and side views
Half cut structure
Central section of
cylindrical average
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HAP2 binding to the filament with symmetry mismatch
Protonic NanoMachine Project, ERATO
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
HAP2
Filament
HAP3
HAP1
Hook
Rod (FliE,FlgB,FlgC,FlgF,FlgG)
L ring
Rotor
FliF
P ring
Outer membrane
MotB
MotA
FliG
FliM
FliN
Peptidoglycan layer
Inner membrane
S ring
M ring
Export apparatus (FlhA,FlhB,FliH,FliI,FliO,FliP,FliQ,FliR)
C ring
Protonic NanoMachine Project, ERATO
The basal body structure
- central section of a cylindrically averaged map -
Francis et al. (1996) J. Mol. Biol.
DeRosier (1998) Cell
Protonic NanoMachine Project, ERATO
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
Tethered cell
Laser Dark Field Photometry
Protonic NanoMachine Project, ERATO
Torque-Speed measurement
Ref: Ryu, Berry & Berg (2000) Nature 403, 444-447
Chen & Berg (2000) Biophys. J. 78, 1036-1041
Optical trap
Latex bead
1
0
Flagellum
Cell
11ºC
-1
-100
0
100
16ºC
200
23ºC
300
400
Speed (Hz)
Protonic NanoMachine Project, ERATO
Single motor rotation measurement
through 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 photomultipliers
for the position sensitive sensor
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Optical path in the optics
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Data from single motor measurements
Protonic NanoMachine Project, ERATO
Member of the Protonic NanoMachine Project, ERATO, JST（0ctober
2001）
Protonic NanoMachine Project, ERATO
Protonic NanoMachine Project, ERATO, JST
(1997-2002)
Protonic NanoMachine Project, ERATO