Exploiting Amyloid Fibril Lamination for Exploiting Amyloid Fibril Lamination for
Nanotube Self-AssemblyNanotube Self-Assembly
Presenter: Kun LuAdvisors: David Lynn Vince Conticello
Third Year Progress Report:
What’s amyloid?
rod-like
non-branched
8-10nm in diameter
Curr. Opinion in Struct. Biol., 2000, 10, 60-68
DAEFRHDSG10YEVHHQKLVF20FAEDVGSNKG30AIIGLMVGGV40VIA
A(1-42), A(1-40):
10YEVHHQKLVF20FAEDVGSNKG30AIIGLM
A(10-35):
Amyloid- (A ) Protein
J. Am. Chem. Soc. 2000, 122, 7883
Solid State NMR:
A(10-35) in parallel, in register orientation
Small Angle Neutron Scattering (SANS)
mass per unit length: 3453 340 Da/Å
M. W. of A (10-35): 2855 Da
distance between adjacent -strands: 5 Å
one -sheet: 572 Da/Å
6 laminated -sheets
Structural Model: 10YEVHHQKLVF20FAEDVGSNKG30AIIGLM
J. Am. Soc. Chem. 2000(122):7887
amplify
top view
Molecular Simulation suggested fluidity of A(10-35) fibril
only short stretches of 5-6 residues maintain H-bonding
J. Am. Chem. Soc. 2002, 124, 15150-15151
60 ps
Designed System:
Solvent: 40% acetonitrile/Water with 0.1% TFA (pH=2.1)
A(16-22) CH3CO-K L V F F A E-NH2
DAEFRHDSG10YEVHHQKLVF20FAEDVGSNKG30AIIGLMVGGV40VIA
10YEVHHQKLVF20FAEDVGSNKG30AIIGLM
A(1-42)
A(10-35)
acidic condition: ensure amphiphilicity
40% acetonitrile: increase solubility slow down the assembly process
E
-sheet structureCD change:
-2.5 105
-2 105
-1.5 105
-1 105
-5 104
0
210 220 230 240 250 260 270
wavelength (nm)
0 hr
67 hr
0 10 20 30 40 50 60 70-2.5x10
5
-2.0x105
-1.5x105
-1.0x105
-5.0x104
0.0
time (hr)
Transmission Electron Microscopy (TEM)
Uniform width: 80 5 nmLength: usually longer than 10 m
equilibrium:
Ribbon-like structure
at 30hr:
Atomic Force Microscopy (AFM)
AFM time course study:
fast
A (16-22) monomer
Round particles~30nm by AFM
no -sheet structure
0-20min
Phase image Phase image
Assembled particlesLength varies
no -sheet structure
further
Large size particles~180nm in length~80nm in width
Within 11hr
assemble
(bent sheet)
around 180 nm wide-sheet structure appears
Within 17hr
Phase image
Sheet twists
super helical ribbons-sheet structure
Within 23hr
Topography image
Coil to Tubes
~90nm wide, 8nm high tubes Significant -sheet structure
after 48hr
Topography image
Small Angle Neutron Scattering (SANS)
Small Angle X-ray Scattering (SAXS)
scattering vector: Q = (4π/λ) sinθ
I(q) (contrast)P(q)
P(q): form factor shape, dimensions of isolated particles
Contrast: difference in scattering length density between particles and solvent
differential neutron scattering cross-section (in a diluted system):
0.001
0.01
0.1
1
10I(
Q) (
cm-1
)
4 5 6 7 8 90.01
2 3 4 5 6 7 8 90.1
2
Q (Å-1)
neutron X-ray
SANS:
outer R1=259.37 1.33 Åinner R2= 216.03 0.71 Åwall thickness= 43.3 Å
outer R1=266.01 0.01 Åinner R2= 224.64 0.03 Åwall thickness= 41.4 Å
SAXS:
dxQHx
QHxSin
xQR
xQRJR
R
xQR
xQRJ
RR
QP
2
2
2
2
1
0 5.0212
5.021212
2
12
5.0211
5.021112
2
2
121
1)(
hollow cylinder
form factor:
Fig. 1. Comparison of the actual fit (red curve) with the calculated scattering profile for a solid cylinder of the same outer radius (265 Å) (green curve)
Fig. 2. Comparison of actual fit (red curve) with calculated scattering profiles for two hollow cylinders with the same outer radius but different wall thickness.
Bilayer model for self-assembly of the peptide nanotubes
pitch calculation:
nmP
nmP
w
P
inner
outer
outer
383
2.70
cos/130cos/sin2214.32
214
7.52
cos/130cos/sin2614.32
cos/tan2
assume they have same number of laminates
+K L V F F A E E A F F V L K+
laminates
increase
16-22:
N N
10-35:
right-handed: left-handed:
absence of helical chirality
AFM:
stereo-TEM:
Ionized C-terminus disrupted the whole structure
+K L V F F A E E A F F V L K+
+K L V F F A E E A F F V L K+
stable interface
pH2:
pH8:+K L V F F A E- - E A F F V L K+
+K L V F F A E- - E A F F V L K+
interface destabilized
180 190 200 210 220 230 240 250 260 270
-3x105
-2x105
-1x105
0
1x105
2x105
3x105
4x105
5x105
Mea
n R
esid
ue E
llipt
icity
(de
g.cm
2/dm
ole)
wavelength(nm)
180 190 200 210 220 230 240 250 260 270
-1.4x104
-1.2x104
-1.0x104
-8.0x103
-6.0x103
-4.0x103
-2.0x103
0.0
2.0x103
Mea
n R
esid
ue E
llipt
icity
(de
g.cm
2/dm
ole)
wavelength (nm)
Mutagenesis study:
K L V F F A E
D (no assembly)
Q free N-E free N-Q
R
Hside chain charge
charge onbackbone
free N-Q, E, G, C
QKchargeburied
C-terminus is critical in self-assembly&
N-terminus can accommodate greater diversity
C-terminus is critical in self-assembly&
N-terminus can accommodate greater diversity
Conclusion:
Shortening A(10-35) to A(16-22) resulted in the peptide nanotube
formation under designed conditions. Compared with A(10-35) fibril,
the lamination order has significantly increased from 6 to 130.
The resulting structures are similar to those formed by several other
amphiphiles including lipids, suggesting that some intrinsic
characteristic in the self-assembly process are common to various
molecular frameworks.
The formed nanotubes with positively charge surfaces of very different
inner and outer curvature provide an easily accessible scaffold for
nanotechnology.
Acknowledgement
Professor David G. Lynn
Professor Vince P. Conticello
Dr. Pappannan Thiyagarajan
Dr. Jaby Jacob
Dr. Robert Apkarian
Dr. David MorganDr. Ken WalshDr. Teresa Anne HillDr. Lizhi Liang
Rong GaoJustin MareshAmi S. Lakdawala
Jijun Dong
Peng LiuFang fang
Yan LiangAndrew G. PalmerHsiao-Pei Liu
Kaya ErbilNora GoodmanBrooke
Yuri and all other conticello lab members
Argonne National Laboratory:
Electron Microscopy facilities of Emory: