exploiting amyloid fibril lamination for nanotube self-assembly presenter: kun lu advisors: david...

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: