1. Exploiting Amyloid Fibril Lamination for Nanotube Self-Assembly Presenter: Kun Lu Advisors: David Lynn Vince Conticello Third Year Progress Report:

2. What’s amyloid? rod-like non-branched 8-10nm in diameter Curr. Opinion in Struct. Biol., 2000, 10, 60-68

3. DAEFRHDSG 10 YEVHHQKLVF 20 FAEDVGSNKG 30 AIIGLMVGGV 40 VIA A(1-42), A(1-40): 10 YEVHHQKLVF 20 FAEDVGSNKG 30 AIIGLM A(10-35): Amyloid- (A ) Protein

4. 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

5. Structural Model: 10 YEVHHQKLVF 20 FAEDVGSNKG 30 AIIGLM J. Am. Soc. Chem. 2000(122):7887 amplify top view

6. 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

7. Designed System: Solvent: 40% acetonitrile/Water with 0.1% TFA (pH=2.1) A(16-22) CH 3 CO - K L V F F A E - NH 2 DAEFRHDSG 10 YEVHHQKLVF 20 FAEDVGSNKG 30 AIIGLMVGGV 40 VIA 10 YEVHHQKLVF 20 FAEDVGSNKG 30 AIIGLM A(1-42) A(10-35) acidic condition: ensure amphiphilicity 40% acetonitrile: increase solubility slow down the assembly process E 

8. -sheet structure CD change:

9. Transmission Electron Microscopy (TEM) Uniform width: 80  5 nm Length: usually longer than 10 m equilibrium: Ribbon-like structure at 30hr:

10. Atomic Force Microscopy (AFM)

11. AFM time course study: fast A (16-22) monomer Round particles ~30nm by AFM no -sheet structure 0-20min Phase image Assembled particles Length varies no -sheet structure further Large size particles ~180nm in length ~80nm in width Within 11hr assemble

12. (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

13. 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 ):

14. SANS: outer R1=259.37  1.33 Å inner R2= 216.03  0.71 Å wall thickness= 43.3 Å outer R1=266.01  0.01 Å inner R2= 224.64  0.03 Å wall thickness= 41.4 Å SAXS: hollow cylinder form factor:

15. 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.

16. Bilayer model for self-assembly of the peptide nanotubes pitch calculation: assume they have same number of laminates + K L V F F A E E A F F V L K +

17. laminates increase 16-22: N N 10-35:

18. right-handed:left-handed: absence of helical chirality AFM:

19. stereo-TEM:

20. Ionized C-terminus disrupted the whole structure + 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 + interface destabilized

21. Mutagenesis study: K L V F F A E D  (no assembly) Q  free N-E  free N-Q  R H side chain charge charge on backbone free N-Q, E, G, C QKQK charge buried  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

22. 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.

23. Acknowledgement Professor David G. Lynn Professor Vince P. Conticello Dr. Pappannan Thiyagarajan Dr. Jaby Jacob Dr. Robert Apkarian Dr. David Morgan Dr. Ken Walsh Dr. Teresa Anne Hill Dr. Lizhi Liang Rong Gao Justin Maresh Ami S. Lakdawala Jijun Dong Peng Liu Fang fang Yan Liang Andrew G. Palmer Hsiao-Pei Liu Kaya Erbil Nora Goodman Brooke Yuri and all other conticello lab members Argonne National Laboratory: Electron Microscopy facilities of Emory: