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					Third Year Progress Report:




        Exploiting Amyloid Fibril Lamination for
                     Nanotube Self-Assembly



                              Presenter: Kun Lu
                              Advisors: David Lynn
                                         Vince Conticello
What’s amyloid?




                  Curr. Opinion in Struct. Biol., 2000, 10, 60-68




                                                  rod-like
                                              non-branched
                                         8-10nm in diameter
Amyloid- (A ) Protein




 A(1-42), A(1-40):

  DAEFRHDSG10YEVHHQKLVF20FAEDVGSNKG30AIIGLMVGGV40VIA


 A(10-35):
              10YEVHHQKLVF20FAEDVGSNKG30AIIGLM
Solid State NMR:




                                                   J. Am. Chem. Soc. 2000, 122, 7883


          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




                          top view




                            amplify




                                            J. Am. Soc. Chem. 2000(122):7887
Molecular Simulation suggested fluidity of A(10-35) fibril 

           only short stretches of 5-6 residues maintain H-bonding

 60 ps




                                              J. Am. Chem. Soc. 2002, 124, 15150-
                                              15151
 Designed System:

  A(1-42)

  DAEFRHDSG10YEVHHQKLVF20FAEDVGSNKG30AIIGLMVGGV40VIA


  A(10-35)    10YEVHHQKLVF20FAEDVGSNKG30AIIGLM




 A(16-22)          CH3CO-K       L V F F A E-NH2


  Solvent:    40% acetonitrile/Water with 0.1% TFA (pH=2.1)


               acidic condition:    ensure amphiphilicity
               40% acetonitrile: increase solubility
                                 slow down the assembly process
CD change:             -sheet structure




               0                                                        0 hr


                                                                        67 hr
                   4
          -5 10



                   5
          -1 10
                                             0.0

                                                4
                                         -5.0x10
                   5
         -1.5 10                                5
                                         -1.0x10

                                                5
                                         -1.5x10

                   5
          -2 10                          -2.0x105

                                                5
                                         -2.5x10
                                                    0   10    20   30     40    50    60   70

                   5
                                                                   time (hr)
         -2.5 10
                       210   220   230      240         250         260         270

                                   wavelength (nm)
Transmission Electron Microscopy (TEM)

equilibrium:                               at 30hr:




       Uniform width: 80  5 nm                       Ribbon-like structure
       Length: usually longer than 10 m
Atomic Force Microscopy (AFM)
AFM time course study:




             Phase image                                                 Phase image


              fast                      further
                                       assemble

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




                        Sheet twists                            Coil to Tubes

       (bent sheet)


   around 180 nm wide                  super helical ribbons            ~90nm wide, 8nm high tubes
-sheet structure appears                -sheet structure               Significant -sheet structure


       Within 17hr                          Within 23hr                             after 48hr
Small Angle Neutron Scattering (SANS)
Small Angle X-ray Scattering (SAXS)




     scattering vector:   Q = (4π/λ) sinθ
     differential neutron scattering cross-section (in a diluted system):

                          I(q)  (contrast)P(q)

    Contrast: difference in scattering length density between particles and solvent

    P(q): form factor shape, dimensions of isolated particles
                   10                                                                                          neutron
                                                                                                               X-ray
                    1
 I(Q) (cm )
-1




                  0.1

                 0.01

               0.001     4      5 6 7 8 9                                2                3        4    5 6 7 8 9                       2
                                             0.01                                                                  0.1
                                                                                              -1
                                                                                     Q (Å )
                                                                2                                                           2
                                                                                                                         
                                                                                                                   
                                                                                                      2
                                                                                      2 0.5   R2                 2 0.5  
                                                                                                                                               2
                                                                   2 J1 QR1 1  x         R  2 J1 QR2 1  x           Sin QHx  
              hollow cylinder               1                                                                                           
                                                                                              1                     
                                                                                                                                
                                                       1                                                                                  2
                                   P (Q )   
                                                           2                                                                                 dx
               form factor:                 0
                                               1   R2  
                                                R  
                                                                    
                                                                             
                                                                     QR1 1  x       
                                                                                   2 0.5
                                                                                                           
                                                                                                     QR2 1  x 
                                                                                                               2 0.5           QHx
                                                                                                                                      2
                                                                                                                                             
                                                                                                                                             
                                                       1        
                                                                                                                            
                                                                                                                             

                                                                                                       SAXS:
               SANS:
                                                                                                        outer R1=266.01  0.01 Å
                  outer R1=259.37  1.33 Å
                                                                                                        inner R2= 224.64  0.03 Å
                  inner R2= 216.03  0.71 Å
                                                                                                        wall thickness= 41.4 Å
                  wall thickness= 43.3 Å
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




                       +K   LVFFAE     E A F F V L K+
                                     P 
  pitch calculation:                 2 outer tan  w / cos
                                     2  3.14  26  sin / cos  130 / cos
                                       52.7
                                     Pouter  214nm
                                     assume they have same number of laminates
                                     2  3.14  22  sin / cos  130 / cos
                                       70.2
                                     Pinner  383nm
10-35:                   16-22:




  N      N


             laminates
             increase
absence of helical chirality

 AFM:

 right-handed:                 left-handed:
stereo-TEM:
                                           Ionized C-terminus
                                                            disrupted the whole structure
                                                                                              stable interface
                                                                                 +K   LVFFAE                                                       E A F F V L K+
                                                         pH2:                    +K   LVFFAE                                                       E A F F V L K+
Mean Residue Ellipticity (deg.cm2/dmole)




                                               5                                                                                                         3
                                           5x10                                                                                                  2.0x10




                                                                                                      Mean Residue Ellipticity (deg.cm2/dmole)
                                               5
                                           4x10                                                                                                        0.0
                                               5                                                                                                         3
                                           3x10                                                                                                  -2.0x10
                                               5                                                                                                         3
                                           2x10                                                                                                  -4.0x10
                                               5                                                                                                         3
                                           1x10                                                                                                  -6.0x10
                                               0                                                                                                 -8.0x10
                                                                                                                                                         3


                                               5                                                                                                         4
                                           -1x10                                                                                                 -1.0x10
                                               5                                                                                                         4
                                           -2x10                                                                                                 -1.2x10
                                               5                                                                                                         4
                                           -3x10                                                                                                 -1.4x10

                                                   180 190 200 210 220 230 240 250 260 270                                                                 180 190 200 210 220 230 240 250 260 270
                                                                wavelength(nm)                                                                                          wavelength (nm)




                                                                                             interface destabilized
                                                         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+
Mutagenesis study:


                R                       D             (no assembly)


                H
                           side chain
                           charge
                                        Q          
  free N-Q, E, G, C           KLVFFAE
                                                   
                      charge on
                      backbone
                                        free N-E

                                                  
                           charge
               QK          buried
                                        free N-Q




        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

Argonne National Laboratory:
            Dr. Pappannan Thiyagarajan
                Dr. Jaby Jacob
Electron Microscopy facilities of Emory:

                Dr. Robert Apkarian

Dr.   David Morgan                Yan Liang
Dr.   Ken Walsh                   Andrew G. Palmer
Dr.   Teresa Anne Hill            Hsiao-Pei Liu
Dr.   Lizhi Liang
                                  Kaya Erbil
Rong Gao                          Nora Goodman
Justin Maresh                     Brooke
Ami S. Lakdawala
                                  Yuri and all other
Jijun Dong                        conticello lab
                                  members
Peng Liu
Fang fang

				
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posted:2/2/2012
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