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					Kinetics and Thermodynamics of Amyloid Fibril Formation




                    Ron Wetzel

               University of Tennessee
            Energetics of Amyloid Fibril Formation




Fibril assembly equilibria and G fibril elongation

      - Aβ(1-40) amyloid fibrils (Alzheimer’s disease)
      - polyglutamine amyloid (Huntington’s disease)


Kinetics of nucleated growth polymerization and G of nucleus formation

      - polyglutamine amyloid
               Thermodynamics of Amyloid Fibril Formation



• Some amyloidogenic mutations work by weakening native structure
    - transthyretin
    - Ig light chain

• local sequence also affects amyloidogenicity through fibril packing effects




                                                                     N




                         N
      fibril                                            fibril
                               Aβ Amyloid Fibril Formation

                30                                                35


                                                                  30
                25




                                                                       ThT Fluorescence (au)
                                                                  25
[Monomer], μM




                20

                                                                  20
                15
                                                                  15

                10
                                                                  10

                 5                                                5


                 0                                                0                    Cr
                     0 lag phase   2    4            6   8   10

                                       Time (days)
   The experimental Cr is the equilibrium position of fibril elongation


                                                                 100
1. Unpolymerized Aβ at equilibrium:
    - chemically indistinguishable from initial                                       S26P mutant of Aβ(1-40)
    - capable of making fibrils after concentration
                                                                 80

2. Fibrils resuspended in buffer:
    - dissociate to the identical Cr position
                                                                 60




                                                      [Aβ], μM
                                                                 40




                                                                 20




                                                                  0
                                                                       0   2   4 6   8 10 12 14 16 18 20 22 24 26 28 30

                                                                                          Time (Hrs)
                              Amyloid Fibril Elongation Thermodynamics


                                                             Keq
                        Monomer +     FibrilN                                    FibrilN+1


                              Keq = [FibrilN+1] / [FibrilN][Monomer]

                              Keq = 1 / [Monomer]

                              Keq = 1 / Cr
Monomer remaining, μM




                                                              ΔG = - RT ln Keq

                                                              ΔG = - RT ln Keq = - RT ln (1 / 0.0000086)

                                                              ΔG = - 8.6 kcal/mol [wild type Aβ(1-40)]



                                                             Cr

                                    Time
                       Ala scan of Aβ(1-40) fibril elongation thermodynamics

ΔΔG(Ala – WT), kcal/mol


                                                                                       15-21



                2.5
                                                                                                  31-36
                 2
ΔΔG, kcal/mol




                1.5

                 1

                0.5

                 0                                  *
                       4 13 14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 31 32 33 34 35 36 37 38 39 40

                -0.5                              Aβ(1-40) sequence position
Ala scan of Aβ(1-40) fibril stability

                            Petkova et al., 2002




      Guo et al., 2004
Positions 6 and 53 in parallel β-sheet in IgG binding protein G (β1)




                                        6

                                            53
Positions 6 and 53 in parallel β-sheet in IgG binding protein G (β1)




                                        6

                                            53
Positions 6 and 53 in parallel β-sheet in IgG binding protein G (β1)




                                        6


                                              53
        Effect of Ala replacements in Aβ(1-40) amyloid and in Gβ1



                                       ΔΔG(Ala – residue), kcal/mol
15-21

                                   Aβ(1-40 amyloid fibrils                         G (β1)
    Mutation         in     18    19    20     31         32         36             6 / 53
             31-36   out


    Val      Ala                                                                    1.25


    Phe       Ala                                                                    1.5


    Ile      Ala                                                                    1.65

                                                 [Merkel et al., Structure 7, 1333 (1999)
                                                  Williams et al., J. Mol. Biol. 357, 1283 (2006)]
        Effect of Ala replacements in Aβ(1-40) amyloid and in Gβ1



                                       ΔΔG(Ala – residue), kcal/mol
15-21

                                   Aβ(1-40 amyloid fibrils                         G (β1)
    Mutation         in     18    19    20     31         32         36             6 / 53
             31-36   out


    Val      Ala            1.3                                     1.0             1.25


    Phe       Ala                                                                    1.5


    Ile      Ala                                                                    1.65

                                                 [Merkel et al., Structure 7, 1333 (1999)
                                                  Williams et al., J. Mol. Biol. 357, 1283 (2006)]
        Effect of Ala replacements in Aβ(1-40) amyloid and in Gβ1



                                        ΔΔG(Ala – residue), kcal/mol
15-21

                                   Aβ(1-40 amyloid fibrils                          G (β1)
    Mutation         in     18    19     20     31         32         36             6 / 53
             31-36   out


    Val      Ala            1.3                                      1.0             1.25


    Phe       Ala                 1.5    0.8                                          1.5


    Ile      Ala                                                                     1.65

                                                  [Merkel et al., Structure 7, 1333 (1999)
                                                   Williams et al., J. Mol. Biol. 357, 1283 (2006)]
        Effect of Ala replacements in Aβ(1-40) amyloid and in Gβ1



                                        ΔΔG(Ala – residue), kcal/mol
15-21

                                   Aβ(1-40 amyloid fibrils                          G (β1)
    Mutation         in     18    19     20     31         32         36             6 / 53
             31-36   out


    Val      Ala            1.3                                      1.0             1.25


    Phe       Ala                 1.5    0.8                                          1.5


    Ile      Ala                                2.0       1.0                         1.65

                                                  [Merkel et al., Structure 7, 1333 (1999)
                                                   Williams et al., J. Mol. Biol. 357, 1283 (2006)]
                                     Pro scan of Aβ(1-40) fibril stability

                                                                                           ΔΔG(Pro – WT), kcal/mol
                3.5

                3.0

                2.5
ΔΔG, kcal/mol




                2.0

                1.5

                1.0

                0.5

                0.0
                       4 6 9 12 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
                -0.5                               Aβ(1-40) sequence position

                -1.0
                                                                         [Williams et al., J. Mol. Biol. 335, 833-842 (2004)]
             How Does Proline Destabilize β-Sheet?



• Backbone Effects
    - no N-H proton: lost H-bond
    - loss of planarity in extended chain

• Side Chain Packing Effects
     - Pro “side chain” is compact loop that does not extend far out of plane
                                Ala-edited Pro scan of Aβ(1-40) fibril stability


                                                                                  ΔΔG(Pro – Ala), kcal/mol


                2.5


                  2


                1.5
ΔΔG, kcal/mol




                  1


                0.5


                  0
                       4 14 15 16 17 18 19 20 21 22 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

                -0.5
                                             Aβ(1-40) sequence position

                 -1                                                 [Williams et al., J. Mol. Biol. 357, 1283 (2006)]
                           ΔΔG values for Pro vs. Ala replacement in β-sheet
                               Globular Protein (Gβ1) vs. Amyloid (Aβ)

Globular Protein
       Gβ1 position                          ΔΔG, kcal/mol                               Source
                            53                    >4                         Minor and Kim, Nature 367, 660 (1994)
                            44                    >4                         Minor and Kim, Nature 371, 264 (1994)


Amyloid

                          2.5

                           2
          ΔΔG, kcal/mol




                          1.5

                           1

                          0.5

                           0 4 14 15 16 17 18 19 20 21 22 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

                          -0.5
                                                     Aβ(1-40) sequence position
                           -1
  Hydrogen-Deuterium Exchange Experiment

Deuterium-                                     Processing Solvent (pH~2)
 labeled
  fibrils
                 (D)
                          T      (H)
                                                   - quench exchange
                                                   - dissociate fibrils
                                                   - efficient MS analysis

    forward exchange -       back exchange -
      D2O, pD = 7.5          H/D mix, pH ~ 2
                             10% D2O

 Ab
fibrils
                          Mass                                   Data
                       Spectrometer                             Analysis
                                               [Kheterpal, Zhou, Cook & Wetzel, PNAS (2000)]
                               Protected Amide Hydrogens in Proline Mutant Fibrils

     Leu34->Pro, ΔΔG = only 1.5 kcal/mol destabilized …. but it also has 4 more H-bonds than WT

                    18




                                                                                                                          fewer H-bonds
                    16

                    14
Deuterium content




                    12

                    10




                                                                                                                          more H-bonds
                    8

                    6

                    4

                    2

                    0
                         4 6 9 12 14 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 33 34 35 36 37 38 39 WT

                                                    Position of Pro replacement

                                                                            [Williams et al., J. Mol. Biol. 335, 833-842 (2004)]
                Thermodynamics of Amyloid Fibril Formation



Results:

    - Aβ(1-40) fibril growth tends to a reversible equilibrium position with a Keq and ΔG

    - ΔΔGs from Ala mutations agree with data from parallel β-sheet in globular protein

       … propagated structural changes suggest a fundamental difference from globular proteins

    - some ΔΔG effects attributable to energy changes within the monomer ensemble




                                                                                            N




                                                                            fibril
    globular protein                           amyloidogenic peptide


U                                        U




                             G
                                                                A4

                                          A1
                                                    A2     A3
         N




                       Conformational space
CAG (polyglutamine) expanded repeat diseases

   Disease     Largest Normal   Smallest Abnormal

Huntington’s        39               36
Kennedy’s           33               38
SCA-1               39               41
SCA-2               31               35
SCA-3 (MJD)         41               40
SCA-6               18               21
SCA-7               17               38
DRPLA               35               51
SCA-17              44               46
   Polyglutamine flanking sequences in expanded CAG repeat disease proteins



Huntingtin (HD)            MATLEKLMKAFESLKSF-Qn- PPPPPPPPPPPQLPQPPPQA-

Atropin 1 (DRPLA)     -PSTGAQSTAHPPVSTHHHHH-Qn-HHGNSGPPPPGAFPHPLEGG-

Androgen Receptor (SBMA) -GPRHPEAASAAPPGASLLLL-Qn- ETSPRQQQQQQGEDGSPQAH-

Ataxin 1 (SCA1)       -YSTLLANMGSLSQTPGHKAE-Qn- HLSRAPGLITPGSPPPAQQN-

Ataxin 2 (SCA2)       -GCPRPACEPVYGPLTMSLKP-Qn- PPPAAANVRKPGGSGLLASP-

Ataxin 3 (SCA3)       -SGTNLTSEELRKRREAYFEK-Qn- RDLSGQSSHPCERPATSSGA-

CACNA1A (SCA6)        -PRPHVSYSPVIRKAGGSGPP-Qn- AVARPGRAATSGPRRYPGPT-

Ataxin 7 (SCA7)       -RGEPRRAAAAAGGAAAAAAR-Qn- PPPPQPQRQQHPPPPPRRTR-

TBP (SCA17)           -LTPQPIQNTNSLSILEEQQR-Qn- AVAAAAVQQSTSQQATQGTS-
                                 Lag phase aborted by seeding

                   120

                   100
                             20 mM Q28 monomer +
                   80         1% Q28 aggregate
Light Scattering




                   60

                   40

                   20
                                           20 mM Q28 monomer
                    0

                         0          75        150       225     300
                                               Hours
                 Nucleation / Elongation

        Kn*
         k-1
    M            N*             N+1             N+2
         k1               k2              k3              k4
                      M               M               M



                 N*
G                              N+1

                                               N+2
    M                                                                                         [Qn]
                                                                                                      time
               Reaction coordinate
                                                                         = ½ Kn*k+2Cn*+2t2
                                      nucleation equilibrium constant

                                                                 second order fibril elongation rate constant
                                           Nucleation Kinetics Analysis for Q47 Aggregation

                                           time2 plots
                       37                                                    110

                       32                                                    105




                                                                                    [polyGln], M (x 106)
[polyGln], M (x 106)




                       27                                                    100

                       22                                                     95

                       17                    slope = ½ Kn*k+2Cn*+2            90

                       12                                                     85

                        7                                                      80
                       0.0E+00   1.0E+08    2.0E+08   3.0E+08   4.0E+08   5.0E+08
                                               time2 (sec2)                                                                 -11



                                                                                                                            -12
                                                                                                                                                          slope = n* + 2 = 2.87


                                                                                                           log (t2 slope)
                                                                                                                                                             n* = 0.87 ~ 1
                                                                                                                            -13



                                                                                                                            -14

                                                                                                                                                                     log (½ Kn*k+2) = -0.7668
                                                                                                                            -15
                                                                                                                              -4.9   -4.8   -4.7   -4.6    -4.5   -4.4   -4.3   -4.2   -4.1   -4   -3.9
                                                                                                                                                      log ([monomer], M)
Mechanism of polyglutamine aggregation

                         Kn*
                                                   nucleation




       +


                                                  elongation




n* = 1 for Q28, Q36, Q47; Kn* increases from Q28 to Q36 to Q47

                                               [Chen, Ferrone & Wetzel, PNAS (2002)]
Calculated Aggregation Kinetics Curves at Low Concentration
                                                                                                         Q47   Q36      Q28
                              40
                                         [Qn] = 0.1 nM
     Aggregated Peptide (%)
                              30



                              20




                              10




                               0
                                    -4           -3        -2        -1           0        1         2              3        4
                               10           10        10        10           10       10        10             10       10

                                                                     Years
                                                                                               31    141        1,273
                                             = ½ k+2 Kn* c(n*+2) t2

                                                                                      [Chen, Ferrone & Wetzel, PNAS (2002)]
  Pseudo-first order kinetics of seeded polyGln elongation

      = ½ k+2 Kn* c(n*+2) t2
-0.7668 = log (½ Kn*k+2)                             Fibriln + Monomer                     Fibriln+1


                             -10.14                                      Rate = k+ [Fibril][Monomer]
                                                                              = kpseudofirst [Monomer]
                             -10.16
           ln [monomer, M]




                             -10.18                                         k+ = kpseudofirst / [Fibril]
                             -10.20

                             -10.22

                             -10.24

                             -10.26

                             -10.28
                                 0.0E+00   5.0E+03   1.0E+04   1.5E+04    2.0E+04      2.5E+04
                                                      Time (sec)


                                                               [A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]
                                Determination of Kn*


              +




                                                       fmol biotinyl-polyGln bound
                                                                                     35


                                                                                     30


                                                                                     25


                                                                                     20


                                                                                     15
                                                                                          0   1   2      3    4    5     6    7      8   9    10

k+ = kpseudo1st / [aggregate] = 1.14 x 104 liters/mol-sec                                             [biotinyl-polyglutamine], μM


                       -0.7668 = log (½ Kn*k+2)
                                                                                      Kn* = 2.6 x 10-9


[A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]                                                                   ΔG = + 12.2 kcal/mol
Mechanism of polyglutamine aggregation

                    Kn*
                                                  nucleation


          For Q47, Kn* = 2.6 x 10-9 (ΔGnucleation = + 12.2 kcal/mol)


                          k+
    +


                                                 elongation




                               [A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]
                       Acknowledgments



     Aβ Team                             PolyGln Team


Angela Williams                     Anusri Bhattacharyya
Shankari Shivaprasad                Ashwani Thakur

Brian O’Nuallain                    Songming Chen
Indu Kheterpal
Eric Portelius




                                    Frank Ferrone (Drexel Univ.)
                                    Trevor Creamer (Univ. Kentucky)
                                    Veronique Hermann (Univ. Kentucky)
                      Polyproline dampens polyglutamine aggregation


            100
                            Q40P10     H 2N   KKQ 40 CKK COOH
            90                                      |
                                                    S CH 2CONH G 3 P KK COOH
                                                                  10
            80                Q40
            70
% Monomer




            60
            50
                            P10Q40
            40
            30
            20
            10
             0
                  0    50       100   150            200           250           300           350
                                      Time (hrs)
                                                                         [A Bhattacharyya et al., J. Mol. Biol. 2006]
                      Polyproline dampens polyglutamine aggregation


            100
            90              Q40P10

            80                Q40
            70
% Monomer




            60
            50
            40
            30                                      Cr = 4.5 μM
            20                                                           ΔΔG ≥
            10                                                          3 kcal/mol
                                                    Cr ≤ 50 nM
             0
                  0    50       100   150   200    250     300    350
                                      Time (hrs)
                      Polyproline dampens polyglutamine aggregation


            100
            90              Q40P10

            80                Q40
            70
% Monomer




            60
            50
                            P10Q40
            40
            30
            20
            10
             0
                  0    50       100   150   200    250    300    350
                                      Time (hrs)
                      Polyproline dampens polyglutamine aggregation


            100
                            Q40P10     H 2N   KKQ 40 CKK COOH
            90                                      |
                                                    S CH 2CONH G 3 P KK COOH
                                                                  10
            80                Q40
            70
% Monomer




            60
            50
                            P10Q40
            40
            30
            20
            10
             0
                  0    50       100   150            200           250         300   350
                                      Time (hrs)
                               Is the Plateau a Real Thermodynamic Cr?




                12

                10
[Monomer], µM




                8
                          Q40
                6                                       Q40P10

                4

                2                                                      Q40P10
                         Q40
                0
                     0              100        200               300            400
                                           Time (hrs)




                                                                          [A Bhattacharyya et al., J. Mol. Biol. 2006]
A Conformational Correlate to the P10 Connectivity Effect on Aggregation


                             35°C - 5°C difference spectra




                                                  [A Bhattacharyya et al., J. Mol. Biol. 2006]
       A Possible Basis of the OligoProline Effect


                      aggregation-
    aggregation-
                      competent
    incompetent
                       monomer
     monomer




G

                                                      ΔG



                                             fibril




                   Conformational Space
     Transportability of the P10 Effect




    Peptide                                  Cr, μM



Aβ(1-40)                                       0.9 μM

Aβ(1-40)-P10                                  21.5 μM




                                [A Bhattacharyya et al., J. Mol. Biol. 2006]
                Side Chain Packing by Disulfide Formation


          HS                                                                         HS
HS                                                                         HS




     SH                                  [O]                                    SH
          HS                                                                         HS




           HS                                                                            S
          SH                                                                         S




                   [S. Shivaprasad and R. Wetzel, Biochem. 43, 15310 (2004)]
Stability of amyloid fibrils from various double Cys mutants of Aβ(1-40)




               21         32                                        R-SH                            R-S-S-R
20                  19              31                      3
                               33
     18             17
                                                           2.5
                          34

                                         ΔΔG ( kcal/mol)
 16                  36             35                      2
          15
                                                           1.5
                                                            1
                                                           0.5
                                                            0
                                                                 17C-34C        17C-35C     17C-36C
                                                                           Cysteine mutants


                                                                     [S. Shivaprasad and R. Wetzel, Biochem. 43, 15310 (2004)]
HX-MS with in-line pepsin: distribution of protected amide protons

                                                                                       (c)
                       (a)
                                    A              1-40                                              A                20-34
                                                    +6                                                                 +2

                                                                                                                 B




                                              B




                                                                  Relative Intensity
  Relative Intensity




                             723        727       731                                        746         750         754

                       (b)                                                             (d)
                                                   1-19                                                              35-40
                                                    +5                                                                +1




                             461        465       469                                        561         565         569
                                   Mass/Charge                                                     Mass/Charge


                                                          [M. Chen, I. Kheterpal, K. D. Cook and R. Wetzel, unpublished]
        Nucleation / Elongation

                 N*


          k-1
    M             N*           N+1           N+2
          k1             k2            k3            k4
                       Mel           Mel           Mel



                  N*
G                             N+1

                                            N+2
    M
                Reaction coordinate
Q47 Nucleation Kinetics in the Presence of Various Concentrations of Q20



                                                            2 mM Q47 + [Q20 ], mM
                     105

                                                                     0
                     95
    Relative [Q47]




                     85
                                                                     14

                     75                                              24

                                                                     36
                     65

                                                                     44
                     55
                                                                     54
                     45
                           0   1   2       3        4   5       6
                                       Time (hrs)
Q47 Nucleation Kinetics in the Presence of other PolyGln Peptides



                                                 2 mM Q47 + 20 mM ….
                  110

                  100                                 No addn
                                                      Q10
                   90                                 Q15
 Relative [Q47]




                   80
                                                      Q20
                   70

                   60                                 Q25
                                                      Q29
                   50
                                                      Q33
                   40
                                                      Q40
                   30
                        0   2       4        6   8
                                Time (hrs)
                          Determination of Q47 fibril second order elongation rate constant



              20
                                                                                                              +
fmol biotin-Q30




              15


              10


                  5


                  0
                                                                                                -10.14
                      0      5          10        15        20      25   30
                                                                                                -10.16
                                               Time, mins


                                                                              ln [monomer, M]
                                                                                                -10.18

                                                                                                -10.2

                                                                                                -10.22

                                                                                                -10.24

                                                                                                -10.26
                                 k+ = kpseudo1st / [growing ends]                               -10.28

                                                                                                -10.3
                                                                                                         0   5000   10000   15000   20000   25000
                                 k+ = 11,900 moles/liter-sec
                                                                                                                       Time (sec)
           How is amyloid formation initiated? Polyglutamine studies



There are no kinetically relevant intermediates in nucleation of simple polyGln peptides




Results:
     - the nucleus for polyGln aggregation is an energetically unfavorable monomer
     - repeat length dependent nucleation efficiency may help account for ages-of-onset
     - Kn* for a Q47 peptide is ~ 10-9
     - short polyGln peptides in the environment can enhance nucleation efficiency
                 Nucleation / Elongation

        Kn*
         k-1
    M            N*             N+1             N+2
         k1               k2              k3              k4
                      M               M               M



                 N*
G                              N+1

                                               N+2
    M                                                                                         [Qn]
                                                                                                      time
               Reaction coordinate
                                                                         = ½ Kn*k+2Cn*+2t2
                                      nucleation equilibrium constant

                                                                 second order fibril elongation rate constant
Side Chain Orientation and Packing Within the Aβ(1-40) Amyloid Fibril




                                                   21              32


                               20                                                      31
                                                         19

                                                                                33
                                     18                 17

                                                                    34

                                16                                                   35
                                                              36


                                           15




                                          [S. Shivaprasad, J.-T. Guo, Y. Xu and R. Wetzel, unpublished]
     Side Chain Orientation by Cys Accessibility



          SH                                       S-CH2C(O)NH2




                  I-CH2C(O)NH2




SH                                       SH
                                Ala-edited Pro scan of Aβ(1-40) fibril stability

                                                                               ΔΔG(Pro – Ala), kcal/mol



                2.5


                  2


                1.5
ΔΔG, kcal/mol




                  1


                0.5


                  0
                       4 14 15 16 17 18 19 20 21 22 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

                -0.5
                                             Aβ(1-40) sequence position

                 -1
                                     Amyloid Fibril Thermodynamics

                3.5

                3.0

                2.5
ddG, kcal/mol




                2.0

                1.5

                1.0

                0.5

                0.0
                       4 6 9 12 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 P2 P4
                -0.5
                                                          Proline Mutant
                -1.0

                         WT     DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV
                         P2                             P      P
                         P4             P               P      P       P

                                                                              [Williams et al., J. Mol. Biol. 335, 833-842 (2004)]
Alanine mutation ΔΔGs adjust for hydrophobicity effects in Pro series


                          3.5

                            3                                 Proline - WT ΔΔG
 ΔGmut – ΔGwt, kcal/mol




                          2.5                                 Alanine - WT ΔΔG

                            2                                Pro-Ala ΔΔG

                          1.5

                            1

                          0.5

                            0

                          -0.5
                                 19      20          38                   39
                                      Aβ Sequence Position
                                                          [AD Williams & R Wetzel, Ms. in preparation]
                               Additivity in Alanine mutation ΔΔGs




               21         32

20                                    31
                    19
                                 33
     18             17
                          34                              2.5
16                                    35
                     36
                                                           2
          15
                                           ΔΔG, kcal/ml

                                                          1.5


                                                           1


                                                          0.5


                                                           0
                                                                17   34   17+34 17/34           17      25    17+27 17/27
                                                                                   Ala Mutants

                                                                                 [AD Williams & R Wetzel, Ms. in preparation]
                                  Aβ(1-40) monomer seeded with Aβ(1-40) or IAPP fibrils


                                                      All experiments with 10 nM biotinyl-Aβ

                            Aβ amyloid fibrils on plate
                   10                                                                             0.6




                                                                               Fmol biotinyl-Aβ
                   8                                                                              0.5
Fmol biotinyl-Aβ




                                                                                                  0.4
                   6
                                                                                                  0.3
                   4
                                           IAPP amyloid fibrils on plate                          0.2
                                                                                                                                 Collagen on plate
                   2
                                                                                                  0.1

                   0                                                                               0
                        0        1             2            3              4                            0   1     2          3               4
                                           Time (hrs)                                                           Time (hrs)


                                IAPP fibrils are only 1-2% efficient, compared with Aβ, in seeding Aβ elongation.



                                                          [O’Nuallain, Williams, Westermark & Wetzel, J. Biol. Chem. 279, 17490-17499 (2004)]
Rates of Ab Elongation with Various Amyloid Fibrils as Seeds

Seed Fibril                 Elongation Rate (fmol/hour)                    Relative Efficiency


Ab                                     7.5 ± 1.1                                 100 %


IAPP                                  0.086 ± 0.01                                     1.1
Ig light chain LEN (1-30)              0.019 ± 0.001                                  0.3
Ig light chain VL JTO5                0.042 ± 0.006                                  0.6
b2-microglobulin                       0.014 ± 0.001                                  0.2
Ure2p                                 0.069 ± 0.001                                   0.9
Polyglutamine Q30                     0.44 ± 0.01                                     5.9


Collagen                               0.0075 ± 0.001                                0.1
Ovalbumin, reduced/alkylated           0.009 ± 0.003                                 0.1

                     [O’Nuallain, Williams, Westermark & Wetzel, J. Biol. Chem. 279, 17490-17499 (2004)]
Random coil to b-sheet transition in a Q42 peptide incubated at pH 7, 37 °C

                         20000
                                            T = 217 hrs
                         15000


                         10000
     -1
    [Q] degree cmdmole




                                            T = 86 hrs
                           5000
     2




                             0


                                            T = 45 hrs
                          -5000


                         -10000             T = 0 hrs

                                  200     220             240                   260

                                        Wavelength(nm)
                                                                [Chen, Ferrone & Wetzel, PNAS (2002)]
                       Fractionation of an Incomplete Aggregation Reaction

                     20000
                                                                                        No evidence for stable,
                                                                                        b -sheet structure in
                                                resuspended pellet
                     15000                                                              the non-aggregated
                                                                                        fraction
 -1
[Q] degree cmdmole




                     10000
                                                supernatant plus pellet spectra
 2




                       5000
                                                aggregation time point (86 hrs)

                          0


                      -5000

                                                supernatant
                     -10000

                              200         220                  240                260

                                       Wavelength(nm)
                                A Working Model for the Aβ(1-40) Fibril



      2           6
           4
                   8                                         40
                                12
                        10
                                     14                     38
                                                  36
                                     16

                                18                     34

                           20                           32

                         22                             30
                                24        26     28


[Williams et al., J. Mol. Biol. 335, 833-842 (2004)]


                                                                  [Guo, J.T., Wetzel, R. and Xu, Y., Proteins (2004) In press.]
Aggregation of a Q42 Peptide Monitored by Four Parameters

                        100



                        80                                                          b-sheet formation
% Aggregate Formation




                                                                                    proceeds in parallel
                                                                                    with aggregation
                        60



                        40

                                                       HPLC insolubles

                        20                             Thioflavin T fluoresence
                                                       b-sheet (CD)
                                                       Light scattering
                         0

                              0   50   100           150         200              250
                                             Hours
        Protein Deposition in Human Disease

BRAIN       •   Amyloid Plaques (Alzheimer’s)
            •   Amyloid Angiopathy (microvasculature)
            •   Neurofibrillary Tangles (Alzheimer’s; tauopathies)
            •   Lewy Bodies (Parkinson’s; Lewy Body Dementia)
            •   Polyglutamine aggregates (Huntington’s)
            •   Rosenthal Fibers (astrocytes)
            •   Prion Diseases
            •   SOD aggregates (ALS)

PERIPHERY   • Amyloid (heart, kidney, liver, lungs, peripheral nerves, spleen, skin)
               - serum amyloid A
               - transthyretin
               - Ig light chain
               - islet amyloid polypeptide (IAPP)
               - β2-microglobulin
            • Z-form 1-Antitrypsin Deposition (liver)
            • Inclusion Body Myositis (muscle)
            • Mallory Bodies (liver)
                                               Seeded amyloid growth from Aβ(1-40)


                                30

                                                        ThT
                                25
ThT fluorescence or [Aβ] (μM)




                                20


                                15


                                10


                                 5
                                               [Aβ(1-40)]

                                                                                             Cr
                                 0
                                     0   0.5      1         1.5      2       2.5   3   3.5        4
                                                                  Time (h)
                                               Seeded amyloid growth from Aβ(1-40)


                                30

                                                        ThT
                                25
ThT fluorescence or [Aβ] (μM)




                                20


                                15


                                10


                                 5
                                               [Aβ(1-40)]


                                 0
                                     0   0.5      1         1.5      2       2.5   3   3.5   4
                                                                  Time (h)
Seeded amyloid growth from Aβ(1-40) concentrated from Cr plateau


                   8

                   7

                   6
ThT fluorescence




                   5

                   4

                   3

                   2

                   1

                   0
                       0   1   2       3        4    5         6
                                   Time (hrs)
                                               Seeded amyloid growth from Aβ(1-40)


                                30


                                25                           ThT
ThT fluorescence or [Aβ] (μM)




                                                                                          1.2
                                20                                                         1
                                                                                          0.8




                                                                                Cr (uM)
                                                                                          0.6
                                15                                                        0.4
                                                                                          0.2
                                                                                           0
                                10
                                                                                                0       5       10   15    20
                                                [Aβ(1-40)]                                          Time after ThT max (days)

                                 5


                                 0
                                     0   0.5        1          1.5      2            2.5                    3        3.5        4
                                                                     Time (h)
                      Aβ(1-40) fibril dissociation to equilibrium



            1.2

                                                                         20-day fibrils
             1

                                                                         0.5-day fibrils
            0.8
[Aβ] (μM)




            0.6


            0.4


            0.2


             0
                  0      10      20       30        40    50        60
                                       Time (hrs)
CAG REPEAT LENGTHS IN HUNTINGTON’S DISEASE




  penetrance




  24 25 26 27 28   29 30 31 32 33 34 35   36 37 38 39   40 41 42 43 44 45
Repeat Length Dependence of Age of Onset in Huntington’s Disease




                                                 [Courtesy Marcy MacDonald]
                          Concentration Dependence of Nucleation Kinetics



                          0
                                                                                    Q47
                                  slope = n* + 2
                          -1
log [½ k+2 Kn* c(n*+2)]




                                                                                    Q36
                          -2


                          -3
                                                                                    Q28
                          -4


                          -5


                           -0.3    0.0     0.3     0.6      0.9   1.2   1.5      1.8

                                                         log C

                                                                              [Chen, Ferrone & Wetzel, PNAS (2002)]
                                              PolyGln Aggregate Structure
                40

                35                              PGQ8
                                                                                        Q45
                30
                                                                                      PGQ9                             PG              PG            PG
                                                         PGQ7
 Monomer (uM)




                25
                         Q45
                20                                                                  PGQ9(P2)                           PG         P    PG            PG
                               PGQ9
                15                                                                   Q15PQ26                                      P
                10

                 5

                 0

                     0   100          200        300       400          500   600
                                                                                                              35
                30                          Time (hrs)
                                                                                                              30

                25
                                                                                                              25
                                                                                                                                                          PGQ9(P2)




                                                                                               Monomer (uM)
                20                                         PGQ9
                                                                                                              20
Monomer (uM)




                15
                                                                                                              15
                                                           PDGQ9                                                                                          Q15PQ26
                10                                                                                            10


                 5                                                                                             5                                          PGQ9

                 0                                                                                             0

                     0         50                100              150         200                                  0        100       200      300    400

                                            Time (hrs)                                                                            Time (hrs)
             PolyGln Aggregate Structure

                                    N
 N                                                   P
                       P
                       G         G                   G
P                               P
G                                                     P
                       P            N
                                                     G
 C                     G        C
                                                      P
 N
                       P         G                    G
                                P
                       G                              P
P
G                                                     G
                       P         C
 C                     G


Anti-parallel b-sheet model      Parallel b-helix model
         Effect of flanking sequences on polyglutamine aggregate stability


 Aggregation-incompetent
         monomer
(polyproline type II helix??)


                                         Aggregation-competent
                                               monomer



                                                                             Aggregate
              Wetzel and Creamer labs



                                                              Wetzel lab



                  Computer simulations: Rohit Pappu, Washington University
                                     Summary


• as predicted by theory, in vitro amyloid fibrils can achieve an equilibrium with monomer

• the position of this equilibrium is proportional to the free energy of fibril formation

• measurement of shifted equilibria allows quantitation of mutational effects

• amyloid fibrils exhibit a remarkable structural plasticity

• in ideal cases, aggregation kinetics can be interpreted mechanistically

• the kinetic nucleus for polyglutamine aggregation is an alternatively folded monomer

• accumulated sequence changes strongly diminish cross-seeding efficiency
Mutagenesis and Kinetics/Thermodynamics in Globular Protein Structure


  • studies on “natural” mutants of globular proteins (1970s)
        - Gary Ackers (human hemoglobin variants)
        - Mike Laskowski (ovomucoid variants)

  • protein engineering approaches to globular protein folding stability (1984->)
        - Ron Wetzel (T4 lysozyme disulfide bonds)
        - Brian Matthews (T4 lysozyme point mutations)
        - Robert Matthews, Alan Fersht (folding kinetics)

  • protein folding stability and amyloidogenicity (1993->)
        - Jeff Kelly (transthyretin / TTR amyloidosis)
        - Ron Wetzel (light chain FV domain / Ig light chain amyloidosis)
        - Chris Dobson (lysozyme amyloidosis)

  • amyloid fibril assembly kinetics and thermodynamics….landscape continuity?
       - kinetics complicated by protofibrils and by secondary nucleation
       - can fibril formation reach true equilibrium positions in vitro?
Aggregation and Packing Interactions




                           [R. Wetzel, Trends Biotech. 12, 193-198 (1994)]
                               ACKNOWLEDGMENTS

      UTMCK                         UT Main Campus                           UGA
• Indu Kheterpal                    • Maolian Chen                    • Juntao Guo
• Angela Williams                   • Erik Portelius                  • Ying Xu
• Shankaramma Shivaprasad           • David Kaleta
• Israel Huff                       • Shaolian Zhou                     Harvard Med
• Tina Richey                       • Kelsey Cook                    • Hilal Lashuel
• Kimberley Salone                                                   • Peter Lansbury
• Matt Sega                         • Neil Whittemore                • Prasanna Venkatraman
• Brian Bledsoe                     • Rajesh Mishra                  • Fred Goldberg
• Valerie Berthelier                • Engin Serpersu
• Lezlee Dice                                                                Cal Tech
• Brian O’Nuallain                  • Guangyao Gao                    • Jan Ko
• Anusri Bhattacharyya Mitra        • Ying Chen                       • Susan Ou
• Songming Chen                     • Peter Zhang                     • Paul Patterson
• Wen Yang
• Brad Hamilton                     • Anna Gardberg
• Ashwani Thakur
                                                                             Uppsala
                                    • Chris Dealwis
• Geetha Thiagarajan                                                  • Per Westermark
• Roopa Kenoth                      • Liz Howell
• Merav Geva                                                                 Drexel
• Alex Osmand                       • John Dunlap                     • Frank Ferrone
• Erica Johnson Rowe
• Erin Newby
                  FUNDING: NIH (NIA, NINDS); Hereditary Disease Foundation
                Thermodynamics of Amyloid Fibril Formation


• In globular proteins, some amyloidogenic mutations work by weakening native structure
         - transthyretin (Kelly)
         - Ig light chain (Wetzel)

• local sequence also affects amyloidogenicity through fibril packing effects

• simplest systems are where the starting monomer is in coil, …..
         - no overlay of a stable native state
         - reasonable assumption that mutation minimally affects native state G

• ….. and where there is an easily and accurately measured Cr

• Results:
         -   Aβ(1-40) fibril growth tends to an easily measured, reversible equilibrium position
         -   ΔG = - 8.6 kcal/mol
         -   ΔΔGs from Ala mutations agree with data from parallel β-sheet in globular protein
         -   Ala-edited Pro scan reveals sequence segments in rigid structure, …..
         -   … but propagated structural changes in H-bonding complicate interpretation
                               Many Pro-destabilized Aβ(1-40) fibrils gain H-bonds



                    18




                                                                                                                          fewer H-bonds
                    16

                    14
Deuterium content




                    12

                    10




                                                                                                                          more H-bonds
                    8

                    6

                    4

                    2

                    0
                         4 6 9 12 14 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 33 34 35 36 37 38 39 WT

                                                    Position of Pro replacement

                                                                            [Williams et al., J. Mol. Biol. 335, 833-842 (2004)]
Normal globular proteins generally have only one stable state

				
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