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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
Nfibril fibril
N
Time (days)
Aβ Amyloid Fibril Formation
lag phase
Cr
[Mo
no
mer
], μ
M
0
5
10
15
20
25
30
0 2 4 6 8 100
5
10
15
20
25
30
35
Th
T F
luo
resc
ence
(au
)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 300
20
40
60
80
100
Time (Hrs)
[Aβ
], μ
M
S26P mutant of Aβ(1-40)
The experimental Cr is the equilibrium position of fibril elongation
1. Unpolymerized Aβ at equilibrium: - chemically indistinguishable from initial - capable of making fibrils after concentration 2. Fibrils resuspended in buffer: - dissociate to the identical Cr position
Monomer + FibrilN FibrilN+1
Keq
Amyloid Fibril Elongation Thermodynamics
Keq = [FibrilN+1] / [FibrilN][Monomer]
Keq = 1 / [Monomer]
Keq = 1 / Cr
ΔG = - RT ln Keq
ΔG = - RT ln Keq = - RT ln (1 / 0.0000086)
ΔG = - 8.6 kcal/mol [wild type Aβ(1-40)]
CrMon
omer
rem
ain
ing,
μM
Time
-0.5
0
0.5
1
1.5
2
2.5
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
0
0.5
1
1.5
2
2.5
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
*
Aβ(1-40) sequence position
ΔΔ
G,
kcal/
mol
Ala scan of Aβ(1-40) fibril elongation thermodynamics
ΔΔG(Ala – WT), kcal/mol
15-21
31-36
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
Mutation 18 19 20 31 32 36 6 / 53
Val Ala 1.25
Phe Ala 1.5
Ile Ala 1.65
Aβ(1-40 amyloid fibrils G (β1)
15-21
31-36
[Merkel et al., Structure 7, 1333 (1999) Williams et al., J. Mol. Biol. 357, 1283 (2006)]
inout
Effect of Ala replacements in Aβ(1-40) amyloid and in Gβ1
ΔΔG(Ala – residue), kcal/mol
Mutation 18 19 20 31 32 36 6 / 53
Val Ala 1.3 1.0 1.25
Phe Ala 1.5
Ile Ala 1.65
Aβ(1-40 amyloid fibrils G (β1)
15-21
31-36
[Merkel et al., Structure 7, 1333 (1999) Williams et al., J. Mol. Biol. 357, 1283 (2006)]
inout
Effect of Ala replacements in Aβ(1-40) amyloid and in Gβ1
ΔΔG(Ala – residue), kcal/mol
Mutation 18 19 20 31 32 36 6 / 53
Val Ala 1.3 1.0 1.25
Phe Ala 1.5 0.8 1.5
Ile Ala 1.65
Aβ(1-40 amyloid fibrils G (β1)
15-21
31-36
[Merkel et al., Structure 7, 1333 (1999) Williams et al., J. Mol. Biol. 357, 1283 (2006)]
inout
Effect of Ala replacements in Aβ(1-40) amyloid and in Gβ1
ΔΔG(Ala – residue), kcal/mol
Mutation 18 19 20 31 32 36 6 / 53
Val Ala 1.3 1.0 1.25
Phe Ala 1.5 0.8 1.5
Ile Ala 2.0 1.0 1.65
Aβ(1-40 amyloid fibrils G (β1)
15-21
31-36
[Merkel et al., Structure 7, 1333 (1999) Williams et al., J. Mol. Biol. 357, 1283 (2006)]
inout
Pro scan of Aβ(1-40) fibril stability
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4 6 9 12 1415161718 19202122 23242526 27282930 3132333435 36373839
Aβ(1-40) sequence position
ΔΔ
G,
kcal/
mol
ΔΔG(Pro – WT), kcal/mol
[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
Aβ(1-40) sequence position
ΔΔ
G, k
cal/m
ol
-1
-0.5
0
0.5
1
1.5
2
2.5
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
[Williams et al., J. Mol. Biol. 357, 1283 (2006)]
ΔΔG values for Pro vs. Ala replacement in β-sheetGlobular Protein (Gβ1) vs. Amyloid (Aβ)
Gβ1 position ΔΔG, kcal/mol Source
53 > 4 Minor and Kim, Nature 367, 660 (1994)44 > 4 Minor and Kim, Nature 371, 264 (1994)
Aβ(1-40) sequence position
ΔΔ
G, k
cal/m
ol
-1
-0.5
0
0.5
1
1.5
2
2.5
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
Amyloid
Globular Protein
T
Hydrogen-Deuterium Exchange Experiment
Deuterium- labeled fibrils
Processing Solvent (pH~2) - quench exchange - dissociate fibrils - efficient MS analysis
Afibrils
forward exchange - D2O, pD = 7.5
back exchange - H/D mix, pH ~ 210% D2O
( D ) ( H )
Data Analysis
MassSpectrometer
[Kheterpal, Zhou, Cook & Wetzel, PNAS (2000)]
Protected Amide Hydrogens in Proline Mutant Fibrils
[Williams et al., J. Mol. Biol. 335, 833-842 (2004)]
0
2
4
6
8
10
12
14
16
18
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
Deu
teri
um
co
nte
nt
few
er H
-bon
dsm
ore
H-b
onds
Leu34->Pro, ΔΔG = only 1.5 kcal/mol destabilized …. but it also has 4 more H-bonds than WT
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
fibril
N
Conformational space
G
globular protein amyloidogenic peptide
N
U U
A1
A3A2
A4
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
AVAAAAVQQSTSQQATQGTS--LTPQPIQNTNSLSILEEQQR-Qn-
PPPPQPQRQQHPPPPPRRTR--RGEPRRAAAAAGGAAAAAAR-Qn-
AVARPGRAATSGPRRYPGPT--PRPHVSYSPVIRKAGGSGPP-Qn-
RDLSGQSSHPCERPATSSGA--SGTNLTSEELRKRREAYFEK-Qn-
PPPAAANVRKPGGSGLLASP--GCPRPACEPVYGPLTMSLKP-Qn-
HLSRAPGLITPGSPPPAQQN--YSTLLANMGSLSQTPGHKAE-Qn-
ETSPRQQQQQQGEDGSPQAH--GPRHPEAASAAPPGASLLLL-Qn-
HHGNSGPPPPGAFPHPLEGG--PSTGAQSTAHPPVSTHHHHH-Qn-
PPPPPPPPPPPQLPQPPPQA- MATLEKLMKAFESLKSF-Qn-
TBP (SCA17)
Ataxin 7 (SCA7)
CACNA1A (SCA6)
Ataxin 3 (SCA3)
Ataxin 2 (SCA2)
Ataxin 1 (SCA1)
Androgen Receptor (SBMA)
Atropin 1 (DRPLA)
Huntingtin (HD)
0 75 150 225 300
0
20
40
60
80
100
120
Lig
ht S
catt
erin
g
Hours
20 M Q28 monomer
20 M Q28 monomer + 1% Q28 aggregate
Lag phase aborted by seeding
Nucleation / Elongation
M N*k1
k-1
G
Reaction coordinate
Mk3
MMk2
M
N*
k4
N+1
N+2
N+1 N+2
Kn*
nucleation equilibrium constant
second order fibril elongation rate constant
= ½ Kn*k+2Cn*+2t2
[Qn] time
Nucleation Kinetics Analysis for Q47 Aggregation
time2 plots
slope = ½ Kn*k+2Cn*+2
-15
-14
-13
-12
-11
-4.9 -4.8 -4.7 -4.6 -4.5 -4.4 -4.3 -4.2 -4.1 -4 -3.9
log ([monomer], M)
log
(t2
slo
pe)
slope = n* + 2 = 2.87
n* = 0.87 ~ 1
log (½ Kn*k+2) = -0.7668
time2 (sec2)
[po
lyG
ln],
M (
x 10
6 )
7
12
17
22
27
32
37
0.0E+00 1.0E+08 2.0E+08 3.0E+08 4.0E+08 5.0E+08
80
85
90
95
100
105
110
[po
lyG
ln],
M (
x 10
6 )
+
nucleation
elongation
Mechanism of polyglutamine aggregation
Kn*
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
10-4
10-3
10-2
10-1
100
101
102
103
104
0
10
20
30
40
Years
Agg
rega
ted
Pep
tid
e (%
)[Qn] = 0.1 nM
31 141 1,273
= ½ k+2 Kn* c(n*+2) t2
[Chen, Ferrone & Wetzel, PNAS (2002)]
Time (sec)
ln [
mo
no
mer
, M
]
-10.28
-10.26
-10.24
-10.22
-10.20
-10.18
-10.16
-10.14
0.0E+00 5.0E+03 1.0E+04 1.5E+04 2.0E+04 2.5E+04
Pseudo-first order kinetics of seeded polyGln elongation
[A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]
Fibriln + Monomer Fibriln+1
Rate = k+ [Fibril][Monomer] = kpseudofirst [Monomer]
k+ = kpseudofirst / [Fibril]
= ½ k+2 Kn* c(n*+2) t2
-0.7668 = log (½ Kn*k+2)
15
20
25
30
35
0 1 2 3 4 5 6 7 8 9 10
15
20
25
30
35
0 1 2 3 4 5 6 7 8 9 100 1 2 3 4 5 6 7 8 9 10
[biotinyl-polyglutamine], μM
fmo
l b
ioti
ny
l-p
oly
Gln
bo
un
d
+
Determination of Kn*
k+ = kpseudo1st / [aggregate] = 1.14 x 104 liters/mol-sec
-0.7668 = log (½ Kn*k+2)
Kn* = 2.6 x 10-9
ΔG = + 12.2 kcal/mol[A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]
nucleation
+
elongation
Mechanism of polyglutamine aggregation
Kn*
For Q47, Kn* = 2.6 x 10-9 (ΔGnucleation = + 12.2 kcal/mol)
k+
[A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]
Acknowledgments
Aβ Team PolyGln Team
Angela Williams Anusri BhattacharyyaShankari Shivaprasad Ashwani Thakur
Brian O’Nuallain Songming ChenIndu KheterpalEric Portelius
Frank Ferrone (Drexel Univ.) Trevor Creamer (Univ. Kentucky) Veronique Hermann (Univ. Kentucky)
0
10
20
30
40
50
60
70
80
90
100
0 50 100 150 200 250 300 350
Time (hrs)
% M
on
om
erPolyproline dampens polyglutamine aggregation
Q40
Q40P10
P10Q40
H2NKKQ
40CKKCOOH
|SCH
2CONHG
3P
10 COOHKK
[A Bhattacharyya et al., J. Mol. Biol. 2006]
0
10
20
30
40
50
60
70
80
90
100
0 50 100 150 200 250 300 350
Time (hrs)
% M
on
om
erPolyproline dampens polyglutamine aggregation
Q40
Q40P10
Cr = 4.5 μM
Cr ≤ 50 nM
ΔΔG ≥
3 kcal/mol
0
10
20
30
40
50
60
70
80
90
100
0 50 100 150 200 250 300 350
Time (hrs)
% M
on
om
erPolyproline dampens polyglutamine aggregation
Q40
Q40P10
P10Q40
0
10
20
30
40
50
60
70
80
90
100
0 50 100 150 200 250 300 350
Time (hrs)
% M
on
om
erPolyproline dampens polyglutamine aggregation
Q40
Q40P10
P10Q40
H2NKKQ
40CKKCOOH
|SCH
2CONHG
3P
10 COOHKK
0
2
4
6
8
10
12
0 100 200 300 400
Time (hrs)
[Mo
no
me
r],
µM
Is the Plateau a Real Thermodynamic Cr?
Q40
Q40
Q40P10
Q40P10
[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
Conformational Space
G
fibril
aggregation- incompetent monomer
aggregation- competent monomer
ΔG
Transportability of the P10 Effect
Peptide C r, 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
[O]HS
HS
SH
HS
SH
HS
HS
S
SH
HS
S
[S. Shivaprasad and R. Wetzel, Biochem. 43, 15310 (2004)]
Stability of amyloid fibrils from various double Cys mutants of Aβ(1-40)
R-SH R-S-S-R
Cysteine mutants
0
0.5
1
1.5
2
2.5
3
17C-34C 17C-35C 17C-36C
ΔΔ
G (
kca
l/m
ol)
[S. Shivaprasad and R. Wetzel, Biochem. 43, 15310 (2004)]
16
1718
1920
21
31
32
33
343536
15
Re
lati
ve
In
ten
sit
y
20-34+2
A
B
(c)
746 750 754
Mass/Charge
(d)35-40
+1
561 565 569
727
(a)1-40+6
A
B
723 731
(b)
1-19+5
461 465 469
Re
lati
ve
In
ten
sit
y
Mass/Charge
HX-MS with in-line pepsin: distribution of protected amide protons
[M. Chen, I. Kheterpal, K. D. Cook and R. Wetzel, unpublished]
M N*k1
k-1
Mel
k3
MelMel
k2 k4
G
Reaction coordinate
M
N*
N+1
N+2
N+1 N+2
Nucleation / Elongation
N*
45
55
65
75
85
95
105
0 1 2 3 4 5 6
Time (hrs)
Rel
ati
ve
[Q47
]
Q47 Nucleation Kinetics in the Presence of Various Concentrations of Q20
2 M Q47 + [Q20 ], M
0
14
24
36
44
54
30
40
50
60
70
80
90
100
110
0 2 4 6 8
Time (hrs)
Re
lati
ve
[Q
47
]
Q47 Nucleation Kinetics in the Presence of other PolyGln Peptides
2 M Q47 + 20 M ….
No addnQ10
Q15
Q20
Q25
Q29
Q33
Q40
+
0
5
10
15
20
0 5 10 15 20 25 30
Time, mins
fmo
l b
ioti
n-Q
30
-10.3
-10.28
-10.26
-10.24
-10.22
-10.2
-10.18
-10.16
-10.14
0 5000 10000 15000 20000 25000
Time (sec)
ln [
mo
no
mer
, M]
Determination of Q47 fibril second order elongation rate constant
k+ = kpseudo1st / [growing ends]
k+ = 11,900 moles/liter-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
M N*k1
k-1
G
Reaction coordinate
Mk3
MMk2
M
N*
k4
N+1
N+2
N+1 N+2
Kn*
nucleation equilibrium constant
second order fibril elongation rate constant
= ½ Kn*k+2Cn*+2t2
[Qn] time
16
1718
1920
21
31
32
33
34
3536
15
Side Chain Orientation and Packing Within the Aβ(1-40) Amyloid Fibril
[S. Shivaprasad, J.-T. Guo, Y. Xu and R. Wetzel, unpublished]
Side Chain Orientation by Cys Accessibility
SH
SH SH
I-CH2C(O)NH2
S-CH2C(O)NH2
Ala-edited Pro scan of Aβ(1-40) fibril stability
ΔΔG(Pro – Ala), kcal/mol
Aβ(1-40) sequence position
ΔΔ
G, k
cal/m
ol
-1
-0.5
0
0.5
1
1.5
2
2.5
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
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
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
Proline Mutant
dd
G,
kcal
/mo
l
Amyloid Fibril Thermodynamics
WT DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVVP2 P PP4 P P P P
[Williams et al., J. Mol. Biol. 335, 833-842 (2004)]
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
19 20 38 39
Alanine mutation ΔΔGs adjust for hydrophobicity effects in Pro series
Proline - WT ΔΔG
Alanine - WT ΔΔG
Pro-Ala ΔΔG
Aβ Sequence Position
ΔG
mu
t –
ΔG
wt,
kcal/
mol
[AD Williams & R Wetzel, Ms. in preparation]
Additivity in Alanine mutation ΔΔGs
16
1718
1920
21
31
32
33
343536
15
0
0.5
1
1.5
2
2.5
17 34 17+34 17/34 17 25 17+27 17/27
Ala Mutants
ΔΔ
G,
kc
al/
ml
[AD Williams & R Wetzel, Ms. in preparation]
Aβ(1-40) monomer seeded with Aβ(1-40) or IAPP fibrils
0
2
4
6
8
10
0 1 2 3 4Time (hrs)
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1 2 3 4Time (hrs)
All experiments with 10 nM biotinyl-Aβ
Fm
ol b
ioti
nyl-
Aβ
Fm
ol b
ioti
nyl-
Aβ
Aβ amyloid fibrils on plate
IAPP amyloid fibrils on plate
IAPP fibrils are only 1-2% efficient, compared with Aβ, in seeding Aβ elongation.
Collagen on plate
[O’Nuallain, Williams, Westermark & Wetzel, J. Biol. Chem. 279, 17490-17499 (2004)]
Rates of A Elongation with Various Amyloid Fibrils as Seeds
Seed Fibril Elongation Rate (fmol/hour) Relative Efficiency
A 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
2-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)]
Wavelength(nm)
Random coil to -sheet transition in a Q42 peptide incubated at pH 7, 37 °C
-10000
-5000
0
5000
10000
15000
20000
200 220 240 260
[]
deg
ree
cm2 d
mol
e-1
T = 0 hrs
T = 217 hrs
T = 86 hrs
T = 45 hrs
[Chen, Ferrone & Wetzel, PNAS (2002)]
Fractionation of an Incomplete Aggregation Reaction
200 220 240 260
-10000
-5000
0
5000
10000
15000
20000
Wavelength(nm)
[]
deg
ree
cm2 d
mol
e-1
aggregation time point (86 hrs)
resuspended pellet
supernatant
supernatant plus pellet spectra
No evidence for stable, -sheet structure in the non-aggregated fraction
14
1210
8
6
4
2
40
38
16
18
282624
22
20
36
34
32
30
14
1210
8
6
440
38
40
38
16
18
282624
22
20
36
34
32
30
A Working Model for the Aβ(1-40) Fibril
[Williams et al., J. Mol. Biol. 335, 833-842 (2004)]
[Guo, J.T., Wetzel, R. and Xu, Y., Proteins (2004) In press.]
0 50 100 150 200 250
0
20
40
60
80
100
Hours
% A
ggre
gate
For
mat
ion
Aggregation of a Q42 Peptide Monitored by Four Parameters
-sheet formation proceeds in parallel with aggregation
HPLC insolubles
Thioflavin T fluoresence
-sheet (CD)
Light scattering
Protein Deposition in Human Disease
• 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)
• 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)
BRAIN
PERIPHERY
0
5
10
15
20
25
30
0 0.5 1 1.5 2 2.5 3 3.5 4Time (h)
Th
T f
luo
resc
ence
or
[Aβ
] (μ
M)
Seeded amyloid growth from Aβ(1-40)
ThT
[Aβ(1-40)]
Cr
0
5
10
15
20
25
30
0 0.5 1 1.5 2 2.5 3 3.5 4Time (h)
Th
T f
luo
resc
ence
or
[Aβ
] (μ
M)
Seeded amyloid growth from Aβ(1-40)
ThT
[Aβ(1-40)]
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
Time (hrs)
Th
T f
luo
res
ce
nc
e
Seeded amyloid growth from Aβ(1-40) concentrated from Cr plateau
0
5
10
15
20
25
30
0 0.5 1 1.5 2 2.5 3 3.5 4Time (h)
Th
T f
luo
resc
ence
or
[Aβ
] (μ
M)
Seeded amyloid growth from Aβ(1-40)
ThT
[Aβ(1-40)]
Aβ(1-40) fibril dissociation to equilibrium
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30 40 50 60
Time (hrs)
[Aβ
] (μ
M)
0.5-day fibrils
20-day fibrils
CAG REPEAT LENGTHS IN HUNTINGTON’S DISEASE
29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 4525 26 27 2824
penetrance
Repeat Length Dependence of Age of Onset in Huntington’s Disease
[Courtesy Marcy MacDonald]
log C
-0.3 0.0 0.3 0.6 0.9 1.2 1.5 1.8
-5
-4
-3
-2
-1
0
Q28
Q36
Q47
Concentration Dependence of Nucleation Kinetics
log
[½ k
+2 K
n* c
(n*+
2)]
slope = n* + 2
[Chen, Ferrone & Wetzel, PNAS (2002)]
0
5
10
15
20
25
30
35
40
0 100 200 300 400 500 600
Time (hrs)
Mo
no
me
r (u
M)
Q45
PGQ9
PGQ8
PGQ7
0
5
10
15
20
25
30
0 50 100 150 200
Time (hrs)
Mo
no
mer
(u
M)
PGQ9
PDGQ9
PolyGln Aggregate Structure
0
5
10
15
20
25
30
35
0 100 200 300 400
Time (hrs)
Mo
no
me
r (u
M) PGQ9(P
2)
Q15PQ26
PGQ9
Q45
PGQ9 PG PG PG
PG PG PGPGQ9(P2) P
PQ15PQ26
PolyGln Aggregate Structure
PG
PG
PG
PG
PG
PG
N
N
C
C
PG
PG
PG
PGG
PG
P
N
N
C
C
Anti-parallel -sheet model Parallel -helix model
Aggregation-competentmonomer
Aggregation-incompetentmonomer
(polyproline type II helix??)
AggregateWetzel and Creamer labs
Wetzel lab
Computer simulations: Rohit Pappu, Washington University
Effect of flanking sequences on polyglutamine aggregate stability
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• Indu Kheterpal• Angela Williams• Shankaramma Shivaprasad• Israel Huff• Tina Richey• Kimberley Salone• Matt Sega• Brian Bledsoe• Valerie Berthelier• Lezlee Dice• Brian O’Nuallain• Anusri Bhattacharyya Mitra• Songming Chen• Wen Yang• Brad Hamilton• Ashwani Thakur• Geetha Thiagarajan• Roopa Kenoth• Merav Geva• Alex Osmand• Erica Johnson Rowe• Erin Newby
UGA• Juntao Guo• Ying Xu
UT Main Campus
• Maolian Chen• Erik Portelius• David Kaleta• Shaolian Zhou• Kelsey Cook
• Neil Whittemore• Rajesh Mishra• Engin Serpersu
• Guangyao Gao• Ying Chen• Peter Zhang
• Anna Gardberg• Chris Dealwis
• Liz Howell
• John Dunlap
Harvard Med• Hilal Lashuel• Peter Lansbury• Prasanna Venkatraman• Fred Goldberg
FUNDING: NIH (NIA, NINDS); Hereditary Disease Foundation
Cal Tech• Jan Ko• Susan Ou• Paul Patterson
Uppsala
• Per Westermark
Drexel • Frank Ferrone
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
[Williams et al., J. Mol. Biol. 335, 833-842 (2004)]
0
2
4
6
8
10
12
14
16
18
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
Deu
teri
um
co
nte
nt
few
er H
-bon
dsm
ore
H-b
onds
Normal globular proteins generally have only one stable state