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
fibril
N
fibril
Aβ Amyloid Fibril Formation
35
30
[Monomer], μM
25
20
20
15
15
10
10
5
5
0
0
0 lag phase
2
4
Time (days)
6
8
10
ThT Fluorescence (au)
30
25
Cr
The experimental Cr is the equilibrium position of fibril elongation
100
1. Unpolymerized Aβ at equilibrium:
- chemically indistinguishable from initial
- capable of making fibrils after concentration
S26P mutant of Aβ(1-40)
80
2. Fibrils resuspended in buffer:
- dissociate to the identical Cr position
[Aβ], μM
60
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]
Monomer remaining, μM
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)]
Cr
Time
Ala scan of Aβ(1-40) fibril elongation thermodynamics
ΔΔG(Ala – WT), kcal/mol
15-21
2.5
31-36
ΔΔG, kcal/mol
2
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
Mutation
31-36
in
out
18
19
20
31
32
G (β1)
36
6 / 53
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
Mutation
31-36
in
out
18
1.3
19
20
31
32
G (β1)
36
6 / 53
1.0
1.25
Val
Ala
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
Mutation
31-36
Val
Ala
Phe
Ala
Ile
Ala
in
out
18
19
20
1.3
1.5
0.8
31
32
G (β1)
36
6 / 53
1.0
1.25
1.5
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
Mutation
31-36
Val
Ala
Phe
Ala
Ile
Ala
in
out
18
19
20
31
32
1.3
1.5
0.8
G (β1)
36
6 / 53
1.0
1.25
1.5
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
ΔΔG, kcal/mol
2.5
2.0
1.5
1.0
0.5
0.0
4 6 9 12 14 15 16 1718 19 20 21 22 23 24 25 26 2728 29 30 3132 33 34 35 3637 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
ΔΔG, kcal/mol
2
1.5
1
0.5
0
-0.5
-1
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
Aβ(1-40) sequence position
[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
53
44
ΔΔG, kcal/mol
>4
>4
Source
Minor and Kim, Nature 367, 660 (1994)
Minor and Kim, Nature 371, 264 (1994)
Amyloid
2.5
ΔΔG, kcal/mol
2
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
-1
Aβ(1-40) sequence position
Hydrogen-Deuterium Exchange Experiment
Deuteriumlabeled
fibrils
T
(D)
forward exchange D2O, pD = 7.5
Processing Solvent (pH~2)
(H)
- quench exchange
- dissociate fibrils
- efficient MS analysis
back exchange H/D mix, pH ~ 2
10% D2O
Ab
fibrils
Mass
Spectrometer
Data
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
fewer H-bonds
18
14
12
10
more H-bonds
Deuterium content
16
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
amyloidogenic peptide
globular protein
U
U
G
A4
A1
A2
N
Conformational space
A3
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)
Atropin 1 (DRPLA)
MATLEKLMKAFESLKSF-Qn- PPPPPPPPPPPQLPQPPPQA-PSTGAQSTAHPPVSTHHHHH-Qn-HHGNSGPPPPGAFPHPLEGG-
Androgen Receptor (SBMA) -GPRHPEAASAAPPGASLLLL-Qn- ETSPRQQQQQQGEDGSPQAHAtaxin 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
Light Scattering
100
20 mM Q28 monomer +
1% Q28 aggregate
80
60
40
20
20 mM Q28 monomer
0
0
75
150
Hours
225
300
Nucleation / Elongation
Kn*
M
k-1
k1
N*
k2
N+1
M
k3
N+2
k4
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
37
110
32
105
27
100
22
95
slope = ½ Kn*k+2Cn*+2
85
12
7
0.0E+00
90
1.0E+08
2.0E+08
3.0E+08
4.0E+08
80
5.0E+08
time2 (sec2)
-11
-12
log (t2 slope)
17
[polyGln], M (x 106)
[polyGln], M (x 106)
time2 plots
slope = n* + 2 = 2.87
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
log ([monomer], M)
-4.2
-4.1
-4
-3.9
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
Aggregated Peptide (%)
[Qn] = 0.1 nM
30
20
10
0
10
-4
10
-3
10
-2
10
-1
Years
10
0
10
1
10
31
2
141
10
3
10
4
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
Fibriln +
-0.7668 = log (½ Kn*k+2)
Monomer
Rate = k+ [Fibril][Monomer]
= kpseudofirst [Monomer]
-10.14
-10.16
ln [monomer, M]
Fibriln+1
k+ = kpseudofirst / [Fibril]
-10.18
-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
+
k+ = kpseudo1st / [aggregate] = 1.14 x 104 liters/mol-sec
35
30
25
20
15
0
1
2
3
4
5
6
7
8
9
[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
10
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
Shankari Shivaprasad
Anusri Bhattacharyya
Ashwani Thakur
Brian O’Nuallain
Indu Kheterpal
Eric Portelius
Songming Chen
Frank Ferrone (Drexel Univ.)
Trevor Creamer (Univ. Kentucky)
Veronique Hermann (Univ. Kentucky)
Polyproline dampens polyglutamine aggregation
100
Q40P10
90
% Monomer
80
H 2N
KKQ 40 CKK COOH
|
S CH 2CONH G 3 P KK COOH
10
Q40
70
60
50
P10Q40
40
30
20
10
0
0
50
100
150
200
Time (hrs)
250
300
350
[A Bhattacharyya et al., J. Mol. Biol. 2006]
Polyproline dampens polyglutamine aggregation
100
Q40P10
90
% Monomer
80
Q40
70
60
50
40
30
Cr = 4.5 μM
20
ΔΔG ≥
10
3 kcal/mol
Cr ≤ 50 nM
0
0
50
100
150
200
Time (hrs)
250
300
350
Polyproline dampens polyglutamine aggregation
100
Q40P10
90
% Monomer
80
Q40
70
60
50
P10Q40
40
30
20
10
0
0
50
100
150
200
Time (hrs)
250
300
350
Polyproline dampens polyglutamine aggregation
100
Q40P10
90
% Monomer
80
H 2N
KKQ 40 CKK COOH
|
S CH 2CONH G 3 P KK COOH
10
Q40
70
60
50
P10Q40
40
30
20
10
0
0
50
100
150
200
Time (hrs)
250
300
350
Is the Plateau a Real Thermodynamic Cr?
12
[Monomer], µM
10
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
aggregationincompetent
monomer
aggregationcompetent
monomer
G
ΔG
fibril
Conformational Space
Transportability of the P10 Effect
Peptide
Aβ(1-40)
Aβ(1-40)-P10
Cr, μM
0.9 μM
21.5 μM
[A Bhattacharyya et al., J. Mol. Biol. 2006]
Side Chain Packing by Disulfide Formation
HS
HS
HS
HS
SH
HS
[O]
HS
SH
SH
HS
S
S
[S. Shivaprasad and R. Wetzel, Biochem. 43, 15310 (2004)]
Stability of amyloid fibrils from various double Cys mutants of Aβ(1-40)
33
17
18
2.5
34
16
36
15
R-S-S-R
3
31
19
35
ΔΔG ( kcal/mol)
20
R-SH
32
21
2
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)
20-34
+2
A
1-40
+6
A
B
723
727
731
(b)
1-19
+5
461
465
Mass/Charge
469
Relative Intensity
Relative Intensity
B
746
750
754
(d)
35-40
+1
561
565
569
Mass/Charge
[M. Chen, I. Kheterpal, K. D. Cook and R. Wetzel, unpublished]
Nucleation / Elongation
N*
M
k-1
k1
N*
k2
N+1
Mel
k3
N+2
k4
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
Relative [Q47]
95
85
14
24
75
36
65
44
55
54
45
0
1
2
3
Time (hrs)
4
5
6
Q47 Nucleation Kinetics in the Presence of other PolyGln Peptides
2 mM Q47 + 20 mM ….
110
No addn
Q10
Q15
100
Relative [Q47]
90
80
Q20
70
Q25
Q29
60
50
Q33
40
Q40
30
0
2
4
Time (hrs)
6
8
Determination of Q47 fibril second order elongation rate constant
+
fmol biotin-Q30
20
15
10
5
0
5
10
15
20
Time, mins
k+ = kpseudo1st / [growing ends]
25
-10.14
30
-10.16
ln [monomer, M]
0
-10.18
-10.2
-10.22
-10.24
-10.26
-10.28
-10.3
k+ = 11,900 moles/liter-sec
0
5000
10000
15000
Time (sec)
20000
25000
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*
M
k-1
k1
N*
k2
N+1
M
k3
N+2
k4
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
32
21
20
31
19
33
17
18
34
16
36
35
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
ΔΔG, kcal/mol
2
1.5
1
0.5
0
-0.5
-1
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
Aβ(1-40) sequence position
Amyloid Fibril Thermodynamics
3.5
3.0
ddG, kcal/mol
2.5
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
P2
P4
DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV
P
P
P
P
P
P
[Williams et al., J. Mol. Biol. 335, 833-842 (2004)]
Alanine mutation ΔΔGs adjust for hydrophobicity effects in Pro series
ΔGmut – ΔGwt, kcal/mol
3.5
3
Proline - WT ΔΔG
2.5
Alanine - WT ΔΔG
Pro-Ala ΔΔG
2
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
32
21
20
31
19
33
17
18
2.5
34
16
36
35
2
ΔΔG, kcal/ml
15
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
0.6
Fmol biotinyl-Aβ
Fmol biotinyl-Aβ
10
8
6
4
IAPP amyloid fibrils on plate
2
0.5
0.4
0.3
0.2
Collagen on plate
0.1
0
0
0
1
2
Time (hrs)
3
4
0
1
2
3
4
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
IAPP
0.086 ± 0.01
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
100 %
1.1
[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
-1
5000
[Q] degree cmdmole
10000
2
15000
T = 86 hrs
0
T = 45 hrs
-5000
T = 0 hrs
-10000
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
the non-aggregated
fraction
resuspended pellet
10000
supernatant plus pellet spectra
2
[Q] degree cmdmole
-1
15000
5000
aggregation time point (86 hrs)
0
-5000
supernatant
-10000
200
220
Wavelength(nm)
240
260
A Working Model for the Aβ(1-40) Fibril
2
6
4
8
10
40
12
38
14
36
16
34
18
32
20
30
22
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
% Aggregate Formation
100
b-sheet formation
proceeds in parallel
with aggregation
80
60
40
HPLC insolubles
Thioflavin T fluoresence
20
b-sheet (CD)
Light scattering
0
0
50
100
150
Hours
200
250
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
ThT fluorescence or [Aβ] (μM)
25
20
15
10
[Aβ(1-40)]
5
Cr
0
0
0.5
1
1.5
2
Time (h)
2.5
3
3.5
4
Seeded amyloid growth from Aβ(1-40)
30
ThT
ThT fluorescence or [Aβ] (μM)
25
20
15
10
[Aβ(1-40)]
5
0
0
0.5
1
1.5
2
Time (h)
2.5
3
3.5
4
Seeded amyloid growth from Aβ(1-40) concentrated from Cr plateau
8
7
ThT fluorescence
6
5
4
3
2
1
0
0
1
2
3
Time (hrs)
4
5
6
Seeded amyloid growth from Aβ(1-40)
ThT
25
1.2
20
1
0.8
Cr (uM)
ThT fluorescence or [Aβ] (μM)
30
15
0.6
0.4
0.2
0
10
0
[Aβ(1-40)]
5
10
15
20
Time after ThT max (days)
5
0
0
0.5
1
1.5
2
Time (h)
2.5
3
3.5
4
Aβ(1-40) fibril dissociation to equilibrium
1.2
20-day fibrils
1
0.5-day fibrils
[Aβ] (μM)
0.8
0.6
0.4
0.2
0
0
10
20
30
Time (hrs)
40
50
60
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
log [½ k+2 Kn* c(n*+2)]
-1
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
PGQ8
35
Q45
Monomer (uM)
30
PGQ7
25
Q45
20
PGQ9
PG
PGQ9(P2)
PG
P
PG
PG
PG
PG
PGQ9
15
Q15PQ26
P
10
5
0
0
100
200
300
400
500
600
35
Time (hrs)
30
30
25
PGQ9
20
Monomer (uM)
Monomer (uM)
25
15
PDGQ9
10
PGQ9(P2)
20
15
10
5
5
0
0
0
50
100
Time (hrs)
150
200
Q15PQ26
PGQ9
0
100
200
Time (hrs)
300
400
PolyGln Aggregate Structure
N
N
P
G
C
N
P
G
C
P
G
P
PG
G
P
P
G
C
N
G
P
P
G
G
P
G
P
G
P
G
Anti-parallel b-sheet model
C
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
UGA
UT Main Campus
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
• Maolian Chen
• Erik Portelius
• David Kaleta
• Shaolian Zhou
• Kelsey Cook
• Neil Whittemore
• Rajesh Mishra
• Engin Serpersu
• Juntao Guo
• Ying Xu
Harvard Med
• Hilal Lashuel
• Peter Lansbury
• Prasanna Venkatraman
• Fred Goldberg
Cal Tech
• Guangyao Gao
• Ying Chen
• Peter Zhang
• Anna Gardberg
• Chris Dealwis
• Jan Ko
• Susan Ou
• Paul Patterson
Uppsala
• Per Westermark
• Liz Howell
• John Dunlap
Drexel
• Frank Ferrone
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
fewer H-bonds
18
14
12
10
more H-bonds
Deuterium content
16
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