1. Kinetics and Thermodynamics of Amyloid Fibril Formation Ron Wetzel University of Tennessee

2. 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

3. 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

4. Time (days) Aβ Amyloid Fibril Formation lag phase CrCr

5. 024681012141618202224262830 0 20 40 60 80 100 Time (Hrs) [Aβ], μM S26P mutant of Aβ(1-40) The experimental C r 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 C r position

6. Monomer + Fibril N Fibril N+1 K eq Amyloid Fibril Elongation Thermodynamics K eq = [Fibril N+1 ] / [Fibril N ][Monomer] K eq = 1 / [Monomer] K eq = 1 / C r ΔG = - RT ln K eq ΔG = - RT ln K eq = - RT ln (1 / 0.0000086) ΔG = - 8.6 kcal/mol [wild type Aβ(1-40)] CrCr Monomer remaining, μM Time

7. Ala scan of Aβ(1-40) fibril elongation thermodynamics ΔΔG (Ala – WT), kcal/mol 15-21 31-36

8. Ala scan of Aβ(1-40) fibril stability Petkova et al., 2002 Guo et al., 2004

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

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

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

12. 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)] in out

13. 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)] in out

14. 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)] in out

15. 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)] in out

16. Pro scan of Aβ(1-40) fibril stability -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 469121415161718192021222324252627282930313233343536373839 Aβ(1-40) sequence position ΔΔG, kcal/mol ΔΔG (Pro – WT), kcal/mol [Williams et al., J. Mol. Biol. 335, 833-842 (2004)]

17. 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

18. Ala-edited Pro scan of Aβ(1-40) fibril stability ΔΔG (Pro – Ala), kcal/mol Aβ(1-40) sequence position ΔΔG, kcal/mol -0.5 0 0.5 1 1.5 2 2.5 414151617181920212224252627282930313233343536373839 [Williams et al., J. Mol. Biol. 357, 1283 (2006)]

19. ΔΔG values for Pro vs. Ala replacement in β-sheet Globular 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 ΔΔG, kcal/mol -0.5 0 0.5 1 1.5 2 2.5 414151617181920212224252627282930313233343536373839 Amyloid Globular Protein

20. T Hydrogen-Deuterium Exchange Experiment Deuterium- labeled fibrils Processing Solvent (pH~2) - quench exchange - dissociate fibrils - efficient MS analysis A  fibrils forward exchange - D 2 O, pD = 7.5 back exchange - H/D mix, pH ~ 2 10% D 2 O ( D ) ( H ) Data Analysis Mass Spectrometer [Kheterpal, Zhou, Cook & Wetzel, PNAS (2000)]

21. Protected Amide Hydrogens in Proline Mutant Fibrils [Williams et al., J. Mol. Biol. 335, 833-842 (2004)] fewer H-bonds more H-bonds Leu34->Pro, ΔΔG = only 1.5 kcal/mol destabilized …. but it also has 4 more H-bonds than WT

22. Thermodynamics of Amyloid Fibril Formation Results: - Aβ(1-40) fibril growth tends to a reversible equilibrium position with a K eq 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

23. Conformational space G globular protein amyloidogenic peptide N U U A1A1 A3A3 A2A2 A4A4

24. 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

25. Polyglutamine flanking sequences in expanded CAG repeat disease proteins AVAAAAVQQSTSQQATQGTS- -LTPQPIQNTNSLSILEEQQR-Q n - PPPPQPQRQQHPPPPPRRTR- -RGEPRRAAAAAGGAAAAAAR-Q n - AVARPGRAATSGPRRYPGPT- -PRPHVSYSPVIRKAGGSGPP-Q n - RDLSGQSSHPCERPATSSGA- -SGTNLTSEELRKRREAYFEK-Q n - PPPAAANVRKPGGSGLLASP- -GCPRPACEPVYGPLTMSLKP-Q n - HLSRAPGLITPGSPPPAQQN- -YSTLLANMGSLSQTPGHKAE-Q n - ETSPRQQQQQQGEDGSPQAH- -GPRHPEAASAAPPGASLLLL-Q n - HHGNSGPPPPGAFPHPLEGG- -PSTGAQSTAHPPVSTHHHHH - Q n - PPPPPPPPPPPQLPQPPPQA- MATLEKLMKAFESLKSF-Q n - TBP (SCA17) Ataxin 7 (SCA7) CACNA1A (SCA6) Ataxin 3 (SCA3) Ataxin 2 (SCA2) Ataxin 1 (SCA1) Androgen Receptor (SBMA) Atropin 1 (DRPLA) Huntingtin (HD)

26. 075150225300 0 20 40 60 80 100 120 Light Scattering Hours 20  M Q 28 monomer 20  M Q 28 monomer + 1% Q 28 aggregate Lag phase aborted by seeding

27. Nucleation / Elongation MN* k1k1 k -1 G Reaction coordinate M k3k3 M M k2k2 M N* k4k4 N +1 N +2 N +1 N +2 K n* nucleation equilibrium constant second order fibril elongation rate constant  = ½ K n* k + 2 C n*+2 t 2 [Q n ] time

28. Nucleation Kinetics Analysis for Q 47 Aggregation time 2 plots slope = ½ K n* k + 2 C n*+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 (t 2 slope) slope = n* + 2 = 2.87 n* = 0.87 ~ 1 log (½ K n* k + 2 ) = -0.7668 time 2 (sec 2 ) [polyGln], M (x 10 6 ) 7 12 17 22 27 32 37 0.0E+001.0E+082.0E+083.0E+084.0E+085.0E+08 80 85 90 95 100 105 110 [polyGln], M (x 10 6 )

29. + nucleation elongation Mechanism of polyglutamine aggregation K n* n* = 1 for Q 28, Q 36, Q 47 ; K n* increases from Q 28 to Q 36 to Q 47 [Chen, Ferrone & Wetzel, PNAS (2002)]

30. Calculated Aggregation Kinetics Curves at Low Concentration Q 47 Q 36 Q 28  = ½ k + 2 K n* c (n*+2) t 2 [Chen, Ferrone & Wetzel, PNAS (2002)]

31. Time (sec) ln [monomer, M] -10.28 -10.26 -10.24 -10.22 -10.20 -10.18 -10.16 -10.14 0.0E+005.0E+031.0E+041.5E+042.0E+042.5E+04 Pseudo-first order kinetics of seeded polyGln elongation [A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005] Fibril n + Monomer Fibril n+1 Rate = k + [Fibril][Monomer] = k pseudofirst [Monomer] k + = k pseudofirst / [Fibril]  = ½ k + 2 K n* c (n*+2) t 2 -0.7668 = log (½ K n* k + 2 )

32. 15 20 25 30 35 012345678910 15 20 25 30 35 0123456789100123456789 [biotinyl-polyglutamine], μM fmol biotinyl-polyGln bound + Determination of K n* k + = k pseudo1st / [aggregate] = 1.14 x 10 4 liters/mol-sec -0.7668 = log (½ K n* k + 2 ) K n* = 2.6 x 10 -9 ΔG = + 12.2 kcal/mol [A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]

33. nucleation + elongation Mechanism of polyglutamine aggregation K n* For Q 47, K n* = 2.6 x 10 -9 (ΔG nucleation = + 12.2 kcal/mol) k+k+ [A Bhattacharyya, AK Thakur and R Wetzel, PNAS 2005]

34. 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)

36. Polyproline dampens polyglutamine aggregation Q 40 Q 40 P 10 P 10 Q 40 H 2 N KKQ 40 CKK COOH | S CH 2 CONH G 3 P 10 COOH KK [A Bhattacharyya et al., J. Mol. Biol. 2006]

37. Polyproline dampens polyglutamine aggregation Q 40 Q 40 P 10 C r = 4.5 μM C r ≤ 50 nM ΔΔG ≥ 3 kcal/mol

38. Polyproline dampens polyglutamine aggregation Q 40 Q 40 P 10 P 10 Q 40

39. Polyproline dampens polyglutamine aggregation Q 40 Q 40 P 10 P 10 Q 40 H 2 N KKQ 40 CKK COOH | S CH 2 CONH G 3 P 10 COOH KK

40. Is the Plateau a Real Thermodynamic C r ? Q 40 Q 40 P 10 [A Bhattacharyya et al., J. Mol. Biol. 2006]

41. A Conformational Correlate to the P 10 Connectivity Effect on Aggregation 35°C - 5°C difference spectra [A Bhattacharyya et al., J. Mol. Biol. 2006]

42. A Possible Basis of the OligoProline Effect Conformational Space G fibril aggregation- incompetent monomer aggregation- competent monomer ΔGΔG

43. Transportability of the P 10 Effect Peptide C r, μM Aβ(1-40) 0.9 μM Aβ(1-40)-P 10 21.5 μM [A Bhattacharyya et al., J. Mol. Biol. 2006]

44. Side Chain Packing by Disulfide Formation HS [O] HS SH HS SH HS S SH HS S [S. Shivaprasad and R. Wetzel, Biochem. 43, 15310 (2004)]

45. 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-34C17C-35C 17C-36C ΔΔ G ( kcal/mol) [S. Shivaprasad and R. Wetzel, Biochem. 43, 15310 (2004)] 16 17 18 19 20 21 31 32 33 34 35 36 15

46. Relative Intensity 20-34 +2 A B (c) 746750754 Mass/Charge (d) 35-40 +1 561565569 727 (a) 1-40 +6 A B 723 731 (b) 1-19 +5 461465469 Relative Intensity Mass/Charge HX-MS with in-line pepsin: distribution of protected amide protons [M. Chen, I. Kheterpal, K. D. Cook and R. Wetzel, unpublished]

47. MN* k1k1 k -1 M el k3k3 k2k2 k4k4 G Reaction coordinate M N* N +1 N +2 N +1 N +2 Nucleation / Elongation N*

48. Q 47 Nucleation Kinetics in the Presence of Various Concentrations of Q 20 2  M Q47 + [Q 20 ],  M 0 14 24 36 44 54

49. Q 47 Nucleation Kinetics in the Presence of other PolyGln Peptides 2  M Q 47 + 20  M …. No addn Q 10 Q 15 Q 20 Q 25 Q 29 Q 33 Q 40

50. + -10.3 -10.28 -10.26 -10.24 -10.22 -10.2 -10.18 -10.16 -10.14 0500010000150002000025000 Time (sec) ln [monomer, M] Determination of Q 47 fibril second order elongation rate constant k + = k pseudo1st / [growing ends] k + = 11,900 moles/liter-sec

51. 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 - K n* for a Q 47 peptide is ~ 10 -9 - short polyGln peptides in the environment can enhance nucleation efficiency

52. Nucleation / Elongation MN* k1k1 k -1 G Reaction coordinate M k3k3 M M k2k2 M N* k4k4 N +1 N +2 N +1 N +2 K n* nucleation equilibrium constant second order fibril elongation rate constant  = ½ K n* k + 2 C n*+2 t 2 [Q n ] time

53. 16 17 18 19 20 21 31 32 33 34 35 36 15 Side Chain Orientation and Packing Within the Aβ(1-40) Amyloid Fibril [S. Shivaprasad, J.-T. Guo, Y. Xu and R. Wetzel, unpublished]

54. Side Chain Orientation by Cys Accessibility SH I-CH 2 C(O)NH 2 S-CH 2 C(O)NH 2

55. Ala-edited Pro scan of Aβ(1-40) fibril stability ΔΔG (Pro – Ala), kcal/mol

56. -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 469121415161718192021222324252627282930313233343536373839P2P4 Proline Mutant ddG, kcal/mol Amyloid Fibril Thermodynamics WT DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV P2 P P P4 P P P P [Williams et al., J. Mol. Biol. 335, 833-842 (2004)]

57. Alanine mutation ΔΔGs adjust for hydrophobicity effects in Pro series Proline - WT ΔΔG Alanine - WT ΔΔG Pro-Ala ΔΔG Aβ Sequence Position ΔG mut – ΔG wt, kcal/mol [AD Williams & R Wetzel, Ms. in preparation]

58. Additivity in Alanine mutation ΔΔGs 16 17 18 19 20 21 31 32 33 34 35 36 15 0 0.5 1 1.5 2 2.5 173417+3417/34172517+2717/27 Ala Mutants ΔΔ G, kcal/ml [AD Williams & R Wetzel, Ms. in preparation]

59. Aβ(1-40) monomer seeded with Aβ(1-40) or IAPP fibrils 0 2 4 6 8 10 01234 Time (hrs) 0 0.1 0.2 0.3 0.4 0.5 0.6 01234 Time (hrs) All experiments with 10 nM biotinyl-Aβ Fmol biotinyl-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)]

60. 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 V L JTO5 0.042 ± 0.006 0.6  2-microglobulin 0.014 ± 0.001 0.2 Ure2p 0.069 ± 0.001 0.9 Polyglutamine Q 30 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)]

61. Wavelength(nm) Random coil to  -sheet transition in a Q 42 peptide incubated at pH 7, 37 °C [Chen, Ferrone & Wetzel, PNAS (2002)]

62. Fractionation of an Incomplete Aggregation Reaction aggregation time point (86 hrs) resuspended pellet supernatant supernatant plus pellet spectra No evidence for stable,  -sheet structure in the non-aggregated fraction

63. 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.]

64. 050100150200250 0 20 40 60 80 100 Hours % Aggregate Formation Aggregation of a Q 42 Peptide Monitored by Four Parameters  -sheet formation proceeds in parallel with aggregation HPLC insolubles Thioflavin T fluoresence  -sheet (CD) Light scattering

65. 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) - β 2 -microglobulin Z-form  1 -Antitrypsin Deposition (liver) Inclusion Body Myositis (muscle) Mallory Bodies (liver) BRAIN PERIPHERY

66. Seeded amyloid growth from Aβ(1-40) ThT [Aβ(1-40)] CrCr

67. Seeded amyloid growth from Aβ(1-40) ThT [Aβ(1-40)]

68. Seeded amyloid growth from Aβ(1-40) concentrated from C r plateau

69. Seeded amyloid growth from Aβ(1-40) ThT [Aβ(1-40)]

70. Aβ(1-40) fibril dissociation to equilibrium 0 0.2 0.4 0.6 0.8 1 1.2 0102030405060 Time (hrs) [Aβ] (μM) 0.5-day fibrils 20-day fibrils

71. CAG REPEAT LENGTHS IN HUNTINGTON’S DISEASE 29303132333435363738394041424344452526272824 penetrance

72. Repeat Length Dependence of Age of Onset in Huntington’s Disease [Courtesy Marcy MacDonald]

73. log C -0.30.00.30.60.91.21.51.8 -5 -4 -3 -2 0 Q 28 Q 36 Q 47 Concentration Dependence of Nucleation Kinetics log [½ k + 2 K n* c (n*+2) ] slope = n* + 2 [Chen, Ferrone & Wetzel, PNAS (2002)]

74. 0 5 10 15 20 25 30 050100150200 Time (hrs) Monomer (uM) PGQ 9 P D GQ 9 PolyGln Aggregate Structure 0 5 10 15 20 25 30 35 0100200300400 Time (hrs) Monomer (uM) PGQ 9 (P 2 ) Q 15 PQ 26 PGQ 9 Q 45 PGQ 9 PG PGQ 9 (P 2 ) P P Q 15 PQ 26

75. PolyGln Aggregate Structure PGPG PGPG PGPG PGPG PGPG PGPG N N C C P G P G P G P G G P G P N N C C Anti-parallel  -sheet model Parallel  -helix model

76. Aggregation-competent monomer Aggregation-incompetent monomer (polyproline type II helix??) Aggregate Wetzel and Creamer labs Wetzel lab Computer simulations: Rohit Pappu, Washington University Effect of flanking sequences on polyglutamine aggregate stability

77. 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

78. 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 F V 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?

79. Aggregation and Packing Interactions [R. Wetzel, Trends Biotech. 12, 193-198 (1994)]

80. 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

81. 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 C r 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

82. Many Pro-destabilized Aβ(1-40) fibrils gain H-bonds [Williams et al., J. Mol. Biol. 335, 833-842 (2004)] fewer H-bonds more H-bonds

83. Normal globular proteins generally have only one stable state