1. Simulations of Peptide Aggregation Joan-Emma Shea Department of Chemistry and Biochemistry The University of California, Santa Barbara

2. Protein and Peptide Aggregation

3. PROTEIN AGGREGATION AND DISEASE Alzheimer Huntington Parkinson Prion (“Mad Cow”) Proteins not associated with a specific disease can also aggregate to form amyloid fibrils

4. APPLICATION TO BIOMATERIALS Nano-tube and nano-sphere fabrication using aromatic di- peptides. [Gazit et al., Nano Lett, 4 (2004) 581] Use of peptides to form nanoscale-ordered monolayers, COSB 2004, 14: 480

5. Time scales Side-chain rotations Loop closure Helix formation Folding of  -hairpins Protein folding Protein aggregation

6. All-atom: Molecular Dynamics (MD) With EXPLICIT Solvent MULTISCALE APPROACH Off-lattice minimalist: Langevin dynamics All-atom: Molecular Dynamics (MD) with IMPLICITsolvent COARSE GRAINING

7. 1. Dimerization Mechanisms of 4 tetrapeptides KXXE 2. Design of Inhibitors of aggregation of the Alzheimer Amyloid-beta (A  ) peptide. Fibrils of KFFE

8. KXXE peptides Tjernberg et al., JBC 277 (2002) 43243 KAAE KVVEKFFE KLLE Fibrils!

9. PEPTIDE MODEL CHARMM19 Force Field and Generalized Born Implicit Solvent R e p =1 kCal/mol,  =1Å, R=17Å Confining Sphere:

10. NVT T1T1 TNTN T2T2 Simulation protocol: Replica Exchange MD 6 replicas for monomer: total simulation time: 40 ns 12 replica for dimers: total simulation time: 400 ns

11. Heterogeneous dimers RMSD (Å) E (kCal/mol)

12. Association temperature T a KFFE > KVVE > KLLE > KAAE Thermodynamic stability of dimers:

13. What determines transition temperature T a ? Propensity for  structure Salt bridges Hydrophobic contacts Aromatic interactions

14. T=325K T=240KT=275K Free energy as a function of interaction energy and radius of gyration T=285K U (kCal/mol) Rg (Å) U U U

15. T=325K T=240KT=275K Free energy as a function of interaction energy and radius of gyration T=285K  U (kCal/mol) Rg (Å) UU U U Interaction energy: KFFE > KLLE ~ KVVE > KAAE Most favorable Least favorable

16. Entropy loss due to dimerization Monomers, Rg>5A    -s tr and Random Coil Helix

17. Entropy loss due to dimerization Monomers, Rg>5A    -s tr and Random Coil Helix

18. Entropy loss due to dimerization Monomers, Rg>5ADimers, Rg<5A    -s tr and Random Coil Helix

19. Entropy loss due to dimerization Monomers, Rg>5ADimers, Rg<5A    -s tr and Random Coil Helix  -s tr and basin more populated for monomer of KVVE than KLLE:  S L >  S V

20. Association temperature Energetic effect Entropic effect

21. KFFE KLLE Kinetic accessibility of dimers E (Kcal/mol) Rg (Å) KFFEKLLE “DOWNHILL” DIMERIZATION FOR KFFE FREE ENERGY BARRIER FOR KLLE

22. Different mechanisms of dimerization PHE-PHE come together first (stabilized by vdw interactions), followed by the overall collapse of the structure with formation of peptide backbones contacts. Rg over C  Rg over PHE atoms Rg over LEU atoms LEU side chain formation and overall collapse with formation of peptide backbone interactions occur simultaneously. KFFEKLLE

23. CONCLUSIONS for dimerization of the KXXE peptides 1. 2. Strong sequence dependence of free energy landscapes for dimerization, with KFFE experiencing a barrierless transition. 3. KFFE dimers are the most thermodynamically stable and kinetically accessible. 4. Dimer trends match experimental trends observed for fibrils.

24. Aβ40: DAEFRHDSGYEVHHQ 16 KLVFFA 22 EDVGSNKGAIIGLMVGGVV Aβ42: DAEFRHDSGYEVHHQ 16 KLVFFA 22 EDVGSNKGAIIGLMVGGVVIA LARGER SYSTEMS: OLIGOMERIZATION OF THE ALZHEIMER AMYLOID BETA (A  ) PEPTIDES AMYLOID BETA (A  ) PEPTIDES AGGREGATE TO FORM TOXIC OLIGOMERS AND FIBRILS

25. Aβ40: DAEFRHDSGYEVHHQKLVFFAEDV 25 GSNKGAIIGL 35 M VGGVV FRAGMENT 25-35 OF THE (A  ) PEPTIDE APPEARS TO BE TOXIC IN MONOMERIC, SMALL OLIGOMERIC AND FIBRILLAR FORMS NO STRUCTURE OF THE MONOMERIC PEPTIDE AVAILABLE IN AQUEOUS SOLVENT PEPTIDE ADOPTS A HELICAL STRUCTURE IN APOLAR ORGANIC SOLVENT (SUCH AS HEXAFLUOROISOPROPANOL HFIP)

26. EFFECTS OF SOLVENT ON FREE ENERGY LANDSCAPE OF THE MONOMER REPLICA EXCHANGE SIMULATION IN: 1)HFIP/WATER CO-SOLVENT 2)PURE WATER GROMOS96 FORCE FIELD, EXPLICIT SOLVENT 40 REPLICAS, 16 NS EACH, TOTAL SIMULATION TIME OF 640 NS

27. FREE ENERGY SURFACE IN HFIP/WATER COSOLVENT: HELIX STABILIZATION T=300 K 45 % 8% N N N N C C C C

28. HFIP DISPLACES WATER NEAR THE PEPTIDE, AND FORMS A “COAT” AROUND THE PEPTIDE

29. FREE ENERGY SURFACE IN PURE WATER FORMATION OF COLLAPSED-COILS and  -HAIRPINS T=300K N N N N N C C C C C

30. Possible importance of turn in toxicity of monomer

31. REPLICA EXCHANGE MD ON DIMERS IN WATER: HETEROGENEOUS DIMERS

32. TWO TYPES OF ORDERED DIMERS Experimentally: two different protofilament of diameters: 1.41 +/- 0.48 nm 3.58 +/- 1.53 nm

33. PEPTIDE INHIBITORS OF ALZHEIMER A  AGGREGATION Alzheimer’s disease (AD) is a neurodegenerative disease of the central nervous system.

34. ALZHEIMER DISEASE IS CHARACTERIZED BY THE PRESENCE OF NEUROFIBRILLAR TANGLES AND AMYLOID PLAQUES IN THE BRAIN

35. Aβ40: DAEFRHDSGYEVHHQ 16 KLVFFA 22 EDVGSNKGAIIGLMVGGVV Aβ42: DAEFRHDSGYEVHHQ 16 KLVFFA 22 EDVGSNKGAIIGLMVGGVVIA AMYLOID PLAQUES CONSIST OF AMYLOID BETA (A  ) PEPTIDES GENERATED FROM THE PROTEOLYTIC CLEAVAGE OF THE APP TRANSMEMBRANE PROTEIN

36. Amyloid Plaques Aβ40: DAEFRHDSGYEVHHQ 16 KLVFFA 22 EDVGSNKGAIIGLMVGGVV Aβ42: DAEFRHDSGYEVHHQ 16 KLVFFA 22 EDVGSNKGAIIGLMVGGVVIA In healthy individuals, the A  peptides are broken down and eliminated. In AD, these peptides self-assemble into amyloid fibrils Fibril

37. Both small soluble oligomers and fibrils appear to be toxic to cells. Native Protein Misfolded Protein Soluble Oligomer Protofibrils Fibril

38. N-methylated A  (16-20)m peptides can: 1)prevent the aggregation of full length A  peptide 2)disassemble existing fibrils and possibly small oligomers. Aβ40: DAEFRHDSGYEVHHQ 16 KLVFFA 22 EDVGSNKGAIIGLMVGGVV N-METHYLATED PEPTIDE INHIBITORS 16 K(me)LV(me)FF Fibrils of A  (1-40) peptides After Incubation with A  (16-20)m peptides Meredith and co-workers, J. Pep. Res. (2002) 60, 37-55

39. Fragment A  (16-22) KLVFFAE aggregates to form fibrils Tycko et al. Biochemistry, 39 (45), 13748 -13759, 2000 Antiparallel arrangements from solid state NMR MODEL SYSTEM Aβ40: DAEFRHDSGYEVHHQ 16 KLVFFA 22 EDVGSNKGAIIGLMVGGVV

40. A  (16-22) KLVFFAE PROTOFIBRIL INITIAL STRUCTURE: Two parallel bilayers Peptides in layer antiparallel lys 16 and glu 22 point to solvent leu 17, phe 19, ala 21 point inside core

41. A  (16-22) KLVFFAE PROTOFIBRIL INITIAL STRUCTURE: Two parallel bilayers Peptides in layer antiparallel lys 16 and glu 22 point to solvent leu 17, phe 19, ala 21 point inside core GROMOS96 FORCE FIELD EXPLICIT SPC WATER (23000 atoms) REACTION FIELD/ PME TWO 20 NS SIMULATIONS

42. REPRESENTATIVE A  (16-22) PROTOFIBRIL (LAST 7 NS OF SIMULATIONS) Distance between bilayers: 0.93 nm (Tycko: 0.99nm) Distance between peptides: 0.44-0.52 nm (Tycko: 0.47 nm)

43. Structure of N-methylated A  (16-20)m Inhibitor Peptide Replica Exchange Molecular Dynamics, 30 replicas, 20 ns per replica A  (16-20)m more rigid than A  (16-22), with  -strand content: This pre-organization may allow A  (16-20)m to successfully compete with free A  (16-22) for binding to fibril.

44. Interaction of A  (16-20)m Inhibitor Peptide with protofibril INITIAL STRUCTURE A  (16-20)m with N-methyl groups pointing away fibril A  (16-20)m with N-methyl groups pointing toward fibril

45. Interaction of A  (16-20)m Inhibitor Peptide with protofibril AFTER 50 ns A  (16-20)m with N-methyl groups pointing toward fibril Intercalates between layers A  (16-20)m with N-methyl groups pointing away fibril Forms hydrogen bonds with fibril (antiparallel)

46. POSSIBLE MECHANISM OF FIBRIL DISRUPTION Inhibitor drifts from edge of fibril to side and inserts in fibril (between strands 6 and 7) with Lys pointing to solvent and hydrophobic residues inserted in fibril.

47. POSSIBLE MECHANISM OF INHIBITION OF FIBRIL GROWTH Inhibitor forms hydrogen bond with fibril, with antiparallel alignment, possibly preventing additional A  (16-22) peptides from binding.

48. DESTABILIZATION OF FIBRIL WHEN INHIBITOR INSERTED IN FIBRIL Inhibitor inserted in fibril affects the “twist” of the fibril and the distance between strands.

49. CONCLUSIONS AND FUTURE DIRECTIONS 1.Results suggest possible mechanisms of fibril inhibition and disassembly 2.Extend the simulations to consider other orientations of the inhibitor peptides 3.Study and design new inhibitors

50. ACKNOWLEDGEMENTS Dr. P. Soto Other group members: M. Friedel, M. Griffin, A. Jewett, W. B. Lee and E. Zhuang Funding: NSF Career, David and Lucile Packard Foundation, A. P. Sloan Foundation, Army Research Office. Collaborator: Prof. Stephen Meredith, University of Chicago Dr. G. WeiDr. A. Baumketner

51. University of California Santa Barbara