1. Thermodynamic and Kinetic Origins of Alzheimer's and Related Diseases: a Chemical Engineer's Perspective Thermodynamic and Kinetic Origins of Alzheimer's and Related Diseases: a Chemical Engineer's Perspective Carol K. Hall Department of Chemical & Biomolecular Engineering North Carolina State University http://turbo.che.ncsu.edu

2. Protein Folding: The ABCs A. A protein is a chain of amino acid residues arranged in a unique sequence.

3. B. There are 20 possible sidechains.

4. C. Physiological proteins exist in the folded or “native” state, the state with the lowest free energy. Villin headpiece protein

5. D.Proteins unfold into a “random coil” if temperature raised or denaturant (urea, GuHCl) added. E.Of all the forces thought to govern protein folding, hydrophobicity and hydrogen bonding are considered most important. Unfolded (high T or high denaturant) Folded (moderate T or low denaturant) www.sas.upenn.edu

6. Amyloidoses: Diseases characterized by the abnormal aggregation of proteins into ordered structures, called “fibrils” or “amyloid.” Disease Pick’s Alzheimer’s Parkinson’s Prion disease (e.g. Mad Cow) Amyloid Lateral Sclerosis ( Lou Gehrig’s) Huntington’s Disease Protein tau A-beta alpha synuclein prion protein TDP-43 Huntingtin

7. Alzheimer’s Disease 100 years ago --Dr. Alois Alzheimer described abnormal clumps in brain of deceased dementia patient, Auguste D. Clinical symptoms: severe dementia, loss of memory & motor skills----> death Late onset disease : 5-10% of 65-74 year olds, 50% of 85+ year olds 4.5 million Americans Costs $100 billion/year US Research Budget $650 million/year.

8. Structure of Amyloid Fibrils Fibrils are ordered aggregates of peptides characterized by cross- beta structure Protofilament structure  -sheets in a protofilament AFM on fibrils of A-ß protein

9. Issues in Amyloid Disease Research –Identity of toxic species--- early oligomers or fibrils? –Kinetics of fibril nucleation and growth –Structure of fibrils –Interactions with inhibitors

10. Objective To develop a computational tool that : allows investigation (particularly visualization) of spontaneous fibril formation. reveals the basic physical principles underlying fibril formation. Six Blind Men and Elephant

11. Polyalanine– A Model System for Studying Protein Fibrillization Speculation - fibril formation is natural consequence of peptide geometry, hydrogen-bonding capability and hydrophobic interactions under slightly-denatured, concentrated conditions. Polyalanine peptides form fibrils in vitro at high concentrations (C > 1.5 mM) and high temperature (T > 40 o C) (Blondelle et al., Biochem. 1997). Peptide Sequence: KA 14 K alpha-helixbeta-sheets in a fibril

12. Molecular Dynamics Simulations of Protein Folding Packages: Amber, CHARMm, ENCAD, ECEPP, Discover, UNRES, etc. Force fields:describe interactions between all atoms on protein and in solvent at atomic resolution Desired Output: “folding” trajectory of a protein Limitation: very difficult to simulate folding of a single protein even with the fastest computers Implications : sacrifice details to study protein aggregation

13. Discontinuous Molecular Dynamics Discontinuous Molecular Dynamics Traditional MD: Forces based on Lennard Jones (LJ) potential. Follow particle trajectories by numerically integrating Newton’s 2nd law every picosecond. Discontinuous MD: Forces field based on square- well potential. Follow particle trajectories by analytically integrating Newton’s 2nd law Particles move linearly between collisons, capture or bounce

14. (Protein Intermediate Resolution Model): PRIME (Protein Intermediate Resolution Model): United atom: NH, C a H, CO, R R= CH 3 for alanine Steric Interactions: hard spheres with realistic diameters Pseudo-bonds maintain: ideal backbone bond angles trans-configuration residue L-isomerization Covalent bond and pseudo-bond lengths set to ideal experimental values CH 3 CaHCaH CO NH Nguyen et al. Protein Sci (2004) 13 2909-2924 Smith and Hall. PROTEINS (2001) 44 344

15. NH i CH 3,i CHiCHi CO j NH j CO i CHjCHj Square-well attraction Hydrogen bonds between backbone amine and carbonyl groups are modeled with a directional square-well attraction of strength  H-bonding. Model Forces: Hydrogen Bonding Define reduced temperature as: T*=k B T/ε H-bonding

16. Model Forces: Hydrophobic Interactions Solvent effects captured implicitly. Hydrophobic side chains cluster together to avoid water Hydrophobic interaction modeled as square-well attraction between side chains. R= ε hydrophobicity /ε H-bonding

17. Folding of Single KA14K Chain Nguyen,Marchut & Hall Biophys. J (2004)

18. A Constant-Temperature Simulation: 48 Peptides at c=10.0mM, T*=0.14 Nguyen & Hall, PNAS (2005)

19. Equilibrium Simulations: 96 Peptides Use the replica- exchange method to simulate 96-peptide systems at different temperatures and peptide concentrations. These trends qualitatively agree with experimental data (Blondelle 1997) Nguyen & Hall Biophys. J. (2004 )

20. Intra-sheet distance: 4.92 ± 0.01A, comparable to experimental values of 4.76A (Shinchuk et al., Proteins, 2005) Fibril Structure: Intra-sheet Distance

21. Inter-sheet distance: 7.52 ± 0.23A, comparable to experimental values of 5.4A (Shinchuck et al., Proteins, 2005) Fibril Structure: Inter-sheet Distance

22. Fibril Structure: Peptide Orientation Most peptides are in-register, same as experimental results for the A-ß (10-35) peptide (Benzinger et al., PNAS 1998)

23. Forming Various Structures versus t* c=5mM, T*=0.14  Amorphous aggregates form instantaneously, followed by ß-sheets, and then fibrils after a delay, called the lag time.  Appearance of a lag time indicates that this is a nucleated phenomenon. all aggregates Nguyen & Hall, J. Biol. Chem (2005)

24. Fibril Formation in Seeded and Unseeded Systems at T*=0.14, c=2mM Adding a seed eliminates the fibril formation lag time, as found experimentally.

25. In Conclusion---Technical  First intermediate resolution simulations of spontaneous “fibril” formation  Our results qualitatively agree with experimental data in general, and specifically with those obtained by Blondelle et al. (Biochemistry, 1997) on polyalanines.  Next step: Extending PRIME to all 20 amino acids. Which road to take?????

26. Acknowledgements Dr. Hung D. Nguyen Dr. Alexander J. Marchut Dr. Anne V. Smith Dr. Hyunbum Jang Dr. Andrew J. Schultz National Institutes of Health National Science Foundation