Why Does Polyglutamine Aggregate? Insights from studies of monomers

1. Why Does Polyglutamine Aggregate?Insights from studies of monomers Xiaoling Wang, Andreas Vitalis, Scott Crick, Rohit Pappu Biomedical Engineering & Center for Computational Biology, Washington University in St.Louis pappu@biomed.wustl.edu http://lima.wustl.edu

2. Expanded CAG Repeat Diseases and Proteins DISEASE GENE PRODUCT NORMAL CAG MUTANT CAG REPEAT RANGE REPEAT RANGE Huntington’s huntingtin 6 - 39 36-200 DRPLA atrophin 1 – 35 3 49 - 88 SBMA androgen rec. – 33 9 38 - 65 SCA1 ataxin - 1 6 – 44 39 - 83 SCA2 ataxin - 2 13 – 33 32 - 200 SCA3/MJD ataxin - 3 3 – 40 54 - 89 SCA6 CACNA1A 4 – 19 20 - 33 SCA7 ataxin - 7 4 – 35 37 - 306 SCA17 TBP 24 – 44 46 - 63 Bates, et al., Eds. (2002) Huntington's Disease, Oxford University Press

3. Basic physics of aggregation n: denotes the number of peptide molecules in the system (concentration) N: Length of each peptide molecule in the system

4. n* Work done to grow a cluster • In vitro aggregation studies of synthetic polyglutamine peptides • Evidence for nucleation-dependent polymerization • Rates of elongation versus concentration are fit to a pre-equilibrium model • And fits to the model suggests that n*=1 for Q28, Q36, Q47 • See Chen, Ferrone, Wetzel, PNAS, 2002

5. UV-CD data: Q5(-), D2Q15K2(-.-), Q28(…), Q45(---); Chen et al. JMB, 311, 173 (2001) • No major difference between different chain lengths • CD spectra for polyglutamine resemble those of denatured proteins

6. For given N, there is a concentration (n) for which ∆ < 0. Why? • Hypothesis: Water is a poor solvent for polyglutamine: • Chain flexibility and attractions overwhelm chain-solvent interactions • Polymers form internally solvated collapsed globules • Rg and other properties scale with chain length as N0.34 • Most chains aggregate and fall out of solution • CD data and heuristics counter our hypothesis: • For denatured proteins, Rg~ N0.59 - polymers in good solvents • Polyglutamine is polar – suggests that water is a good solvent • Requires new physics to explain polyglutamine aggregation Let’s test our hypothesis

7. MRMD – the “algorithm” • Using a series of “short” simulations, estimate the time scale  over which : • Autocorrelation of “soft” modes decay • There are recurrent transitions between compact and swollen conformations • Use the estimate for , the time scale for each “elementary simulation” is tS~10 • 60-100 independent simulations, each of “length” ts • Pool data from all simulations and construct conformational distributions using bootstrap methods

8. Simulation engine • Forcefield: OPLSAA for peptides and TIP4P for water • Constant pressure (P), constant temperature (T): NPT • T = 298K, P = 1atm • Thermostat and barostat: Berendsen weak coupling • Long-range interactions: Twin range spherical cutoffs • Periodic boundary conditions in boxes that contain > 4000 water molecules • Peptides: ace-(Gln)N-nme, N=5,15,20,… • Cumulative simulation times > 5s • We have an internal control – the excluded volume (EV) limit – to quantify conformational equilibria in good solvents

9. Top row in water, bottom row in EV limit Q5 Q15 Q20 In water EV Limit

10. Scaling of internal distances is consistent with behavior of chain in a poor solvent Q5 Q15 Q20 Data for polyglutamine in EV limit Data for polyglutamine in water

11. Can we test our “prediction”? Yes • Using Fluorescence Correlation Spectroscopy (FCS) • Peptides studied: -Gly-(Gln)N-Cys*-Lys2 • * indicates fluorescent label, which is Alexa488 • Solution conditions: • PBS: pH 7.3, 8.0g NaCl, 0.2g KCl, 1.15g Di-sodium orthophosphate, 0.2g Potassium di-hydrogen orthophosphate, dissolved in pure H2O • Approximately one molecule in beam volume • Is diffusion time, D N0.33 or is ln(D )  0.33ln(N)?

12. Evidence for poor solvent scaling

13. Polyglutamine: Compact albeit disordered Observation of disorder is consistent with CD data

14. Quantifying topology What is the length scale over which spatial correlations decay? Compute <cos(θij)> as a function of |j-i| residue i C  θ i N C i i+1 C  C j j+1 N n residue j

15. Up-down topology for collapsed polyglutamine

16. Q15 Q20

17. Hydrogen bonding patterns

18. Why collapse and what does it mean? • Summary – The ensemble for polyglutamine in water: • Is disordered albeit collapsed • Has a preferred up-down average topology • With a strong propensity for forming beta turns • And little to no long-range backbone hydrogen bonds • What drives collapse in water: Generic backbone? • Is there anything special about polyglutamine? • What does all this mean for nucleation of aggregation?

19. Distributions for polyglycine Water 8M Urea EV Limit Mimics of polypeptide backbones prefer to be collapsed in water, which appears to be a universal poor solvent for polypeptides Polyglutamine is a chain of two types of amides: secondary and primary

20. Primary and secondary amides Propanamide (PPA) N-methylformamide (NMF)

21. Amides in water • Pure (primary or secondary) Amides in water: • N =nW + nA • NPT Simulations with varying nA implies varying A • T=300K, P = 1atm • OPLSAA forcefield for amides, TIP4P for H2O • nA = 16, 32, 64, etc. for 1, 2, 3, … molal solutions; • nW = 800 • Amide (ternary) mixtures: Primary and secondary amides • N = nW + nP + nS • Keep nW and nP fixed and vary nS or nW and nS fixed, vary nP • Will show data for nP = nS = 32

22. Pair correlations • NMF prefers water-separated contacts over hydrogen bonded contacts • PPA prefers hydrogen bonded contacts over water-separated contacts • PPA donor - NMF acceptor hydrogen bonds are preferred in mixtures

23. Cluster statistics

24. Typical large cluster in PPA:NMF mixtures Consistent with data of Eberhardt and Raines, JACS, 1994

25. In polyglutamine, sidechains “solvate” the backbone in compact geometries Q20: Rg=8.86Å,  =0.096 Q20: Rg=8.11Å,  =0.13 Q20: Rg=8.49Å, =0.16

26. Hypothesis – part I: Why is aggregation spontaneous? • For a system of peptides of length N: • There is a finite concentration (n) for which ∆ < 0 • ∆ < 0 if: • Aggregated state of intermolecular solvation via glutamine sidechains is preferred to the disordered state of intramolecular solvation whereby sidechains solvate their own backbones • It is our hypothesis that: • Peptide concentration at which ∆ becomes negative will decrease “rapidly” with increasing chain length

27. Hypothesis – part II: Nucleation • Ensemble of nucleus is species of highest free energy for monomer • Nucleation must involve the following penalties: • DESOLVATION: Replace favorable sidechain-backbone contacts and residual water-backbone contacts with unfavorable backbone-backbone contacts • ENTROPIC BOTTLENECK: Replace disordered ensemble with ordered nucleus • Conformations in the nucleus ensemble? • β-helix-like (see work of Dokholyan group, PLoS, 2005) • -pleated sheet (see work of Daggett group, PNAS, 2005) • Antiparallel β-sheet (see fiber diffraction data)

28. Thanks to… THE LAB • Xiaoling Wang • Andreas Vitalis • Scott Crick • Hoang Tran • Alan Chen • Matthew Wyczalkowski Collaborations • Ron Wetzel – UTK • Murali Jayaraman – UTK • Carl Frieden – WUSTL

29. Ongoing work… • Monomer distributions for N > 25 • Free energies of nucleating intramolecular beta sheets • Influence of sequence context: In vivo, its not just a polyglutamine • Quantitative characterization of oligomer landscape • Generalizations to aggregation of other intrinsically disordered proteins rich in polar amino acids • Experiments: New FCS methods to study oligomers and nucleation kinetics