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_at_biomed.wustl.edu
• http//lima.wustl.edu

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Expanded CAG Repeat Diseases and Proteins
DISEASE

GENE PRODUCT
NORMAL CAG
MUTANT CAG

REPEAT RANGE
REPEAT RANGE




Huntingtons
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

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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
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 n1 for Q28,
  Q36, Q47
• See Chen, Ferrone, Wetzel, PNAS, 2002

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UV-CD data Q5(-), D2Q15K2(-.-), Q28(),
Q45(---) Chen et al. JMB, 311, 173 (2001)

1. No major difference between different chain
   lengths
2. CD spectra for polyglutamine resemble those of
   denatured proteins

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For given N, there is a concentration (n) for
which ?? lt 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

Lets test our hypothesis
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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 tS10?
• 60-100 independent simulations, each of length
  ts
• Pool data from all simulations and construct
  conformational distributions using bootstrap
  methods

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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 gt 4000 water molecules
• Peptides ace-(Gln)N-nme, N5,15,20,
• Cumulative simulation times gt 5?s
• We have an internal control the excluded volume
  (EV) limit to quantify conformational
  equilibria in good solvents

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Top row in water, bottom row in EV limit
Q5
Q15
Q20
In water
EV Limit
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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
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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)?

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Evidence for poor solvent scaling
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Polyglutamine Compact albeit disordered
Observation of disorder is consistent with CD data
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Quantifying topology
What is the length scale over which spatial
correlations decay? Compute ltcos(?ij)gt as a
function of j-i
residue i
C
?
?
i
N
C
i
i1
C
?
C
j
j1
N
n
residue j
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Up-down topology for collapsed polyglutamine
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Q15
Q20
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Hydrogen bonding patterns
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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?

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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 Polyglutam
ine is a chain of two types of amides secondary
and primary
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Primary and secondary amides
Propanamide (PPA)
N-methylformamide (NMF)
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Amides in water

• Pure (primary or secondary) Amides in water
• N nW nA
• NPT Simulations with varying nA implies varying
  ?A
• T300K, 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

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Pair correlations

1. NMF prefers water-separated contacts over
   hydrogen bonded contacts
2. PPA prefers hydrogen bonded contacts over
   water-separated contacts
3. PPA donor - NMF acceptor hydrogen bonds are
   preferred in mixtures

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Cluster statistics
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Typical large cluster in PPANMF mixtures
Consistent with data of Eberhardt and Raines,
JACS, 1994
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In polyglutamine, sidechains solvate the
backbone in compact geometries
Q20 Rg8.86Å, ? 0.096
Q20 Rg8.11Å, ? 0.13
Q20 Rg8.49Å, ?0.16
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Hypothesis part I Why is aggregation
spontaneous?

• For a system of peptides of length N
• There is a finite concentration (n) for which ??
  lt 0
• ?? lt 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

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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?
• ß-helix-like (see work of Dokholyan group, PLoS,
  2005)
• ?-pleated sheet (see work of Daggett group, PNAS,
  2005)
• Antiparallel ß-sheet (see fiber diffraction data)

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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

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Ongoing work

1. Monomer distributions for N gt 25
2. Free energies of nucleating intramolecular beta
   sheets
3. Influence of sequence context In vivo, its not
   just a polyglutamine
4. Quantitative characterization of oligomer
   landscape
5. Generalizations to aggregation of other
   intrinsically disordered proteins rich in polar
   amino acids
6. Experiments New FCS methods to study oligomers
   and nucleation kinetics