Phase Behavior of ProteinPrion Conversion and Aggregation

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Phase Behavior of Protein-Prion Conversion and
Aggregation
Committee Members William
Gelbart Tom Chou Charles Knobler Giovanni
Zocchi Joseph Loo

• Li Tai Fang
• Department of Chemistry and Biochemistry
• University of California, Los Angeles

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What Are Prions

• cause of transmissible spongiform
  encephalopathies (TSE)
• rogue conformers (PrPSc) of the cellular proteins
  (PrPC)

Native Conformer
Misfolded Conformer
Picture Credit Fred E. Cohen
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Properties of Prions

• Protein-only Hypothesis
• Seeding Model
• Prions aggregate
• Highly hydrophobic

Weismann C, Nature Review Microbiology, 2,
861-871, (2004)
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Prion Phenomenon Wide Spread?

• Prion-like proteins discovered in
• Yeast
• Sup35
• Ure2
• Fungi
• Het-S

Electron Micrograph of Yeast Sup35N amyloid King
CY et al, PNAS, 94, 6618-6622, (1997)
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Motivation

• Rare
• vCJD occurs one in a million
• Sup35 prion occurs one in 1-10 million
• Incurable
• Irreversible
• Transmissible
• Kuru
• Mad cow disease

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Research Proposal

• Ising Model
• Prion model with mathematical formalism
• Experiments using yeast prions
• manipulating the protein-prion equilibrium by
  adjusting
• Strength of hydrophobic interaction
• Relative stability of the monomeric proteins

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Ising Model

• N lattice sites, each with 2 possible values
• e.g. 0 or 1
• For the simplest case, only the nearest-neighbors
  interact
• Phase transition in 2-D or higher
• Monte Carlo solution using Metropolis method
• Try to change one thing at a time, and see if the
  change got the system closer to equilibrium

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Ising Model

• 3-D cubic lattice setup
• 0 native or anything
• 1 prion
• field strength ? F
• coupling constant ? -J
• Total Energy
• Change in Energy

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Metropolis Method
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Metropolis Method

• Randomly select one particle

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Metropolis Method

• Randomly select one particle

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Metropolis Method

• Randomly select one particle
• Switch the Conformation

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Metropolis Method

• Randomly select one particle
• Switch the Conformation

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Metropolis Method

• Randomly select one particle
• Switch the Conformation
• The switch will be accepted if
• ?E lt 0, or
• e-?E a random
• iterate many cycles

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Metropolis Method

• Equilibrium configuration
• 1 wants to be next to 1
• 1 does not want to be alone
• Transition from 0 dominated to 1-dominated
  systems
• Phase diagram with respect to F and J .

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Ising Phase Diagram
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Experimental Work
Klug, A. et al, Nature, 229, 37-42, (1971)

• Phase diagram tobacco mosaic virus capsid
  protein self-assembly
• not a conventional phase-diagram

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Phase Behavior

• Phase diagram for prion formation?
• Thermodynamic process
• Reversible
• Experimental method
• distinguish native and prion conformers in
  solution

Hypothetical Protein-Prion Phase Diagram
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Circular Dichroism

• Chiral molecules absorb left- and right-
  circularly polarized light differently
• Light comes out elliptic (CD) and rotated (ORD)
• Widely used to estimate protein secondary
  structures

Animations adapted from András Szilágyi URL
http//www.enzim.hu/szia/cddemo/
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Circular Dichroism

• CD at far UV range is sensitive to protein
  secondary structures
• Native proteins and prions differ in secondary
  structures

CD spectra of native (black) and prion (white)
conformers of yeast Sup35N King CY et al, PNAS,
94, 6618-22, (1997)
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Protein Concentration

• Micelle formation
• monomers N ? micelles
• Prion formation
• PrPC N ? PrPScN
• critical prion concentration

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Critical Aggregation Concentration

• Phase-transition-like
• sharp transition
• thermodynamic behavior
• Not a true phase-transition
• CMC is a geometric argument
• Slope is continuous

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Hydrophobicity of Prions

• Strength of hydrophobic interaction
• increases with temperature
• increases with ionic strength

Graph plotted with data from Ben-Naim, A. et al.,
J. of Phy. Chem., 78, 170-175, (1973)
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Ionic Strength

• Ionic strength increases the strength of
  hydrophobic interaction
• Lowers the critical prion concentration?

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Temperature

• Hydrophobic interaction increases with
  temperature
• Prusiner et al. kept his prion fraction at 4oC to
  prevent aggregation

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pH relative stability
Native Conformer
Misfolded Conformer

• Alters the chemical properties of the amino acid
  residues
• Alters the amino acid interactions
• Alters the relative stability

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pH relative stability
Native Conformer
Misfolded Conformer

• Alters the chemical properties of the amino acid
  residues
• Alters the amino acid interactions
• Alters the relative stability

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Experiments and Ising Model

• Experimental phase diagram vs.
  Theoretical phase diagram

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Concluding Remarks

• Experimentally, can you change the favored state
  by changing environment?
• Is Ising model a good model
• Phase behavior captured?
• Learn the physics of prion formation

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Acknowledgement
Committee

• Charles Knobler
• William Gelbart
• Joseph Loo
• Giovanni Zocchi
• Tom Chou

Dept. of Chemistry and Biochemistry Dept. of
Chemistry and Biochemistry Dept. of Chemistry and
Biochemistry Dept. of Physics and Astronomy Dept.
of Biomathematics

• Ajay Gopal
• Yufang Hu
• Ajay Gopal, Aron Yoffe, Glenna Sowa, Odisse
  Azizgolshani

Written proposal
Oral Presentation