Kinetics of the Prion Protein: Structure, Misfolding , Disease, and Stability

1. Rhiannon Aguilar HONR299J Final Presentation Spring 2014 Kinetics of the Prion Protein: Structure, Misfolding, Disease, and Stability

2. “Proteinaceous Infectious Particle” • Stanley Prusiner, 1982 • Amyloid disease: visible protein deposits that can be stained • Plaques found in 10% of CJD, higher percentage in other TSEs • Two distinct forms, PrP-C and PrR-res • Membrane-bound protein, 254 amino acids, 2 glycosylation sites • Conserved between species, but with slight changes resulting in a disease species barrier • PrP 27-30, fragment created by digestion, can form amyloid • Highly expressed in CNS, lymphatic tissue Background on the Prion

3. α-Helix: 3.6 amino acids/turn, right-handed spiral β-pleated sheet: parallel or anti-parallel sheets with a kinked shape, connected by a loop Helices and Pleated Sheets http://www.mun.ca/biology/scarr/MGA2_03-18b.html

4. Cellular PrP Lots of alpha helices Point mutations can cause slight changes in structure that make misfolding favorable Biologically interesting fragment: 108-218 Prion Structure Huang, Prusiner, and Cohen 1996

5. Predominantly beta-pleated sheets Presumably, this structure is more likely to form aggregates Same biologically interesting fragment (108-218) Misfolded Structure Huang, Prusiner, and Cohen 1996

6. Refolding: Conversion is very slow normally • Misfolded protein acts as enzyme to re-fold normal • Seeding: Conversion is in constant equilibrium • Seeds form when Sc form accumulates, prevents return to normal state Disease Mechanism: refolding vs Seeding http://www.nature.com/nri/journal/v4/n9/images/nri1437-f1.jpg

7. http://learn.genetics.utah.edu/content/molecules/prions/ • (Slide 7) Animation of “refolding” model

8. PrP can form a covalent dimer Third helix swaps position to form a covalent bond with a second molecule Forms a β-sheet at the interface Possibly a precursor to aggregation in disease PrPDimerization http://www.nature.com/nsmb/journal/v8/n9/full/nsb0901-770.html

9. Top: Green/Pink are the two halves of the dimer, Blue is the monomer superimposed Bottom: Left is the two halves of the dimer, pulled apart, and right is two monomers More Pictures of Dimerization http://www.nature.com/nsmb/journal/v8/n9/full/nsb0901-770.html

10. 17 amino acids present in familiar SE’s are located on the flipped helix Mutations may make this flipping easier, facilitate protein conformational change Covalent dimers present in hamster scrapie brains Formation of new covalent linkages = protein unfolding/refolding Significance of this Dimerization? Red: Amino acids mutated in familial SE’s Bottom left: Met129, site of the Val mutation that is a marker for CJD http://www.nature.com/nsmb/journal/v8/n9/full/nsb0901-770.html

11. Fast-folding • Easily folds incorrectly • Has mutations which perturb folding but do not change stability • Important “nucleus” located between helices 2 and 3 • (3rd helix is moved in dimer formation) Folding Kinetics

12. Folded + GnHCL Unfolded Plot relates to reverse reaction (Unfolded  Folded) Native protein is most stable at ~285K (11.85°C) Kinetics: Effect of Temperature on Stability http://www.pnas.org/content/106/14/5651.full

13. J. Biol. Chem. (2002) • Mutate Trp to Phe gives fluorescence to folded protein • Experiments done at 5°C b/c too fast at 25°C • Results: Prion folds/unfolds with a kinetic intermediate • First conclusive evidence for a folding intermediate • Prev. results say that mouse PrP does not have an intermediate • Possible reason for species barrier? Kinetics: Folding of Native PrP

14. Folding/unfolding goes through a partially-folded intermediate state • In most familial mutant versions, the intermediate is extra stable • Intermediate is highly stabilized by 7/9 mutations • The intermediate is likely to aggregate • Native protein needs PrP-res seed, but maybe mutant intermediate states can aggregate on their own? Kinetics: Effect of Mutations http://www.jbc.org/content/279/17/18008.long

15. Amyloid analogue synthesized: KFFEAAAKKFFE  Aromatic pi-pi stacking (phenylalanine) Charge attraction Β-sheet interactions similar to silk Stability of Aggregates: Amyloid Stability http://www.pnas.org/content/102/2/315.full http://www.pearsonhighered.com/mathews/ch06/fi6p12.htm

16. Differences in structure at aggregate core results in differential stability • Less stable = shorter incubation time (Prusiner) • Synthesize PrP aggregates in two conditions: 2M GnHCl and 4M GnHCl • Produces 2 different stabilities when denaturation is attempted Structural Stability of Prion Aggregates Open circles: 2M, Closed circles: 4M 4M shows significantly higher stability http://www.jbc.org/content/289/5/2643.long

17. Differential stability seems to only relate to packing arrangement, not the protein secondary structure Tighter packing = protease resistance? Stability of disease amyloid may relate to conformation of amyloid innoculated Structural Stability of Prion Aggregates http://www.jbc.org/content/289/5/2643.long

18. Thermal decomposition results in loss of mass and release of gas • Occurs at lower temperature for native molecule than for amyloid Thermodynamic Stability: Studies of Insulin Amyloid http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0086320

19. Incubation at increasing temperatures decomposes fibril structure • Incubation at 100°C shows little effect on structure (if anything, may be more stable?) • Might be unfolding/refolding to a more stable structure? • Autoclaves at 120-130°C not sufficient! • Higher temperatures seem very effective Thermodynamic Stability: Studies of Insulin Amyloid http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0086320