Rhiannon Aguilar HONR299J Final Presentation Spring 2014

 “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

 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 18b.html

 Cellular PrP  Lots of alpha helices  Point mutations can cause slight changes in structure that make misfolding favorable  Biologically interesting fragment: Huang, Prusiner, and Cohen 1996

 Predominantly beta-pleated sheets  Presumably, this structure is more likely to form aggregates  Same biologically interesting fragment ( ) Huang, Prusiner, and Cohen 1996

 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 37-f1.jpg

 les/prions/ les/prions/  (Slide 7)

 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 html

 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 html

 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 Red: Amino acids mutated in familial SE’s Bottom left: Met129, site of the Val mutation that is a marker for CJD html

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

 Folded + GnHCL  Unfolded  Plot relates to reverse reaction (Unfolded  Folded)  Native protein is most stable at ~285K (11.85°C)

 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?

 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?

 Amyloid analogue synthesized: KFFEAAAKKFFE  Aromatic pi-pi stacking (phenylalanine)  Charge attraction  Β-sheet interactions similar to silk m/mathews/ch06/fi6p12.htm

 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 Open circles: 2M, Closed circles: 4M 4M shows significantly higher stability 5/2643.long

 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 3.long

 Thermal decomposition results in loss of mass and release of gas  Occurs at lower temperature for native molecule than for amyloid 2F %2Fjournal.pone

 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 °C not sufficient!  Higher temperatures seem very effective info%3Adoi%2F %2Fjou rnal.pone