KINETICS OF THE PRION PROTEIN: STRUCTURE, MISFOLDING, DISEASE, AND STABILITY
Rhiannon AguilarHONR299JFinal Presentation Spring 2014
BACKGROUND ON THE PRION “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
HELICES AND PLEATED SHEETS 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
http://www.mun.ca/biology/scarr/MGA2_03-18b.html
PRION STRUCTURE Cellular PrP Lots of alpha helices Point mutations can
cause slight changes in structure that make misfolding favorable
Biologically interesting fragment: 108-218
Huang, Prusiner, and Cohen 1996
MISFOLDED STRUCTURE Predominantly beta-
pleated sheets Presumably, this
structure is more likely to form aggregates
Same biologically interesting fragment (108-218)
Huang, Prusiner, and Cohen 1996
DISEASE MECHANISM: REFOLDING VS SEEDING 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
http://www.nature.com/nri/journal/v4/n9/images/nri1437-f1.jpg
ANIMATION OF “REFOLDING” MODEL http://learn.genetics.utah.edu/content/
molecules/prions/ (Slide 7)
PRP DIMERIZATION 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
http://www.nature.com/nsmb/journal/v8/n9/full/nsb0901-770.html
MORE PICTURES OF DIMERIZATION 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
http://www.nature.com/nsmb/journal/v8/n9/full/nsb0901-770.html
SIGNIFICANCE OF THIS DIMERIZATION? 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’sBottom left: Met129, site of the Val mutation that is a marker for CJDhttp://www.nature.com/nsmb/journal/v8/n9/full/nsb0901-770.html
FOLDING KINETICS 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)
KINETICS: EFFECT OF TEMPERATURE ON STABILITY
Folded + GnHCL Unfolded
Plot relates to reverse reaction (Unfolded Folded)
Native protein is most stable at ~285K (11.85°C)
http://www.pnas.org/content/106/14/5651.full
KINETICS: FOLDING OF NATIVE PRP 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: EFFECT OF MUTATIONS 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?
http://www.jbc.org/content/279/17/18008.long
STABILITY OF AGGREGATES: AMYLOID STABILITY Amyloid analogue synthesized: KFFEAAAKKFFE Aromatic pi-pi stacking (phenylalanine) Charge attraction Β-sheet interactions similar to silk
http://www.pearsonhighered.com/mathews/ch06/fi6p12.htm
http://www.pnas.org/content/102/2/315.full
STRUCTURAL STABILITY OF PRION AGGREGATES 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 attemptedOpen circles: 2M, Closed circles: 4M4M shows significantly higher stabilityhttp://www.jbc.org/content/289/5/2643.long
STRUCTURAL STABILITY OF PRION AGGREGATES
Differential stability seems to only relate to packing arrangement, not the protein secondary structure
Tighter packing = protease resistance?
Stability of diseaseamyloid may relate toconformation of amyloidinnoculated
http://www.jbc.org/content/289/5/2643.long
THERMODYNAMIC STABILITY: STUDIES OF INSULIN AMYLOID
Thermal decomposition results in loss of mass and release of gas Occurs at lower
temperature for native molecule than for amyloid
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0086320
THERMODYNAMIC STABILITY: STUDIES OF INSULIN AMYLOID
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
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0086320