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