Transcript Presentation

KINETICS OF THE PRION PROTEIN: STRUCTURE,
MISFOLDING, DISEASE, AND STABILITY
Rhiannon Aguilar
HONR299J
Final Presentation Spring 2014
BACKGROUND ON THE PRION

“Proteinaceous Infectious Particle”


Amyloid disease: visible protein deposits that can be
stained






Stanley Prusiner, 1982
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, righthanded spiral
 β-pleated sheet: parallel
or anti-parallel sheets
with a kinked shape,
connected by a loop

http://www.mun.ca/biology/scarr/MGA2_0318b.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 (108218)

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/nri14
37-f1.jpg
ANIMATION OF “REFOLDING” MODEL

http://learn.genetics.utah.edu/content/molecu
les/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/nsb0901770.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/nsb0901770.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’s
Bottom left: Met129, site of the Val mutation
that is a marker for CJD
http://www.nature.com/nsmb/journal/v8/n9/full/nsb090
1-770.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.pnas.org/content/102/2/315.full
http://www.pearsonhighered.co
m/mathews/ch06/fi6p12.htm
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 attempted
Open circles: 2M, Closed
circles: 4M
4M shows significantly
higher stability
http://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 disease
amyloid may relate to
conformation of amyloid
innoculated

http://www.jbc.org/content/289/5/264
3.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
http://www.plosone.org/article/
info%3Adoi%2F10.1371%2Fjou
rnal.pone.0086320


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