MINIREVIEW
Branched chain mechanism of polymerization and
ultrastructure of prion protein amyloid fibrils
Ilia V. Baskakov
1,2
1 Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD, USA
2 Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA
Prion diseases are a group of fatal neurodegenerative
maladies that can arise spontaneously or be inherited,
and that can also be infectious [1]. Despite enormous
investments over the last 30 years in searching for a
prion virus or virion [2–5], no prion-specific nucleic
acids associated with infectious prion particles have
ever been identified [6]. A notable shift has occurred in
the last few years from debating the question of whe-
ther a protein can be infectious to what makes a pro-
tein infectious and how many proteins are infectious
[7–9]. Elucidating the polymerization mechanisms and
structure of misfolded and aggregated isoforms of the
prion protein (PrP) will help solving these long-stand-
ing research problems.
Prion polymerization is a branched-
chain reaction
To model prion conversion, two kinetic models has
been exploited: the nucleation-polymerization [10] and
the template assisted [11]. These models have been
previously discussed in numerous review articles
[12–14] and therefore will not be presented here.
Although these two models have played an important
role in the evolution of our ideas regarding the
mechanism of prion conversion, neither of them

gress has been made in elucidating the mechanisms of polymerization for
several amyloidogenic proteins and peptides linked to conformational dis-
orders and solving their fibrillar 3D structures, studies of prion protein
amyloid fibrils and their polymerization mechanism have proven to be very
difficult. The present minireview introduces the mechanism of branched-
chain reaction for describing the peculiar kinetics of prion polymerization
and summarizes our current knowledge about the substructure of prion
protein amyloid fibrils.
Abbreviations
AFM, atomic force microscopy; GdnHCl, guanidine hydrochloride; PK, proteinase K; PrP, prion protein; rPrP, recombinant prion protein.
3756 FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS
consistent with the mechanism of branched-chain
reactions.
Employing the theory of branched-chain reactions
will greatly benefit our understanding of the prion rep-
lication mechanism. The first branched-chain processes
were described at the beginning of twentieth century
and the branched-chain theory was developed shortly
afterward in the 1920s by Nikolay Semenov [17].
Although this theory had enormous impact on the
developing chemical industry and nuclear sources of
energy, the Nobel Prize for this amazing discovery was
not awarded until 1956, almost half a century later
[18]. A number of odd features including a strong
dependence of the reaction rate on the volume or the
shape of reaction vessel, the presence of a lag-phase,
threshold effects and a strong dependence of the reac-
tion rate on microimpurities observed for this type of
reactions raised serious cautions and even jokes among
conventional chemists. It took more than 30 years for

tion to decay mode or to auto-acceleration mode. The
branched-chain reactions have been known to be
unusually ‘sensitive’ to slight changes in experimental
parameters that might be seen as stochastic behavior,
in which the reaction follows the ‘all or nothing’ rule.
It is important to indicate that the branched-chain
mechanism is consistent with the sigmoidal kinetics of
fibrillation, which has been previously referred to as
‘nucleation-elongation’ kinetics (Fig. 2). According to
the nucleation-polymerization model, the lag-phase in
the fibrillation process corresponds to the nucleation
step, a stage when mature fibrils are not yet formed
(Fig. 2A). By contrast to this prediction, we found that
mature fibril were present at the lag-phase of rPrP
fibrillation [16,19]. This observation is consistent with
the branched-chain mechanism that attributes the lack
of an observable signal during the second part of the
lag-phase to the limitations in detecting small amounts
of the final reaction products (i.e. in this case, fibrils)
(Fig. 2B). As soon as the final reaction products are
formed even in miniscule amounts, the reaction rate
is accelerated due to the branched-chain mechanism
of multiplication of active centers. Therefore, in a
Branched chain reactions are
similar to autocatalytic processes
(multiplication
coefficient)
probability of formation of new active centers
probability of loss of active centers
r

the length of the lag-phase and polymerization rate of
PrP fibrillation reactions that were carried out under
identical solvent conditions, but subjected to different
fragmentation intensities (O. V. Bocharova & I. V.
Baskakov, unpublished results).
The mechanism of the branched-chain reaction pre-
dicts three potential outcomes for prion disease.
Depending on the dynamic balance between the rate of
multiplication versus clearance, prion disease could:
(a) progress very quickly to the clinical form (if >>1,
the kinetics of PrP
Sc
(Sc-scrapie) accumulation follow
the formal mechanism of branched-chain reactions);
(b) develop very slowly and exist at subclinical level
for a long period of time (r ¼ 1, the kinetics of PrP
Sc
formation follow the formal mechanism of enzyme cata-
lysis), or (c) never progress (r < 1, PrP
Sc
is cleared, the
rate of clearance follow apparent first order kinetics). It
has been shown that the concentration of PrP
Sc
in the
brain of experimental animals drops substantially in the
first week after intracerebral inoculation [20,21], indica-
ting that the rate of clearance may exceed the rate of
multiplication during the initial stage of prion transmis-
sion. Despite substantial resistance to proteolytic diges-

amounts (approximately 20%) of PrP
C
supplied to the
reaction mixtures were converted into the PrP-res form
despite a 50-fold molar excess of PrP
Sc
used as a seed.
In subsequent studies, unlimited amplification of PrP
Sc
was achieved in the conversion reactions referred
to as misfolding cyclic amplification by introducing
repetitive cycles of elongation and fragmentation,
ThT lluorescence
The branched chain mechanism
nucle
-ation
elongation and
fragmentation
Time
Time
A
B
C
ThT lluorescence
nucleation
elongation
The nucleation-polymerization model
Time
r >>>1
r >>1

tion of active PrP
Sc
centers? Multiple effects may
contribute to the clearance of PrP
Sc
: strain-specific
intrinsic stability of PrP
Sc
[31,32]; species and tissue-
specific variations in proteolytic activity [33,34];
interactions of PrP
Sc
with cellular cofactors such as
glycosaminoglycans [35–37] or polysaccharides [38]
that stabilize PrP
Sc
. Removal of active PrP
Sc
centers
could also occur due to aggregation of PrP
Sc
into large
plaques or oxidative modification of amino acid resi-
dues on the PrP
Sc
surface that are involved in prion
replication. Our previous studies revealed that sorption
of self-propagating amyloid fibrils to walls of reaction
vessels may account for deactivation of active seeds
in vitro, resulting in dramatic volume-dependent

PrP
Sc
fibrils should control the rate of multiplication.
It is important to note that the fibril elongation does
not result in multiplication of the active or catalytic
centers, unless fibril fragmentation occurs (Fig. 1). Cel-
lular chaperones were found to be involved in frag-
mentation of yeast prion fibrils [43]. Cellular cofactors
participating in fragmentation of mammalian prion
fibrils have yet to be identified. The intrinsic fragility
(i.e. the ability of fibrils to fragment into shorter
pieces) seems to be controlled by the conformational
stability of amyloid fibrils and, specifically, by the
stability of the cross-b-fibrillar structure [8] (Y. Sun &
I. V. Baskakov, unpublished data). Recent studies have
revealed a strong link between conformational stability
and the intrinsic infectivity of fibrils formed by the
yeast prion protein Sup35 [44]. The amyloid fibrils that
displayed low conformational stability exhibited a high
efficiency of infection with the large majority of colon-
ies showing a strong phenotype. Vice versa, fibrils that
had high conformational stability displayed low infec-
tivity and produced ‘weak’ strains that disappeared
fast or that could be easily cured. A similar correlation
between conformational stability and infectivity was
observed for synthetic mammalian prions [45,46]. Both
yeast and mammalian prion studies indicated that the
intrinsic infectivity of fibrils might be controlled, at
least in part, by the conformational stability of the
cross-b-sheet core, an unexpected lesson that we have

microscopy. Light microscopy has been utilized histor-
ically for neuropathological studies and used often for
classification of prion aggregates. Using light micro-
scopy only, it is easy to confuse nonfibrillar oligomers
I. V. Baskakov Branched chain mechanism of polymerization
FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS 3759
with small fibrillar fragments (Fig. 3). In fact, the size
distribution of fibrils is very broad and, at any given
time, includes very small or short fibrillar fragments.
Short fibrils or their fragments can be generated at the
initial stages of fibril elongation, but also produced as
a result of fibril fragmentation. In addition to small
C
A
B
Fig. 3. Fluorescence and electron microscopy of rPrP amyloid fibrils. Amyloid fibrils were produced as described by Bocharova et al. [55] and
(A) stored in Na-acetate buffer, pH 5.5; (B) stored in Na-acetate buffer, pH 5.5, and sonicated for 1 min prior to imaging; and (C) stored in
Tris ⁄ HCl buffer, pH 7.4. All three samples were analyzed in parallel by thioflavine T-fluorescence microscopy (left panels) and by electron
microscopy (right panels). When observed by fluorescence microscopy, the fibrils subjected to 1 min of sonication (B) appeared as small
nonfibrillar oligomers. (A,B) Scale bars ¼ 1 lm; (C) scale bar ¼ 10 lm.
Branched chain mechanism of polymerization I. V. Baskakov
3760 FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS
fragments, fibrils might form aggregates of various
shapes and densities (Fig. 3). Although fibrillar aggre-
gates or plaques are believed to be less pathogenic,
they might serve as repositories of more pathogenic
small fibrillar fragments and therefore are equally
important. Regardless of the fibril size and shape, the
key feature of fibrils is cross- b-sheet structure, which is
essential for the prion self-propagating activity. More-

the highly aggregated, heterogeneous and insoluble
nature of PrP fibrils precluded application of NMR
and other high-resolution techniques. In the absence of
methods to solve structure of PrP fibrils in the near
future, we employed several alternative approaches for
elucidating ultrastructure of fibrils. High resolution
atomic force microscopy revealed that fibrils produced
in vitro from the full-length rPrP consisted of several
laterally assembled filaments [52]. In our recent studies,
we found that the fibrils produced under single growth
conditions varied with respect to the number of consti-
tutive filaments and the manner in which the filaments
were assembled. The high-order fibrils formed through
a highly hierarchical mechanism of lateral assembly.
At each step, filaments were found to associate in pairs
in a pattern resembling dichotomous coalescence
(Fig. 4) [19,52]. Because of alternative modes of lateral
assembly, fibrils produced under a single growth condi-
tion were heterogeneous with respect to the width,
height and twisting morphology.
How many PrP molecules are packed per 1 nm
within an amyloid fibril? As judged from atomic force
microscopy (AFM) measurements and atomic volume
calculations, a single full-length rPrP polypeptide occu-
pied a distance of approximately 1.2 nm within a
single filament (Fig. 5A) [52]. The amyloid fibrils are
Dichotomous mechanism
of lateral assembly
Width (nm)
20 40 60 80

revealed that the PK resistant core of the amyloid
fibrils consisted of residues 138 ⁄ 141–230, 152 ⁄ 153–230
and 162–230, where the fragment 162–230 was the
most resistant to PK digestion (Fig. 5) [53,54]. Upon
treatment with PK, the 152 ⁄ 153–230 and 162–230
PK-resistant fragments maintained fibrillar structure
and preserved a high b-sheet context with strong inter-
molecular hydrogen bonds. Remarkably, the b-sheet
rich fibrillar cores encompassed by residues 152 ⁄ 153–
230 and 162–230 were found to maintain high seeding
activity in vitro despite cleavage of the N-terminal and
central regions [53,55]. Consistent with these studies,
the rPrP regions 159–174 and 224–230 were observed
to be buried in the fibril interior and were the most
resistant to GdnHCl-induced denaturation as judged
from the newly developed dual color immunofluores-
Fig. 5. (A) Three-dimensional AFM image of amyloid fibril. The fibril consists of several filaments assembled laterally in horizontal and vertical
dimensions as seen by a stepwise increase in fibrillar height. Although atomic volume calculations predicted that single PrP molecule occu-
pies the distance of approximately 1.2 nm (52), the precise 3D structure of PrP within amyloid fibrils has yet to be determined. Despite
changes in the shape of the PrP molecule upon conversion from the native a-helical form (inset) into the fibrillar form, the atomic volume
occupied by a single PrP polypeptide chain does not change substantially. (B) Schematic diagram illustrating mapping of PrP regions within
amyloid fibrils. The PK-resistant b-sheet rich core of amyloid fibrils composed of residues 152–230 and 162–230; PK-cleavage sites are
indicated by red arrows. Based on data from [55]. The epitopes to antibodies AH6 and R1 were solvent unaccessible and were the most
resistant to GdnHCl-induced denaturation (highlighted in red); the epitope to antibody D18 was found to be cryptic under native conditions
and solvent exposed under partially denaturing conditions (highlighted in orange), whereas the epitopes to antibodies D13 and AG4 were
solvent-accessible regardless of the solvent conditions (highlighted in green); based on data from [56]. Residues 98, 127, 144, 196 and 230
(blue) showed cooperative unfolding, whereas unfolding at residue 88 (green) was noncooperative; based on data from [58].
Branched chain mechanism of polymerization I. V. Baskakov
3762 FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS
cence microscopy assay (Fig. 5) [56]. The 132–156

infectious particle has a
substructure similar to that of rPrP fibrils generated
in vitro remains to be determined in future studies.
Acknowledgements
I.V.B. is supported by a National Institute of Health
grant, NS045585.
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