Poly l-alanine-dependent fibril formation was studied using the recombinant N-terminal domain of PABPN1.. In the case of the protein fragment with the expanded poly l-alanine sequence [N-
Trang 1extension of seven alanines on the fibrillation properties
of the N-terminal domain of PABPN1
Grit Lodderstedt1, Simone Hess2, Gerd Hause3, Till Scheuermann1,*, Thomas Scheibel2
and Elisabeth Schwarz1
1 Institut fu¨r Biotechnologie, Martin-Luther-Universita¨t Halle-Wittenberg, Halle, Germany
2 Technische Universita¨t Mu¨nchen, Garching, Germany
3 Biozentrum der Martin-Luther-Universita¨t Halle-Wittenberg, Halle, Germany
Protein folding to a conformation distinct from the
native fold gives rise to a wide range of diseases The
most well-known examples are the spongiform
encep-halopathies, Alzheimer’s, Parkinson’s and
Hunting-ton’s diseases [1–4] These various disorders have all
been traced to individual proteins that undergo
alter-native folding to a conformation, the characteristic
feature of which is a b-cross structure formed by
b-strands lying perpendicular to the fibril axis [5]
However, the molecular processes that either directly
or indirectly cause these highly fatal illnesses are still under debate
Huntington’s disease is one of the most prominent examples of neurodegenerative diseases that are caused
by trinucleotide expansions of CAG repeats and thus
an expansion of a run of glutamine residues [3] In the most extreme cases, expansions of up to 180 glutamines have been described Besides Huntington’s disease and
Keywords
AFM; alanine expansions; amyloid-like;
kinetics of fibril formation; OPMD
Correspondence
E Schwartz, Institut fu¨r Biotechnologie,
Martin-Luther-Universita¨t Halle-Wittenberg,
Kurt-Mothes-Str 3, 06120 Halle, Germany
Fax: +49 345 55 27 013
Tel +49 345 55 24 856
E-mail: Elisabeth.Schwarz@biochemtech.
uni-halle.de
*Present address
Roche Diagnostics GmbH, Penzberg,
Germany
(Received 12 September 2006, revised
2 November 2006, accepted 8 November
2006)
doi:10.1111/j.1742-4658.2006.05595.x
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease that usually manifests itself within the fifth decade The most prom-inent symptoms are progressive ptosis, dysphagia, and proximal limb mus-cle weakness The disorder is caused by trinumus-cleotide (GCG) expansions in the N-terminal part of the poly(A)-binding protein 1 (PABPN1) that result
in the extension of a 10-alanine segment by up to seven more alanines In patients, biopsy material displays intranuclear inclusions consisting primar-ily of PABPN1 Poly l-alanine-dependent fibril formation was studied using the recombinant N-terminal domain of PABPN1 In the case of the protein fragment with the expanded poly l-alanine sequence [N-(+7)Ala], fibril for-mation could be induced by low amounts of fragmented fibrils serving as seeds Besides homologous seeds, seeds derived from fibrils of the wild-type fragment (N-WT) also accelerated fibril formation of N-(+7)Ala in a centration-dependent manner Seed-induced fibrillation of N-WT was con-siderably slower than that of N-(+7)Ala Using atomic force microscopy, differences in fibril morphologies between N-WT and N-(+7)Ala were detected Furthermore, fibrils of N-WT showed a lower resistance against solubilization with the chaotropic agent guanidinium thiocyanate than those from N-(+7)Ala Our data clearly reveal biophysical differences between fibrils of the two variants that are likely caused by divergent fibril struc-tures
Abbreviations
AFM, atomic force microscopy; ANS, 8-anilinonaphthalene-1-sulfonate; EM, electron microscopy; OPMD, oculopharyngeal muscular dystrophy; PABPN1, poly(A)-binding protein nuclear; ThT, thioflavine T.
Trang 2several other poly l-glutamine-linked diseases,
exten-sions of poly l-alanine stretches have also been reported
as a cause of congenital disorders However, in contrast
to the massive extensions seen in poly
l-glutamine-based disorders, more than 30 consecutive alanines are
rarely observed upon mutation of GCG repeats in
poly l-alanine-caused illnesses [6] The sensitivity of
protein folds towards poly l-alanine extensions is
evi-dent from genetic analyses, which have revealed that
two additional alanine residues in poly(A)-binding
pro-tein nuclear (PABPN1) are sufficient to elicit dominant
effects [7] These findings have been confirmed using an
animal model for oculopharyngeal muscular dystrophy
(OPMD), in which hemizygous transgenic mice with
three additional alanine residues in PABPN1 displayed
myopathic changes [8]
PABPN1 [nuclear poly(A)-binding protein,
previ-ously, PABP2] is involved in mRNA processing in the
cell nucleus [9,10] Together with cleavage and
poly-adenylation specificity factor, PABPN1 induces
proces-sivity of poly(A)polymerase and controls the length of
poly adenine tails [11–13] PABPN1 is a 306 amino
acid protein with oppositely charged N- and
C-ter-minal domains An RNP-type RNA-binding domain,
which lies in the middle of the protein, is preceded by
an a-helical segment [13,14] The poly l-alanine
exten-sions affect the N-terminal fragment of the protein,
which comprises 125 amino acids In the wild-type
protein, the sequence (Ala)10Gly(Ala)2 follows the
start methionine In OPMD patients, this natural
poly l-alanine sequence is extended by up to seven
additional alanine residues yielding a total of 17
ala-nines in the most extreme case [7] Biochemical
analyses of PABPN1 with extended poly l-alanine
sequences showed that the protein’s activity in poly
adenylation is not affected (B Schulz and E Wahle,
personal communication) Histochemical analysis of
biopsy material from OPMD patients revealed fibrillar
aggregates in muscle fiber nuclei with PABPN1 as a
major constituent [15,16]
The occurrence of aggregates has been confirmed in
both yeast- and cell-culture models of OPMD [17–22] It
is not clear, however, whether the observed aggregates
in the model systems represent amyloid-like deposits
Irrespective of the nature of the aggregates (amorphous
or regular b-cross structures), reduction of aggregate
formation by chemical and⁄ or molecular chaperones
has been shown to reduce cytotoxicity both in cell
cul-ture [17,18,22,23] and in animal models [19,24]
How-ever, the mere fact that no correlation between the
frequency of the inclusions and the severity of the
dis-ease can be observed [25], shows that the molecular
pro-cess(es) that elicits OPMD is to date unknown
We showed previously that recombinant full-length PABPN1 tends to form amorphous aggregates in vitro [26] The formation of amorphous aggregates was inde-pendent of the presence or length of the poly l-alanine sequence In contrast, no amorphous aggregates were observed with the N-terminal domain of PABPN1 The N-terminal fragment of wild-type and the variant carry-ing the most extreme extension observed in man (seven additional alanine residues) formed fibrillar structures with a lag phase that was considerably shorter in the case of the variant with the poly l-alanine extension [26] In this work, we further compare these two N-ter-minal fragments Differences on the level of fibril for-mation kinetics and seeding capacity are observed Furthermore, the two variants also differ in their fibril morphologies and stabilities of the fibrils against solu-bilization
Results Seeding of fibril growth of PABPN1 N-terminal fragment variants
Previous analyses of poly l-alanine-dependent fibril for-mation of PABPN1 have revealed that the full-length protein readily forms amorphous aggregates [26] Although fibril formation also occurred with full-length PABPN1 upon storage (data not shown), the simulta-neous presence of both amorphous aggregates and fibrils hampered the analysis of fibril formation kinetics For this reason, fibril formation was analyzed with the N-terminal domain of PABPN1 consisting of amino acids 1–125 of the wild-type protein (N-WT) Because the aim of this study was to investigate the effect on the fibrillation properties of the most extreme disease-asso-ciated extension of seven additional alanines, fibrillation kinetics and fibril properties of N-WT were compared with those of the corresponding fragment carrying seven additional alanines (N-(+7)Ala) Both proteins were recombinantly produced in Escherichia coli cells and purified as published previously [26]
Fibrillation kinetics were first followed via fluores-cence measurements with thioflavine T (ThT), a dye routinely used to monitor fibril formation [27] How-ever, to obtain fluorescence signals of sufficient ampli-tude, protein concentrations > 10 lm had to be used
We have previously reported unusual tinctorial features
of fibrils of the N-terminal domain of PABPN1 [26] In addition, poor staining of fibrils formed by poly alanine peptides with ThT and resilience against staining with Congo Red were observed by Shinchuk et al [28] Thus, we measured fibril-induced changes of 8-anilino-naphthalene-1-sulfonate (ANS) fluorescence, a method
Trang 3that has been described for monitoring fibril formation,
e.g of the NM domain of the yeast prion protein
Sup35p [29] and also for the detection of a-synuclein
fibrils [30] In fact, ANS signals showed a linear
corre-lation with the concentration of fibrils of N-(+7)Ala in
a range from 1 to 14 lm, whereas no ANS binding was
observed with the monomeric protein (data not shown)
Because ANS fluorescence measurements allowed a
more sensitive quantification of fibrils than with ThT
(data not shown), this spectroscopic detection method
was employed throughout this study
When the kinetics of N-(+7)Ala fibril formation
were monitored in the absence of seeds, fibril
forma-tion started after a lag phase of 10 days (Fig 1A)
In contrast, addition of seeds resulted in an immediate increase in ANS fluorescence as expected As a control, the depletion of the soluble monomeric species was fol-lowed by RP-HPLC, a method by which the decrease
of monomeric species could be shown to be reciprocal
to the increase in ANS fluorescence (Fig 1B) Because
we never observed amorphous aggregates with the N-terminal fragment, we conclude that the increase in ANS fluorescence correlates with the increase in fibril-lar species at the expense of monomeric protein According to the hypothesis that seeded fibril forma-tion in the case of the yeast prion protein Sup35p exhibits a binding equilibrium between soluble interme-diates and seeding molecules [31,32], an increase in seed concentration should accelerate fibril formation
To test this assumption, fibril formation was investi-gated in the presence of increasing concentrations of fragmented N-(+7)Ala fibrils acting as seeds Clearly,
an increase in seed concentration resulted in faster fibrillation rates (Fig 2A) Quantification of fibrillation rates revealed an approximately linear correlation between seed concentration and fibril growth (Fig 2B)
A similar dependence of fibrillation rates on seed con-centration has been demonstrated previously with the
NM domain of Sup35p [31,33]
Induction of fibrillation by seeds was also tested using N-WT In the absence of seeds, an increase in ANS fluorescence was recorded after 30 days (Fig 3A) Seeding with fragmented N-WT fibrils resulted in an immediate increase in ANS fluorescence However, the increase in ANS fluorescence was considerably slower than in the case of seeded reactions with N-(+7)Ala To ensure that the increase in ANS fluorescence reflects fibril growth, quantification of monomeric species of the seeded reaction by RP-HPLC was performed This ana-lysis reveals that monomeric species decreased very slowly over an incubation time of 25 days, indicating that induction of fibril growth of N-WT by seeds is significantly slower than in the case of N-(+7)Ala (Fig 3B) Subsequent analyses using AFM confirmed that N-WT seeds had hardly been elongated to longer fibrils (Fig 5C,D) In contrast, seeded samples of N-(+7)Ala, which revealed small seeds to begin with (data not shown), showed elongated fibrils after 30 days
of incubation (Fig 5G,H) Possibly, in the case of N-WT, conformational change to the fibrillar state is so slow that most of the seeds lose their ability to act as polymerization points for soluble N-WT
Because slow fibril formation of N-WT induced by N-WT seeds may also be due to less active N-WT seeds, cross-seeding experiments were performed As seen upon the addition of homologous seeds, incuba-tion of N-(+7)Ala with N-WT seeds resulted in an
incubation time (d)
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18 20
22
incubation time (d)
0
2
4
6
8
10
12
14
16
A
B
Fig 1 Fibril formation kinetics of N-(+7)Ala The kinetics of fibril
formation were followed using ANS fluorescence in arbitrary units
(AU) (A) Fibril growth of N-(+7)Ala was monitored in the absence
(triangles) and presence (circles) of 0.1% seeds (w ⁄ v) For the
ana-lysis, N-(+7)Ala was incubated at 37 C at a protein concentration
of 1 m M (B) Quantification of the decrease in monomeric species
by RP-HPLC analysis (squares) as indicated in Experimental
proce-dures For comparison, the concomitant increase in ANS
fluores-cence (circles) is shown.
Trang 4increase in ANS fluorescence The increase in the ANS
signal was reciprocal to the decrease in monomeric
species (data not shown) and depended on the
num-bers of seeds added (Fig 4) Comparison of the
fibril-lation rates with homologous and heterologous seeds
(Table 1) indicated that fibril growth rates of
cross-seeded samples are slower by a factor of 2 in
com-parison with experiments using homologous seeds The
large standard deviations of the growth rates indicate
that absolute fibrillation rates have to be interpreted
cautiously We assume that these deviations (in each
experimental set-up, the three different seed concentra-tions originated from an identical seed preparation) are due to the following technical difficulties: our pre-vious experiments have indicated that the seeds quickly lose their activity upon storage (data not shown) For this reason, seeds had to be freshly prepared for each test Seed preparations showed noticeable batch incon-sistencies that may be due to chemical modification(s) caused by the long incubation times which were neces-sary to obtain fibrils
Fibrils of N-WT and N-(+7)Ala differ in morphology and stability
CD analysis of soluble monomeric N-WT and N-(+7)Ala showed that N-(+7)Ala contains more a-helical secondary structures than N-WT [26] Thus,
incubation time (d)
0
2
4
6
8
10
12
14
16
18
A
concentration of seeds (w/v) 0.1% 0.2% 0.4%
-1 )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
B
Fig 2 Fibril formation kinetics of N-(+7)Ala at different seed
con-centrations (A) N-(+7)Ala (1 m M ) was incubated at 37 C with
0.1% (circles), 0.2% (squares) and 0.4% (triangles) seeds (w⁄ v).
The increase in ANS fluorescence was fitted to a first-order
reac-tion to determine the rates of fibril formareac-tion (B) Rates of fibril
for-mation (per day) as a function of the seed concentration Error bars
represent variations from two to four separate experiments
invol-ving separate seed preparations.
incubation time (d)
0 10 20 30 40 50 60 70 80 90 100
0 2 4 6 8 10 12
14
A
incubation time (d)
0 5 10 15 20 25 30 35 40 45 50 0
10 20 30 40 50 60 70 80 90 100
0 2 4 6 8 10 12 14 16 18 20 22
B
Fig 3 Fibril formation kinetics of N-WT (A) N-WT was incubated
at a protein concentration of 1 m M in the absence (triangles) and presence (circles) of 0.1% seeds (w ⁄ v) (B) Loss of monomeric species (squares) in the seeded sample was monitored by RP-HPLC analysis For comparison, the concomitant increase in ANS fluorescence (circles) is shown.
Trang 5in the as yet unfibrillized form, N-WT and N-(+7)Ala
already display different structural features Possibly,
these differences at the structural level are reflected in
the different capacities of the two variants to fibrillize
upon seeding In order to analyze whether structural
differences between N-WT and N-(+7)Ala could also
be detected in the fibrillar forms, fibrils of both
vari-ants were visualized using electron microscopy (EM)
and AFM Both microscopy techniques revealed fibril
diameters of 6 nm Although no differences in fibril
morphology could be detected using EM (Fig 5A,E),
AFM analysis showed a more pronounced fine
struc-ture in N-WT fibrils than in N-(+7)Ala fibrils
(Figs 5B,F and 6) N-WT fibrils resembled a string of
beads not observed for N-(+7)Ala fibrils The
distan-ces between the beads ranged from 27 to 43 nm
(Fig 6) These defined substructures could not be
observed with N-(+7)Ala fibrils We assume that the
different surface morphologies reflect divergent
struc-tural arrangements of the b-strands inside the fibril
Whether the bead-like structure is caused by twist repeats or similar substructures, as reported for the SH3 domain of phosphatidyl inositol-3¢-kinase and lysozyme, remains to be clarified [34] The fact that beaded fibril morphologies of N-(+7)Ala, which was seeded with fragmented N-WT fibrils, were not fre-quently observed (Fig 5H) may indicate that the seed structure does not ‘mold’ newly associating molecules into the existing conformation as has been postulated for Sup35p [35,36]
Because the different structures detected using AFM may correlate with different stabilities against solubili-zation, fibrils of N-WT and N-(+7)Ala were incubated
at increasing concentrations of guanidinium thiocya-nate, the only denaturing agent identified by us that can lead to a partial solubilization of the fibrils First, solubilization was performed at room temperature for 6 h Release of soluble species was quantified by RP-HPLC and is indicated as the percentage of the material that had been previously fibrillar (Fig 7A) With both variants, 40–50% of the fibrillar protein was converted into soluble species Although conver-sion was not complete, a higher amount of N-WT than
of N-(+7)Ala fibrils was solubilized by guanidinium thiocyanate concentrations between 3 and 5 m When solubilization was performed under more stringent conditions (16 h at 50C), maximal conversion to monomeric species was obtained with N-WT fibrils
at 1 m guanidinium thiocyanate, whereas those of N-(+7)Ala required 4 m guanidinium thiocyanate (data not shown)
In order to detect a possible equilibrium between fibrils and soluble monomeric species under solubiliz-ing conditions, the remainsolubiliz-ing undissolved fibrils were reincubated with 6 m guanidinium thiocyanate for
24 h However, no more protein could be dissolved upon this second incubation ruling out equilibrium conditions (data not shown) It is currently unclear whether the remaining guanidinium thiocyanate-resist-ant material reflects fibrillar core structures that cannot
at all be solubilized or whether heterogeneous fibrils display differences in resistance against solubilization When the kinetics of solubilization were monitored
in the presence of 6 m guanidinium thiocyanate, the maximal yield of soluble N-WT was obtained after
1 h, whereas for maximal solubilization of N-(+7)Ala
an incubation period of > 20 h was required (Fig 7B) Clearly, these results indicate differences in stability between N-WT and N-(+7)Ala fibrils Distinct solubi-lization properties in cell culture, depending on the length of the poly l-alanine sequence, have recently been reported for PABPN1 fusion proteins [22] In general, the results of the solubilization experiments
Table 1 Rates of N-(+7)Ala fibril formation upon addition of seeds.
Fibrillation in the presence of different concentrations of N-(+7)Ala
seeds (seeding) and N-WT seeds (cross-seeding) Rate constants
were calculated by assuming a first order reaction Standard
devia-tions are based on three independent experiments.
Concentration
of seeds
(w ⁄ v)
Growth rates with homologous seeds (d)1)
with heterologous seeds
(d)1)
incubation time (d)
0 2 4 6 8 10 12 14 16 18 20
0
5
10
15
20
Fig 4 Cross-seeding of N-(+7)Ala N-(+7)Ala (1 m M ) was incubated
at 37 C with 0.1% (circles) or 0.2% (squares) seeds (w ⁄ v) Seeds
were derived from fibrils of N-WT.
Trang 6underscore the unusually high resistance of the fibrils
against denaturation, which may represent a common
feature of fibrils containing poly l-alanine sequences
Other well-known examples for extreme
mechano-chemical robustness are spider silks which contain
numerous interspersed poly l-alanine repeats [37,38]
Discussion
Previous experiments have shown that fibril formation
of N-(+7)Ala started after a shorter lag phase than
that of N-WT [26] This finding can be interpreted as
a higher propensity of the N-(+7)Ala variant to adopt
the b-cross state Fibrils of N-WT and N-(+7)Ala also
differ with respect to the fibril growth rates In the
case of N-WT, the conversion to the fibrillar
confor-mation is presumably very slow under the applied
conditions This assumption, and the fact that seeds lose their activity upon incubation, may be responsible for the only moderate acceleration of fibril formation
of N-WT by seeds Consequently, an increase in the protein concentration and⁄ or temperature which is known to reduce the lag phases of fibril formation [26] may render N-WT fibril growth rates more susceptible
to seeds
Differences between N-WT and N-(+7)Ala were also observed at the level of fibril morphology A likely interpretation is that the number of alanines deter-mines the arrangement of the b-strands leading to vari-ations in the surface structures as well as fibril stabilities This assumption would be in good agree-ment with recent findings by Kirschner and co-workers who observed poly l-alanine length-dependent differ-ences in the diffraction patterns in fibrillized peptides
Fig 5 Visualization of fibrils using EM (A, E) and AFM (B–D, F–H) (A–D), N-WT fibrils; (E,F), N-(+7)Ala fibrils Fibrils were derived from sam-ples in which 1 m M soluble protein had been either incubated in the absence of seeds (A, B, E, F) or in the presence of 0.1% seeds (w ⁄ v) (C, D, G, H) at 37 C; N-WT (A–D) was incubated for 60 days, N-(+7)Ala (E–H) for 30 days N-WT incubated with N-WT seeds (C), N-(+7)Ala seeds (D), N-(+7)Ala with N-(+7)Ala seeds (G) and N-WT seeds (H) Magnification of EM: 50 000, insets: zoom with a 50 000 magnification The scale bars represent 250 nm; insets in (A) and (E): scale bars ¼ 50 nm.
Trang 7[28] Furthermore, given that even the incubation
tem-perature can influence the conformation and stability
of the Sup-NM domain [35,36], different fibril
struc-tures caused by additional alanines are likely The
manner in which the number of alanines determines
the atomic structure of the fibril and the b-strand
arrangements remains to be determined
Yet, knowledge of the structure and biophysical
analysis of the fibrils alone will not suffice for
understanding the disease causing mechanism, and
in vitro analyses have to be complemented by cell
biological investigations From a recent analysis of
PABPN1 toxicity in Drosophila, the authors
conclu-ded that OPMD symptoms such as nuclear
inclu-sions do not result from adverse effects of the
poly l-alanine sequence [39] Rather, the
RNA-bind-ing function of the protein was suggested to evoke
muscle defects and nuclear inclusions This
conclu-sion was based on investigations employing mutants
in which the RNA-binding domain of PABPN1 had
been either deleted or inactivated by point
muta-tions The absence of an intact RNA-binding domain
should, however, significantly reduce local concentra-tions of PABPN1 close to poly(A) tails Because fibril formation, like other aggregation processes, is known to be a concentration-dependent reaction, the absence of nuclear inclusions in the case of these mutants may simply be due to the fact that crit-ical threshold concentrations of PABPN1 for fibril formation will not be reached at poly(A) tails The argument that high local concentrations of PABPN1 facilitate deposit formation is supported by an earlier study showing that the ability of PABPN1 to form oligomers is crucial for the formation of intranuclear inclusions [20]
Fig 6 Analysis of AFM images to determine substructure widths.
Longitudinal section of N-WT and N-(+7)Ala fibrils obtained using
Nanoscope Section Analysis.
0 10 20 30 40 50
60
A
incubation time (h)
0 20 40 60 80
100
B
Fig 7 Stability of fibrils against solubilization with guanidinium thio-cyanate (A) Solubilization at the indicated guanidinium thiocyanate concentrations was performed at room temperature for 6 h (B) Kin-etics of conversion to monomeric species Solubilized material after the various incubation times is shown as percentage of the max-imal amount of solubilized material The increase in soluble protein was monitored by RP-HPLC analysis Error bars result from two to three independent experiments N-WT, triangles; N-(+7)Ala, circles.
Trang 8However, an alternative scenario for disease
devel-opment can be envisaged: continuous proteolytic
cyto-solic turn-over may be required to keep nuclear levels
of PABPN1 low This degradation may be reduced
by the presence of an extended poly l-alanine tract
Evidence for reduced proteasomal degradation by
glycine–alanine repeats due to impaired substrate
unfolding has been reported recently [40]
Further-more, imbalance in a-synuclein levels due to
muta-tions, overexpression or inefficient proteasomal
removal of a-synuclein has been proposed to induce
fibril formation and thus a-synucleinopathy [41–43]
The observations that the nuclear inclusions found in
OPMD patients colocalize with ubiquitin and the 20S
proteasomal subunit [16,44] and the findings that
pro-teasome inhibitors increase poly l-alanine-induced
cytotoxicity in cell culture [17] would be in accordance
with this disease-causing cascade
Experimental procedures
Recombinant protein production and purification
Recombinant constructs have been described previously
[26] The N-terminal fragments of wild-type (N-WT)
and the variant containing seven additional alanine residues
[N-(+7)Ala] were expressed as fusions with N-terminal
His-tags using the T7 vector system (pET15b) from
Nov-agen (Madison, WI) We showed previously that the
His-tag has no influence on fibril formation [26] As host cells,
the Escherichia coli strain BL21(DE3)Gold with the vector
pUBS520 was employed to circumvent codon usage
prob-lems Culture conditions were described previously [26] with
the exception that bacteria were grown in fermentors of 8 L
culture volumes with 5% yeast extract (Roth, Karlsruhe,
Germany) Feeding with yeast extract was started at
10 mL disruption buffer (50 mm Tris pH 8, 5 mm EDTA,
1 mm phenylmethylsulfonyl fluoride) was added All
subse-quent steps were carried out as described previously [26],
with the exception that elution from the Q Sepharose was
achieved by 300 mm NaCl Relevant fractions were pooled,
diluted 1 : 1 with 8 m guanidinium hydrochloride (NiGU
Ni-NTA column (His Bind Resin, Novagen, Darmstadt,
Germany) equilibrated with 4 m guanidinium
hydrochlo-ride, 50 mm Tris, pH 8.0 The column was washed with
buffer containing 20 mm imidazol, 4 m guanidinium
hydro-chloride, 50 mm Tris, pH 8.0 Protein was eluted by a linear
imidazol gradient from 20 to 250 mm within 12 column
vol-umes Relevant fractions were selected, pooled and further
purified by gel filtration as published [26] Purified protein
was dialyzed against water, lyophillized and stored at )80 C
Fibril formation and fibril analysis by ANS fluorescence
for-mation to occur To determine ANS fluorescence, samples were briefly mixed and then diluted to a final concentration
150 mm NaCl Fluorescence spectra were recorded at an emission wavelength of 480 nm upon excitation at 370 nm
were performed in 1 cm cuvettes with excitation and emis-sion slits widths of 5 nm
Seed preparation Fibrils were formed by incubation of N-(+7)Ala at a con-centration of 1 mm for 30 days and N-WT at a concentra-tion of 2 mm for 100 days When fibril formaconcentra-tion was completed as monitored by maximal ANS fluorescence signals, fibrils without further storage were harvested by centrifugation for 1 h at 260 000 g (OptimaTM TLX
150 mm NaCl, and subjected to pulsed ultrasonification
Seeds were added immediately after preparation to protein solutions
Quantification of soluble (monomeric) protein during fibril formation by RP-HPLC
Samples were diluted 1 : 25 with water to a final protein
260 000 g The supernatant was loaded on a 1.6 mL
pre-equilibrated with 0.05% trifluoroacetic acid in water Protein was eluted by an acetonitrile gradient from 0 to
Pro-tein concentrations were determined by integration of peak areas after calibration with soluble (monomeric) reference protein
EM and AFM For EM analysis, carbonized copper grids (Plano, Wetzlar,
applied for 3 min Subsequently, grids were again air dried
Trang 9uranyl-acetate and visualized in a Zeiss EM 900 electron
microscope operating at 80 kV For AFM, samples were
placed on freshly cleaved mica attached to AFM sample
were rinsed three times with Millipore filtered distilled
water The samples were then allowed to air dry Tapping
mode imaging was performed on a multimode scanning
probe microscope (Veeco, Santa Barbara, CA) by using
Switzerland) Fibril heights and subunit widths were
deter-mined using the nanoscope analysis software
Chemical stability of fibrils
The chemical stability of fibrils was tested after the
fibrilla-tion process was completed (no further rise of ANS
sig-nals) The sample containing fibrils was split into seven
aliquots that were centrifuged for 1 h at 70 000 r.p.m
(OptimaTM TLX ultracentrifuge, Beckman, Fullerton,
NaCl Fibrils were resuspended in the same buffer
contain-ing different concentrations of guanidinium thiocyanate
Samples were incubated under shaking (600 r.p.m.) for
Sup-ernatants obtained after centrifugation were analyzed by
RP-HPLC
Acknowledgements
We thank Elmar Wahle, Christian Lange, Huma
Yonus, Daniel Huster and David Ferrari for critical
comments on the manuscript This work was funded
by the Deutsche Forschungsgemeinschaft through
Sonderforschungsbereich 610, subproject A9 (ES) and
Sonderforschungsbereich 596, subproject B14 (TS)
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