From the Dipartimento di Scienze Neurologiche e della Visione, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università di Verona, Strada Le Grazie 8, I-37134 Verona, Italy
Received for publication, December 23, 2002, and in revised form, January 13, 2003
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ABSTRACT |
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By lyophilizing RNase A from 40% acetic acid
solutions, two dimeric aggregates, the "minor" and "major"
dimers (named here N-dimer and C-dimer, respectively), form by 3D
domain swapping at a ratio of 1:4. Trimeric and tetrameric aggregates
are also obtained. The two dimers and the higher oligomers also form
without a lyophilization step. By keeping RNase A dissolved at a high concentration (generally 200 mg/ml) in various media at temperatures ranging from 23 to 70 °C for times varying from a few minutes to
2 h, various oligomers, in particular the two dimeric conformers, formed in quite different amounts, often inverting their
relative quantities depending on the more or less severe unfolding
conditions. When unfolding mainly concerned the N terminus of the
protein, richer in hydrophilic residues, the N-dimer, formed by 3D
domain swapping of the N-terminal Protein aggregation is presently an event of remarkable interest.
Severe human neurodegenerative diseases are in fact characterized by
the presence of aggregated forms of proteins ( Bovine ribonuclease A is a structurally versatile protein, appropriate
to study the manner in which a protein aggregates. By lyophilization
from 40-50% acetic acid solutions, the protein aggregates in the form
of several types of oligomers, ranging from dimers (2) to pentamers and
possibly higher aggregates, each species existing in at least two
conformational isomers produced in invariable different amounts (3, 4).
The two dimers of RNase A have been well characterized and shown to be
3D domain-swapped oligomers (5, 6), with the domain swapping involving
either the N or C terminus or both of each subunit. For one of the
trimers (the quantitatively major one), a very plausible linear
structure has been proposed (6), whereas the crystal structure of the other shows a circular trimer, similar to a propeller, formed by the
swapping of the C-terminal Interestingly, RNase A also aggregates under relatively mild
conditions. Park and Raines (9) reported that, at 37 and 65 °C and
pH 6.5, it forms domain-swapped dimers, whereas many years ago it was
shown that high substrate concentrations can induce the dimerization of
RNase A (10). It has been suggested that the 3D domain-swapping
mechanism forming RNase A dimers or higher order aggregates may have
implications for the formation of amyloid-like fibrils (5, 6, 11), and
strong support for this idea is given by the recently reported
dimerization through a 3D domain-swapping mechanism of the
amyloidogenic protein cystatin C (12) and the human prion protein (13).
We have studied here the oligomerization of RNase A occurring under
various conditions that favor the unfolding and mobility of the N
and/or C terminus of the protein, and therefore the domain swapping
event, without a lyophilization step. Our aim was to contribute to the
understanding of the 3D domain-swapping mechanism in protein
aggregation. The aggregates, mainly the two dimeric conformers, but
also higher order oligomers, formed in varying relative, often inverted
proportions depending on the experimental conditions.
Materials--
Ribonuclease A from bovine pancreas (Type XII-A)
was from Sigma. All chemicals were of the highest purity available.
Superdex 75 HR10/30 and Source 15S HR10/10 and HR16/10 columns were
from Amersham Biosciences. Chromatographic experiments were performed with an Amersham Biosciences fast protein liquid chromatography system.
Preparation of RNase A Aggregates--
RNase A was dissolved in
various media, and aggregation was performed as described below, always
without a lyophilization step: (a) 20 or 40% ethanol in
water and an RNase A concentration of 200 mg/ml; (b) 20 or
40% ethanol in 0.08 M sodium phosphate buffer (pH 6.7) and
an RNase A concentration of 200 mg/ml; (c) 40%
2,2,2-trifluoroethanol (TFE)1
in water and an RNase A concentration of 200 mg/ml; (d) 40%
acetic acid (pH ~1) and an RNase A concentration of 66.7 mg/ml;
(e) 5, 10, or 30% aqueous acetone and an RNase A
concentration of 200 mg/ml; (f) 40% aqueous acetone
and an RNase A concentration of 170 mg/ml; (g) 0.05 M glycine HCl buffer (pH 3) and an RNase A concentration of
200 mg/ml; (h) double-distilled water (pH 6.0) and an RNase
A concentration of 200 mg/ml; (i) 0.02 M NaCl
(pH 7) and an RNase A concentration of 200 mg/ml; (j) 0.0125 M Na2B4O7 and 0.0152 M HCl buffer (pH 8.5) and an RNase A concentration of 200 mg/ml; (k) 0.05 M
KCl/H3BO3 and 0.0208 M NaOH buffer
(pH 9) and an RNase A concentration of 200 mg/ml; and (l)
0.05 M KCl/H3BO3 and 0.0437 M NaOH buffer (pH 10) and an RNase A concentration of 200 mg/ml. Aliquots (2 µl) of each RNase A solution were maintained for
appropriate times, ranging from minutes to hours, at the chosen temperature. At the end of each incubation time, 200 µl of 0.2 M sodium phosphate buffer (pH 6.7), preheated at the same
temperature (sometimes at room temperature), were added, and the sample
was brought to 0 °C. Aliquots of the RNase A solutions indicated
above were also kept at 23 °C for the same times chosen for the
experiments at 60 °C. At the end of each incubation time, 200 µl
of 0.2 M sodium phosphate buffer (pH 6.7) were added.
Gel Filtration--
After the treatment described above, each
sample was frozen until used or applied to a Superdex 75 HR10/30 column
equilibrated with 0.2 M sodium phosphate buffer (pH 6.7) at
23 °C. Elution was performed at 23 °C with the same buffer at a
flow rate of 0.01-0.2 ml/min. Every aggregated species emerging from
the column was identified by checking its elution position and
comparing it with that of the well characterized oligomers obtained by
the usual lyophilization procedure (3, 4), which were always used as standards.
Ion-exchange Chromatography--
Analysis and separation of the
various RNase A aggregates were performed under conditions identical to
those described elsewhere (3, 4). Their identification was performed by
comparison with the elution pattern of standard aggregates (see above).
Quantification of the Aggregates Formed--
Each aggregate
produced under the various experimental conditions was quantified by
measuring the area of its peak appearing on the gel-filtration or
ion-exchange chromatogram and calculating its percentage relative to
the sum of the areas of the peaks of all RNase A species eluted,
monomeric RNase A included. The values shown in the table and/or
figures are the means ± S.D. of several determinations.
Gel Electrophoresis--
Cathodic gel electrophoresis under
nondenaturing conditions was performed according to Goldenberg (14)
with slight modifications, using Analysis of the Thermal Unfolding of RNase A--
The thermal
unfolding of RNase A was followed spectrophotometrically at 287 nm
using a Beckman DU-650 spectrophotometer equipped with a
thermostatically controlled water bath. The protein was dissolved in
the various media at a concentration of ~1.5 mg/ml. At each
temperature, the absorbance values of the RNase A solution were
determined every 2 min, for a total time of 60 min, until measurements
became constant for at least 10 min.
Lyophilization of RNase A from 40% acetic acid solutions produces
two dimeric conformers at a rather invariable ratio of ~1:4, one
called in fact the "minor" dimer and the other the "major" dimer (3, 4). The minor dimer forms by 3D domain swapping of the
N-terminal By the lyophilization procedure, higher oligomers of RNase A also form:
two trimers in constant, quite invariable amounts (ratio 1.5:1),
formerly called the major and minor trimers (3, 4, 6), as well as two
tetramers, a minor and a major one, forming at a ratio of 1:1.6
(4). However, dimers and larger aggregates of RNase A also form
under conditions that allow the unfolding of the N and/or C terminus of
the protein, but without including a lyophilization step. Moreover, the
relative proportions of the two dimers can change and even be inverted
depending on the experimental conditions. The experiments were
performed by maintaining native monomeric RNase A, dissolved at a high
concentration (usually 200 mg/ml) in the various fluids described under
"Experimental Procedures," at temperatures ranging from 23 to
70 °C for times ranging from a few minutes to 2 h. The
treatment was ended by diluting the incubation mixture 1:100 with 0.2 M sodium phosphate buffer (pH 6.7) and transferring it to
an ice bath. The aggregates formed were separated by gel filtration.
When necessary, each gel-filtered oligomeric species or the incubated
mixtures as such were analyzed by ion-exchange chromatography. In both
cases, identification of the various oligomers formed was carried out
as described under "Experimental Procedures," i.e. by
using the well characterized dimers, trimers, or tetramers (3, 4),
recently prepared, as standards. A control of the nature of the various
oligomers was also performed, when necessary, by gel electrophoresis
under nondenaturing conditions (see "Experimental Procedures"). In
all cases, the relative amounts of the two dimeric conformers and of
the higher order oligomers were calculated as described under "Experimental Procedures."
Aggregation of RNase A Dissolved in Various Media and Incubated at
60 or 23 °C--
As shown in Fig. 1
(A and B, blue lines) and Table
I, a solution of RNase A (200 mg/ml) in
40% aqueous or buffered ethanol heated at 60 °C for 30 min
produced, similar to the lyophilization procedure, the C-dimer in a
higher amount and the N-dimer in a lower amount, the ratio of the
former to the latter being ~2:1 (lower than the 4:1 ratio usually
found with the lyophilization procedure). Similar results were obtained
when RNase A dissolved in 40% aqueous TFE was maintained at 60 °C
for 30 min (C-dimer/N-dimer ratio of 2:1). Under identical conditions,
significant amounts of trimers or tetramers also formed. Because these
oligomers were collected by gel filtration and not by ion-exchange
chromatography, no complete separation of the two conformers of each
species was obtained. The trimers and tetramers indicated here (Table
I) are therefore mixtures of the two trimeric and tetrameric conformers (3), respectively.
By incubating (60 °C, 30 min) RNase A dissolved in either 20%
aqueous or buffered ethanol, as well as in many other fluids (Fig. 1,
A and B, red lines; and Table I), we
found instead that the relative proportions of the two dimers inverted,
the N-dimer often prevailing over the C-dimer. Unexpectedly,
oligomerization of the protein also occurred when incubation was
performed at room temperature (23 °C). Under these conditions, the
prevalence of the N-dimer over the C-dimer was observed in all cases,
including RNase A solutions in 40% ethanol or TFE, although the
aggregates often formed in quite modest amounts (Fig. 1, A
and B, green lines; and Table I).
It is also worth mentioning that incubation of a 10-fold less
concentrated RNase A solution (20 mg/ml) in 40% ethanol or TFE at
60 °C gave only traces of dimers. This points out the importance of
protein concentration in the aggregation process.
Oligomerization of RNase A Dissolved in 40% Aqueous Ethanol and
Heated at 45, 50, or 60 °C--
To analyze the results
described in more detail, we studied the formation of RNase A oligomers
by incubating solutions of the protein in 40% aqueous ethanol for up
to 2 h at 45, 50, or 60 °C. By analyzing aliquots withdrawn
from the incubation mixture at the times indicated, we obtained the
results presented in Fig. 2. Fig.
2A shows RNase A aggregation at 45 °C. At this
temperature, the reaction did not reach equilibrium even after
2 h of incubation. The modest amounts of oligomers formed
regularly increased with time; and the N-dimer was definitely prevalent
over the C-dimer. Small amounts of trimers also appeared after 70 min
of incubation. A notable point is that, at zero time, i.e.
during the short time necessary to prepare the incubation mixture at
room temperature (23 °C) and to transfer an aliquot of it to an ice
bath, RNase A appeared to aggregate quite efficiently, with the N-dimer
and C-dimer reaching amounts of 3.4 and 1.2%, respectively. However, after some minutes at 45 °C, the amount of both aggregates
decreased, as if they were dissociated. It is worth pointing out that,
from these and other results, it appears that RNase A has a remarkable propensity to oligomerize (see also Ref. 9), and this propensity might
even increase after some treatment. This could be the reason why
commercial preparations of RNase A are sometimes found to contain
considerable amounts of aggregated material (15).
At 50 °C (Fig. 2B), oligomerization was more conspicuous,
reaching equilibrium after 60 min of incubation. Although the
experimental data appear to be highly variable between ~5 and 30 min,
an inversion of the relative proportions of the N-dimer and C-dimer
seemed to occur after 35 min.
At 60 °C (Fig. 2C), definitely larger amounts of all
oligomeric species were produced, and the quantity of the C-dimer was always remarkably higher than that of the N-dimer. At 60 min of incubation, the relative amounts were 9.7% for the N-dimer and 16.4%
for the C-dimer. Their ratio (1:1.7) is far from the 1:4 ratio usually
obtained with the lyophilization procedure. The RNase A trimer, already
formed at 45 °C (Fig. 2A), became more abundant at
50 °C (Fig. 2B) and definitely increased at 60 °C (Fig. 2C). After 1 h of incubation, it was present at
9.7%, identical to the N-dimer. At the same time, the quantity of
tetramers formed, negligible at 50 °C (Fig. 2B), was
~5% at 60 °C (Fig. 2C). No significant increase in the
amount of the two dimers and the higher order aggregates occurred after
60 min of incubation.
Oligomerization of RNase A Dissolved in 40% TFE and Heated at
60 °C--
A similar pattern of oligomerization was found upon
incubation at 60 °C of RNase A dissolved in 40% TFE (Fig.
3). After 60 min, the formation of the
C-dimer (~17% at 60 min) was remarkably favored over that of the
N-dimer (7.6%), as well as over that of the trimeric and tetrameric
species (7.8, and 2.7%, respectively), the amounts of which were lower
than the amounts found in 40% ethanol at the same temperature (Fig.
2C).
Oligomerization of RNase A Dissolved in 20% Aqueous Ethanol and
Heated at 60 °C--
The pattern of the oligomerization of RNase A
under these conditions (Fig. 4), which
was rather similar to the pattern observed at 50 °C in 40% ethanol
(Fig. 2B), reveals that these two conditions are
qualitatively equivalent and indeed critical (see also Table I) for the
aggregation process. They appear to be a transition point
between the experimental conditions favoring the prevalence of one or
the other of the two dimers. In conclusion, under these conditions, the
quantities of the two dimers fluctuated around an average value of
~50% of each conformer (see also Fig. 1, A and
B, solid and dashed red lines).
Because of its significance, the analysis of RNase A aggregation in
20% ethanol at 60 °C was repeated, but the two conformers were
separated by ion-exchange chromatography. The results of four different
experiments (data not shown) are in very good agreement with those
obtained by gel filtration.
Analysis of the Thermal Unfolding of RNase A Dissolved in Various
Media--
The transition profiles for the thermal unfolding of RNase
A dissolved in many of the fluids used in the experiments described were determined spectrophotometrically at 287 nm and are shown in Fig.
5. The decrease in absorbance (16, 17) is
ascribable to the increased exposure of tyrosine residues as a function
of the increase in temperature. Recent studies using Tyr-to-Phe mutants have assigned the absorbance change upon unfolding largely to three
buried tyrosines at positions 25, 92, and 97 (18). Therefore, the steep
changes in absorbance detected (Fig. 5) should reasonably be
essentially ascribed to the global unfolding of the whole protein. However, Tyr115, located in the hinge loop of the RNase A C
terminus (residues 112-115) (6), might also be important in the
absorbance changes that occur in the unfolding events leading to
C-dimer formation. The temperatures at which the absorbance inflections
occurred (Fig. 5) varied under different conditions: when RNase A was
dissolved in 40% aqueous TFE or ethanol, the absorbance lowering took
place at lower temperatures, whereas under milder conditions (borate buffer at pH 8.5 or 10, double-distilled water, etc.), the absorbance changes shifted toward higher temperatures. Therefore, the correlation between RNase A unfolding and the prevalence of one or the other dimeric conformer is clear: under conditions in which the absorbance lowering occurs definitely below 60 °C, the C-dimer, formed by the C-terminal end-swapping mechanism (6), is produced in higher amounts. This means that unfolding occurs at both the N and C termini
of the protein. Under milder conditions in which the absorbance lowering occurs above 60 °C, the N-dimer is more abundant,
indicating the prevalent unfolding (and swapping) of the N terminus of
RNase A. Under intermediate conditions (20% ethanol at 60 °C and
40% ethanol at 50 °C) in which absorbance changes in the protein
occur just below or close to 60 °C, the amounts of the two dimeric
conformers become similar, possibly indicating that the unfolding
events occurring at the N and C termini of RNase A are equivalent, as well as the energy requirements for the two aggregation events occurring via 3D domain swapping.
The steep absorbance inflection related to the unfolding of RNase A in
40% TFE occurred at ~32 °C (Fig. 5). To further analyze the
correlation between the thermal unfolding of the protein and the yield
of one or the other dimeric conformer, the oligomerization process was
studied at a concentration of 200 mg/ml RNase A in 40% aqueous TFE
heated at lower temperatures (30-45 °C) for 120 min. The results
obtained are shown in Table II. Although
the amount of the N-dimer was higher at 30 °C than that of the
C-dimer at any time of incubation, an inversion of their relative
proportions possibly occurred between 30 and 32 °C. At 32 °C, as
well as at 35 °C and even more at 45 °C, the amount of the
C-dimer became definitely higher than that of the N-dimer. Moreover, by
comparing the results obtained upon the aggregation of RNase A under
all conditions used (Figs. 1-4 and Tables I-IV) with the relative
absorbance inflections detected (Fig. 5), the correlation mentioned
above appears to be confirmed.
Oligomerization of RNase A in 40% Acetic Acid or in 5-40%
Acetone--
We also analyzed the oligomerization of RNase A in 40%
acetic acid solutions (protein concentration of 66.7 mg/ml) heated at
60 °C for 120 min (data not shown). In aliquots withdrawn from time
to time from the incubation mixture, both dimers were found in very
modest quantities (1-2%), which, however, could partly be also
ascribable to the definitely lower protein concentration used in these
experiments. The amount of the C-dimer was twice that of the N-dimer
over the entire course of the experiment. Moreover, in the solution
prepared at 23 °C to perform the experiment, the C-dimer was found,
once again, in amounts (4-5%) definitely higher than those measured
later during the 2-h incubation at 60 °C. In other words, it
appears, as already mentioned, as if exposure of the RNase A solution
to too drastic conditions could result in the dissociation of the
oligomer(s) formed at room temperature.
We also studied the oligomerization pattern of RNase A dissolved in
5-40% acetone and heated at 60 °C for up to 120 min (Table III). The relative proportions of the two
dimers were similar to those forming under mild unfolding conditions
(all media at 23 °C; borate buffer at pH 8.5, 9, or 10; water; NaCl;
etc.). Under all conditions, the N-dimer formed at a higher amount than
the C-dimer. Unfortunately, the thermal transition curve of RNase A in
acetone could not be determined because of the known strong absorbance
of acetone in UV light.
Oligomerization of RNase A at 70 °C--
To try to understand
the results obtained in the presence of acetone, the temperature of
incubation of RNase A dissolved in several different concentrations of
acetone was increased from 60 to 70 °C. Under these conditions, an
inversion of the proportions of the two dimers took place, with the
prevalence now of the C-dimer over the N-dimer (Table IV). This
result can be explained as due to a more severe unfolding
of RNase A obtained at 70 °C. The next step was to heat (70 °C, 60 min) RNase A dissolved in
double-distilled water or borate or glycine HCl buffer. When these
RNase A solutions were incubated at 23 and 60 °C (Fig. 1,
A and B, green lines; and Table I),
the amounts of the N-dimer were always higher than those of the
C-dimer. Now, at 70 °C (Table IV), the relative proportions of the
two dimeric conformers inverted in all cases, with the quantity of the
C-dimer prevailing over that of the N-dimer. This result could be
explained with the data shown in Fig. 5: the absorbance inflection of
the RNase A thermal unfolding profile occurred below 70 °C in all
media used. The almost identical pattern of RNase A aggregation
occurring in aqueous solvents in the presence or absence of various
concentrations of acetone could be explained on the basis of the
solvation process. Acetone, ethanol, and TFE are strong denaturing
agents, but acetone, unlike ethanol or TFE, is not a hydrogen donor.
Therefore, in the presence of various concentrations of acetone, the
protein domains exposed in the first sphere of solvation are
preferentially solvated by water (hydrogen donor) and not by acetone.
In contrast, solvation by ethanol and TFE (if present in aqueous media)
is possible because they are hydrogen donors like water, but less
structured than water, entropically favoring ethanol and TFE in their
solvating action.
We completed the analysis at 70 °C by a 60-min incubation at this
temperature of RNase A dissolved in 40% ethanol or TFE, i.e. under vigorous unfolding conditions, or in 20%
ethanol, which at 60 °C might be taken as a point of transition
between mild and strong unfolding conditions. The amount of the C-dimer
always prevailed over that of the N-dimer (Table IV). The absolute
amounts of the two dimers found at 70 °C were lower than those
measured at 60 °C, which again suggests that the oligomers can be
destabilized if exposed to too drastic treatments.
RNase A Oligomers Larger than Dimers--
Trimers and tetramers
also formed, as already shown, under various experimental conditions:
at 50 °C in 40% ethanol, at 60 °C in 20% ethanol, as well as at
60 °C in 40% ethanol or TFE and also at 70 °C. It might be
useful to remember that the major trimer (T1) could be a
linear structure, formed via both N- and C-terminal end swapping,
whereas the minor trimer (T2) is a circular structure, formed only by the C-terminal end-swapping mechanism (6, 7). The two
trimers were well separated and quantified, and the ratios of
T1 to T2 were 2.65:1 in 40% ethanol and 2.20:1
in 40% TFE. These values are the closest possible to the 1.5:1 ratio
(typical for the two trimers, T1/T2) constantly
found with the lyophilization procedure (4). The data presented in
Table IV are generally consistent. In conclusion, although the amount
of T1 slightly exceeds that of T2 under severe
unfolding conditions, the equilibrium between the two trimeric
conformers is always definitely shifted toward the major trimer
(T1) under mild unfolding conditions: the gel-filtered
trimers obtained under mild incubation conditions (23 °C,
H2O, NaCl, borate buffer at pH 10, etc.) (see Table I), analyzed by nondenaturing gel electrophoresis, showed in fact a
remarkable prevalence of T1 over T2 (data not shown).
Why Do Different Amounts of the Two Dimers Form under Different
Experimental Conditions?--
The results reported indicate that mild
unfolding conditions allow the production of higher amounts of the
N-dimer, whereas strong unfolding conditions allow the prevailing
production of the C-dimer. This means that, under mild unfolding
conditions, the opening and swapping of the N-terminal peptide
(residues 1-15) definitely prevails over the opening and swapping of
the C-terminal peptide (residues 116-124). The results concerning the
two trimeric conformers, T1 and
T2, as previously discussed, support the view that the
opening of the N terminus of RNase A is easier than the opening of its
C terminus. In this context, it is worth mentioning that the
dimension of the "closed interface" of the C-dimer is 1716 Å2 and that of the N-dimer is 1592 Å2 (6).
This difference is in line with the idea that a higher energy
contribution is necessary for the opening of the C-terminal strand.
Although the opening of the N or C terminus of RNase A can be
considered as a first step in the oligomerization process of the
protein, a second step should be responsible for the swapping and
stabilization of the oligomer(s) formed. According to Chiti et
al. (19), the regions of a protein inducing its aggregation are
those relatively rich in hydrophobic amino acids and prone to form
It is appropriate to mention that the per residue stability of
ribonuclease A has been studied by hydrogen exchange measurements in
folded monomeric RNase A (20). The results obtained revealed that the
C-terminal
It is also worth pointing out that because the global stability of
RNase A increases sharply from pH 2 to 5 and shows a broad maximum over
pH 7-10 (22), it is surprising that the amount of the N-dimer
increases at high pH values (Table I, columns 1 and 2), where the
protein is more stable. A likely explanation for these results could be
the strong ion pair interaction between Asp14
(pK < 2) and His48 (pK = 5.9) (23, 24), which links the N-terminal helix to the protein core and
breaks at neutral and high pH values when His48 titrates.
In conclusion, the results of this work appear to be in line with the
already advanced ideas that every protein can form aggregates, provided
its concentration is high and the environmental conditions are such
that they destabilize its native structure (6, 25-27). These results
contribute to the understanding of the mechanism of RNase A
oligomerization by 3D domain swapping (28) and of the role that 3D
domain swapping has in protein aggregation.
-helix of each monomer, prevailed over the C-dimer. Under more vigorous denaturing conditions, where also
the C terminus of RNase A, richer in hydrophobic amino acids, unfolded,
the C-dimer, formed by 3D domain swapping of the C-terminal
-strand,
prevailed over the other, possibly because of the induction to
aggregation promoted by the hydrophobic residues present in the C
termini of the two monomers.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-amyloid in Alzheimer's disease,
-synuclein (a primary component of Lewy bodies) in Parkinson's disease, a definitely modified form of the
prion protein in various transmissible encephalopathies, etc.), and
protein aggregation also characterizes the so-called polyglutamine expansion diseases (Huntington's disease, Kennedy's disease, etc.) (1).
-strand of each monomer (7). Models for
the putative structures of the two tetrameric conformers have also been
proposed (4, 8).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-alanine/acetic acid buffer (pH 4).
12.5% polyacrylamide gels were run at 20 mA for 100-120 min at
4 °C. Fixing and staining were performed with 12.5% trichloroacetic
acid and 0.1% Coomassie Brilliant Blue. Identification of the
aggregates was performed by comparison of their RF
values with those of the aggregates obtained by the lyophilization
procedure, already well characterized and used here as standards.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix (residues 1-15) of the RNase A monomer (5), and
the major dimer forms by 3D domain swapping of the C-terminal
-strand (residues 116-124) of each RNase A subunit (6). Here we
have simply named them the N-dimer and C-dimer, respectively, on the
basis of the mechanism of their formation (5, 6).
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Fig. 1.
Patterns of RNase A aggregates obtained by
thermal treatment under different environmental conditions.
The RNase A oligomers were obtained as follows. Eppendorf tubes
containing 2 µl (0.4 mg) of each solution were put in a
thermostatically controlled bath at one of the temperatures indicated
for 30 min (dashed red line refers to 60-min kinetics).
Then, 200 µl of 0.2 M sodium phosphate buffer (pH 6.7),
preheated at the same temperature used in the incubations, were added.
Each sample was transferred to an ice-cold bath. After 5 min at
0 °C, the sample was applied to a gel-filtration Superdex 75 HR10/30
column (A) or an ion-exchange Source 15S HR10/30 column
(B) (3). As a control, aliquots of each RNase A solution
were brought to 0 °C immediately after preparation, kept for
10 min at 0 °C, and then chromatographed at 23 °C. No significant
differences were observed compared with the data obtained by incubating
the same solutions at 23 °C. In A and B,
identification of the various oligomers formed was performed by
comparison of their elution properties with those of the already well
characterized dimers, trimers, or tetramers, recently prepared by the
lyophilization procedure (3) and used as standards in this work.
M, monomer; T, trimer; TT, tetramer;
CD, C-dimer; ND, N-dimer.
Dimeric, trimeric, and tetrameric aggregates formed by RNase A
dissolved in various media and kept for 30 min at 60 or 23 °C
20 °C) until
used. Elution was performed, and the amounts of aggregates formed were
calculated as described under "Experimental Procedures." As
controls, aliquots of each RNase A solution were brought to 0 °C
immediately after preparation, kept for 10 min at 0 °C, and
chromatographed at 23 °C. No significant differences were observed
compared with the data obtained by incubating the same solutions at
23 °C and then chromatographing. Values are means ± S.D.
ND and CD, N-dimer and C-dimer, respectively; T, trimer
(mixture of the two conformers). TT, tetramer (mixture of the two
conformers); ddH2O, double-distilled H2O.
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[in a new window]
Fig. 2.
Formation of aggregated dimers, trimers, and
tetramers by RNase A dissolved in 40% aqueous ethanol and incubated at
45, 50, or 60 °C. RNase A was dissolved in 40% ethanol at a
concentration of 200 mg/ml. For each time of incubation, Eppendorf
tubes containing 2-µl aliquots were transferred to a thermostatically
controlled water bath already brought to 45 °C
(A), 50 °C
(B), or 60 °C
(C). At the times indicated, 200 µl
of preheated 0.2 M sodium phosphate buffer (pH 6.7) were
added to each sample, and the tube was transferred to an ice-cold bath.
After 5 min, the sample was applied to a gel-filtration column and
eluted as described under "Experimental Procedures." Otherwise, it
was frozen until used. Each point of the curves represents the value of
the area of the chromatographic peak of the N-dimer
(ND; green), C-dimer
(CD; blue), trimer (T;
black), or tetramer (TT; red). Each
area is expressed as the percentage of the sum of the areas of the
peaks of all eluted RNase A species. Each point is the mean of two to
four experiments for A-C, respectively.
View larger version (22K):
[in a new window]
Fig. 3.
Formation of aggregated dimers, trimers, and
tetramers by RNase A dissolved in 40% aqueous TFE. RNase A was
dissolved in 40% TFE at a concentration of 200 mg/ml. For each
incubation time, Eppendorf tubes containing 2-µl aliquots were
transferred to a thermostatically controlled water bath already brought
to 60 °C. At the times indicated, each sample was processed as
described in the legend to Fig. 1. Each point of the curves represents
the value of the area of the chromatographic peak of the N-dimer
(ND; green), C-dimer
(CD; blue), trimer (T;
black), or tetramer (TT; red). Each
area is expressed as the percentage of the sum of the areas of the
peaks of all eluted RNase A species. Each point of the curves is the
mean of three experiments.
View larger version (23K):
[in a new window]
Fig. 4.
Formation of aggregated dimers, trimers, and
tetramers by RNase A dissolved in 20% aqueous ethanol. RNase A
was dissolved in 20% ethanol at a concentration of 200 mg/ml. For each
time of incubation, 2-µl aliquots were dispensed in Eppendorf tubes
and transferred to a thermostatically controlled water bath already
brought to 60 °C. At the times indicated, each sample was processed
as described in the legend to Fig. 1. Each point of the curves
represents the value of the area of the chromatographic peak of the
N-dimer (ND; green), C-dimer
(CD; blue), trimer (T;
black), or tetramer (TT; red). Each
area is expressed as the percentage of the sum of the areas of the
peaks of all eluted RNase A species. Each point is the mean of
four experiments.
View larger version (24K):
[in a new window]
Fig. 5.
Thermal unfolding of RNase A dissolved in
various media. In each fluid (H2O (red),
borate buffer at pH 8.5 (cyan) or 10 (black),
glycine HCl buffer at pH 3 (pink), 20% (yellow)
or 40% (blue) ethanol, or 40% TFE (green)),
RNase A was dissolved at a concentration of 1.5 mg/ml and progressively
heated within a temperature range of 10-75 °C. At each temperature,
the absorbance at 287 nm was recorded every 2 min for a total time of
40-60 min. When the absorbance remained constant for at least 10 min,
its value was recorded and used for drawing the curve. The absorbance
in H2O, extrapolated to 0 °C, was used as the
A0 value.
Formation of dimeric aggregates by RNase A dissolved in 40% TFE as a
function of time and temperature
Oligomers formed by RNase A dissolved in aqueous acetone as a function
of different acetone concentrations and time of incubation at 60 °C
Oligomers formed by RNase A dissolved in various media and incubated at
70 °C for 60 min
-sheet structures, which, at the same time, however, do not
participate in the formation of the folding nucleus. The N-terminal
-helix (residues 1-15) of RNase A contains a higher number of
hydrophilic than hydrophobic amino acids. In contrast, the C-terminal
-strand (residues 116-124) is richer in hydrophobic amino acids.
Under vigorous unfolding conditions, the hydrophobic residues present
in definitely higher proportion in the C terminus could (becoming
exposed in the partially denatured or unfolded state of the protein)
induce the aggregation event and shift the equilibrium of the process
toward the production of the C-dimer. Moreover, in the C-dimer,
Asn113, present in one strand of the hinge loop (residues
112-115), forms three additional hydrogen bonds with
Asn113 in the other strand (6), further stabilizing the
structure. Under mild experimental conditions, the unfolding of the
N-terminal region, richer in hydrophilic residues, should require a
lower energy contribution, therefore prevailing over the unfolding of the RNase A C terminus. This would then lead to the production of a
higher amount of the N-dimer compared with the C-dimer. Intermediate conditions (20% ethanol at 60 °C or 40% ethanol at 50 °C) may represent a sort of transition between the unfolding conditions favoring the formation of the C-dimer and those favoring the formation of the N-dimer. This interpretation is supported by comparison of the
results originally obtained under mild experimental conditions (all
media at 23 °C, NaCl, water, and borate buffer at 60 °C) (Fig. 1,
A and B, green lines; and Table I)
with those obtained under the same ionic conditions, but with an
increase in the incubation temperature from 60 to 70 °C (Table
IV).
-strand forms multiple strongly protected hydrogen bonds
with the rest of the protein. In contrast, most of the hydrogen bonds
formed by the N-terminal
-helix are local, i.e. within
the helix, and weak, whereas only one or two weak to moderately strong
tertiary hydrogen bonds connect the helix to the rest of the protein.
Moreover, recent kinetic unfolding experiments (21) showed that
the backbone NH groups in the N-terminal
-helix show significant
unfolding in the burst phase and exchange locally, whereas the NH
groups in the C-terminal
-strand are remarkably more resistant to
local unfolding, even under vigorous unfolding conditions (5.2 M guanidine hydrochloride (pH 8) at 10 °C), and break
only when the protein globally unfolds. These data are
consistent with the results presented in this work, viz. that more vigorous unfolding conditions are required to loosen the
C-terminal
-strand for swapping and oligomerization in comparison with the conditions sufficient to unfold the weakly attached N-terminal
-helix.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Douglas V. Laurents, Yanshun Liu, and Stefano Moro for comments and suggestions.
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FOOTNOTES |
---|
* This work was supported by Italian MURST-PRIN 2000-2001.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dedicated to the memory of Eraldo Antonini, eminent biochemist, on the 20th anniversary of his premature death.
To whom correspondence should be addressed. Tel.: 39-45-802-7166;
Fax: 39-45-802-7170; E-mail: massimo.libonati@univr.it.
Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M213146200
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ABBREVIATIONS |
---|
The abbreviation used is: TFE, 2,2,2-trifluoroethanol.
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