 |
INTRODUCTION |
The close relationship between protein oxidation and
susceptibility to proteolysis was documented in a series of
publications from our laboratory and other groups (1-6). These studies
were conducted with erythrocytes and reticulocytes of various species (7-9), with Escherichia coli (10), with primary and
permanent cell culture systems (5, 6, 11-13), and with purified
proteins and proteases (7, 8, 14, 15). A number of publications demonstrated the enhanced degradation of proteins oxidized by exposure
to hydrogen peroxide, to the superoxide anion radical (O
2),
the hydroxyl radical (OH·), or to peroxynitrite
(ONOO
) (1-6, 15). Most of these studies revealed that
the degradation of mildly oxidized proteins seems to be a normal
cellular function, whereas extensively oxidized proteins are poor
substrates for proteases and may accumulate and therefore contribute to
diseases or aging processes (16-18).
In studies using mammalian cells, cell lysates or extracts, strong
evidence was presented for the key role of a single proteolytic complex, the 20 S proteasome, in the selective recognition and degradation of oxidatively damaged proteins (5, 6, 14, 19). The basis
of this recognition still remains unknown. However, some experimental
evidence suggests the selective recognition of hydrophobic moieties at
the protein surface (14, 16, 20-22) or the importance of the
methionine oxidation product, methionine sulfoxide (14, 21). The groups
of Davies (16, 20) and Stadtman and Levine (14, 21) demonstrated
several times the enhanced proteolytic susceptibility of oxidized
proteins with increased surface hydrophobicity using various methods
such as separation on hydrophobic interaction chromatography (16, 20) or the 8-anilino-1-naphtalenesulfonic acid-dependent
fluorescence intensity (21). Levine et al. (14) demonstrated
a correlation between methionine sulfoxide formation and proteolytic
degradation. It was concluded that the oxidative modification of amino
acid side chains disrupts, at least locally, the tertiary protein
structure, which is, in turn, accompanied by exposure of hydrophobic
moieties to the surface of the protein. This increase in surface
hydrophobicity seems to be the recognition signal for the 20 S
proteasome for binding and degradation of the substrate protein (14,
16, 18, 20-22). However, until now it could not be shown directly that
protein oxidation is accompanied by the disruption of secondary and
tertiary protein structure.
The present investigation addresses the question of whether protein
oxidation is followed by amino acid side chain modifications or changes
in secondary and tertiary protein structure and whether these effects
correlate with increased proteolytic susceptibility. For our studies,
we selected the small single domain cytoplasmic protein RNase A
as a model, since this protein is exceptionally well characterized by a
variety of structure-sensitive techniques such as x-ray
crystallography (23), NMR spectroscopy (24), UV-visible and CD
spectroscopy (25), differential scanning calometric (for a
review, see Pace et al. (26)), and Fourier transform infrared (FT-IR)1
spectroscopy (27-30). In particular, FT-IR spectroscopy has proved to
be a sensitive tool for following conformational changes in proteins
(Ref. 31; for a review, see Jackson & Mantsch (32)). Peptide backbone
and side chain infrared "marker" bands can be employed as
conformation-sensitive monitors to derive structural parameters during
refolding of RNase A in solution (33, 34). Recently, time-resolved
infrared spectroscopic techniques have been employed to follow
refolding processes of RNase A in the time range of 50 ms to 15 minutes (29, 35, 36).
In the present paper, we report structural alterations, detected by
FT-IR spectroscopy, of the model protein RNase A upon exposure toward
hydrogen peroxide and upon thermal unfolding. The changes of the
protein structure were related to the results obtained by degradation
measurements of the oxidized RNase A samples by isolated 20 S proteasomes.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Highly purified lyophilized RNase A (from bovine
pancreas) was purchased from Sigma Chemie GmbH (Deisenhofen, Germany).
Proteasome Preparation--
Proteasome was isolated from
erythrocytes of outdated human blood conserves according to Hough
et al. (37). Erythrocytes were lysed in 1 mM
dithiothreitol. After the removal of membranes and nonlysed cells by
centrifugation, the proteasome was isolated by DEAE chromatography,
sucrose density gradient ultracentrifugation, and separation on a Mono
Q column of a fast protein liquid chromatography system.
RNase A Treatment with Hydrogen Peroxide--
All RNase A
treatments were performed under standardized conditions to ensure
reproducibility of the FT-IR spectroscopic and proteasome degradation
experiments. Stock solutions containing RNase A were prepared in a 10 mM cacodylat/D2O-buffer at pH* 7.1 to yield an
enzyme concentration of 20 mg/ml. The protein solutions were heated for
15 min at 60 °C to achieve complete H/D exchange of the amide
protons (28). The different H2O2 solutions (0, 10, 20, 40, and 80 mM) were prepared from a 22 M H2O2 stock solution by dilution
with the respective volume of a 10 mM
cacodylat/D2O buffer, pH* 7.1. The protein and
H2O2 solution were subsequently mixed in a
ratio of 1:1 (by volume) to a yield final protein concentration of 10 mg/ml in H2O2 concentrations of 0 (control), 5, 10, 20, and 40 mM, respectively. Therefore, the maximal
1H content of the final solutions never exceeded 0.2% (40 mM H2O2).
Measurement of Proteolytic Susceptibility--
Prior to the
measurements of the proteolytic susceptibility, all RNase A solutions
were diluted to 1 mg/ml. The degradation of native and oxidized RNase A
by the 20 S proteasome was measured by incubation of 40 µg of
substrate protein with 0.6 µg of proteasome in a proteolysis buffer
containing 50 mM HEPES (pH 7.8), 20 mM KCl, 5 mM magnesium acetate, and 1 mM dithiothreitol
for 2 h at 37 °C. The reaction was stopped by the addition of
an equal volume of trichloroacetic acid (20%). After centrifugation
(15 min, 12,000 × g) the supernatants containing
primary amines were neutralized with 1 M HEPES (pH 7.8).
Fluorescamine (0.3 mg/ml in acetone) was added under vigorous shaking,
and the fluorescence was determined at 390-nm excitation/470-nm
emission. Leucine was used as a standard. Proteolysis rates were
calculated as the difference between the measured value and the blank
value (sum of incubated substrate protein without protease and of
incubated protease only).
Mass Spectroscopy of Oxidized RNase A--
To exclude the
possibility of hydrogen peroxide-induced RNase A backbone
fragmentation, RNase A samples (10 mg/ml) were incubated for 48 h
with H2O2 (0, 1, 2, and 4 µmol of
H2O2/mg of protein) and subsequently analyzed
by matrix-assisted laser desorption ionization-time of flight mass
spectroscopy. A comparative analysis of the mass spectra indicated that
no new peaks of the oxidized RNase A occurred between 300 and 60,000 Da. Thus, it could be experimentally confirmed that mild
oxidation of RNase A does not cause protein backbone fragmentation.
However, small shifts of RNase A peaks were observed. We interpreted
these experimental findings as a result of covalent side chain
modification of RNase A.
FT-IR Spectroscopic Measurements--
Infrared spectra were
recorded using a Bruker IFS28B FT-IR spectrometer equipped with a
computer-controlled sample changer and a deuterated triglycin sulfate
detector. To eliminate spectral contributions due to atmospheric water
vapor, the instrument was continuously purged with dry air. After
mixing, the solutions of RNase A and H2O2 were
quickly transferred to an IR sample cell consisting of a pair of
CaF2 windows separated by a 50-µm spacer. FT-IR
measurements were carried out at a temperature of 30 °C. For each
spectrum, 1024 interferograms were coadded and Fourier-transformed employing a Happ-Genzel apodization function and a zero filling factor
of 4. Nominal resolution was 4 cm
1. For
Fourier self-deconvolution, the software routines implemented in the
manufacturer software package OPUS (Bruker) were used. Second
derivative spectra were evaluated applying a Savitzky-Golay algorithm
with seven smoothing points. To visualize and to interpret small
spectroscopic changes, difference spectra Dt were calculated in the following way,
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(Eq. 1)
|
where Dt is the difference spectrum at a
given time t, while Alast and
At are absorbance spectra recorded at the end of a
measurement series or at time t. Therefore, negative bands
of the difference spectra indicate decreasing amounts of the respective
structures, and vice versa.
FT-IR Temperature Gradient Measurements--
Temperature
profiles were carried out on a Bruker IFS66 FT-IR spectrometer, which
was equipped with a deuterated triglycin sulfate detector. Again, a
Happ-Genzel apodization function, a zero filling factor of 4, and a
nominal resolution of 4 cm
1 was used. For
temperature profile measurements, infrared spectra were collected
continuously in a thermostated IR cuvette with an optical path length
of 50 µm. For this type of measurement, RNase A was dissolved in 10 mM cacodylat/D2O buffer at pH* 7.1 to a
concentration of 20 mg/ml. The protein was unfolded and refolded in two
consecutive cycles by applying a linear temperature gradient of 0.5 K/min between 20 and 80 °C and back to 20 °C. Complete H/D
exchange of the amide protons was obtained after the first heating
cycle. For data evaluation, only spectra of the second heating run
(unfolding) were utilized. Infrared spectra were corrected for spectral
contributions of buffer and water vapor as described previously (33).
Difference spectra were calculated in the same way as outlined above.
Calculation of the Accessible Surface Area--
The accessible
surface areas (ASA) were calculated using the program Naccess version
2.1 (38), which is an implementation of the Lee and Richards method
(39). The program calculates the atomic accessible surface defined by
rolling a probe of given size around a van der Waals surface. A slice
thickness of 0.05 Å and a probe size of 1.4 Å for H2O and
2.1 Å for H2O2 was used. The latter value was
estimated on the basis of the bond length and the atom radii of
O2 and F2. We utilized an output file
containing summed atomic ASA over each residue. For these calculations,
the coordinates from the Protein Data Bank ID 1rbx (RNase A without ligand) by J. L. H. Dunbar and G. K. Farber (40) were used.
 |
RESULTS |
To obtain new insights into the recognition process of oxidized
proteins by the proteasome, we combined two different techniques: structural changes of the model protein RNase A upon oxidation by
hydrogen peroxide were followed by FT-IR spectroscopy, while the
changes of susceptibility to proteasome degradation were tested by
biochemical methods. For this purpose, stock solutions of RNase A and
H2O2 were prepared and used for both kinds of
experiments. Although the use of D2O buffers or fully
H/D-exchanged proteins is not essential for the proteasome degradation
characterizations, we tried to maintain the experimental protocols as
much as possible. Therefore, the first steps of proteasome digestion
were performed in D2O buffers after a complete H/D exchange
of the protein.
Proteolytic Susceptibility of RNase A--
The degradation of
various proteins by the 20 S proteasome was already measured by several
groups for a number of different proteins (5, 14, 15). Since most of
these experiments were performed under standardized conditions, we used
the same experimental protocol to study the proteolytic susceptibility
of RNase A toward the 20 S proteasome. The results of the
investigations are demonstrated in Fig.
1. When RNase A is treated with hydrogen
peroxide concentrations up to 4 µmol/mg of protein (approximately 55 molecules of H2O2 per molecule of RNase A), an
increase in proteolytic susceptibility of the resulting oxidized
proteins is detected. At higher concentrations of hydrogen peroxide, a
decrease of the proteolytic susceptibility was measured. As previously
postulated and shown earlier (5, 13, 14, 41, 42), this decline is due
to the irreversible formation of protein aggregates. These protein
aggregates are poor substrates for the protease or may even be able to
inhibit the 20 S proteasome (15, 18, 41, 42). One of the objectives of
the present work was to investigate the structural rearrangements of
the protein induced by oxidation, which causes the recognition and the
degradation of the protein by the 20 S proteasome. Consequently, hydrogen peroxide concentrations below 4 µmol/mg of protein were applied in all experiments. Since FT-IR spectroscopy requires relatively high protein concentrations (~10 mg/ml), the hydrogen peroxide concentration was varied from 0 (control) to 40 mM. The hydrogen peroxide/protein concentration ratio was
therefore identical to that utilized in the proteasome degradation
experiments (0-4 µmol of H2O2/mg of
protein).

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Fig. 1.
Degradation of hydrogen peroxide-treated
RNase A by the 20 S proteasome. RNase A (1 mg/ml) was treated with
the indicated concentrations of hydrogen peroxide for 2 h and then
extensively washed by ultrafiltration and incubated with the isolated
20 S proteasome. Proteolysis was measured by analyzing the acid-soluble
supernatant for free amines. The data represent the mean ± S.D.
of three independent experiments.
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FT-IR Spectroscopic Characterization of Oxidized RNase
A--
FT-IR spectroscopic studies of proteins in aqueous solutions
are frequently performed in D2O buffers for the following
reasons. The amide I band (1620-1690 cm
1),
arising predominantly from the C=O oscillators of the secondary amide
function of protein backbone, is superimposed on the deformation band
at 1643 cm
1 of the solvent H2O.
Thus, in many FT-IR spectroscopic studies on protein structure,
H2O is substituted by 2H2O
(D2O), which exhibits its deformation band at 1210 cm
1. Thus, the use of D2O instead
of H2O as the solvent makes it possible to analyze spectral
features in the secondary structure-sensitive amide I region without
interference of bulk water.
The interpretation and quantification of structural changes of RNase A
during oxidation by hydrogen peroxide was carried out, comparing these
data with infrared spectra for thermally unfolded protein. It is known
that RNase A can be reversibly unfolded thermally, giving rise to a
characteristic spectral unfolding pattern, particularly in the amide I
region (29, 33, 35, 43). In the present study, FT-IR spectra were
collected between 20 and 80 °C, applying two consecutive heating and
cooling cycles with a linear temperature gradient of 0.5 K/min. The
FT-IR spectrum of native RNase A is shown in Fig.
2. This spectrum was recorded at a
temperature of 30 °C after one heating and one cooling cycle
(i.e. after complete thermal unfolding and refolding), which
accelerates the exchange of the amide protons by deuterons (in the
following, this procedure is called H/D exchange). The
dotted line in Fig. 2A displays the absorbance spectrum corrected for the spectral contributions of the
buffer, while the solid line represents the
corresponding Fourier self-deconvolution spectrum (Fig. 2A)
to demonstrate the fine structure of the amide I band. A second
derivative spectrum (Fig. 2B) was also calculated from the
absorbance spectrum (positive bands of an absorbance spectrum appear in
second derivative spectra as negative bands). From the literature, it
is known that the energy of the C=O oscillators of the protein backbone
depends on the coupling to adjacent C=O oscillators and the strength of the hydrogen bonds. Furthermore, the strength of these bonds and the
symmetry of hydrogen bond patterns is characteristic for distinct secondary structure elements of proteins. It is therefore possible to
distinguish various secondary structures from the experimentally observed band components of the amide I' band. For RNase A, the band
assignment was carried out according to literature data (27, 29, 43,
44). In good agreement with these studies, IR marker bands for
antiparallel
-pleated sheets were found at 1631 and 1680 cm
1, for
-helix at 1651 cm
1, and for unordered turn structures at
1665 cm
1 (cf. Fig. 2). Also,
infrared absorption bands of the amino acid side chains such as the
"tyrosine band" (aromatic tyrosine ring vibration at 1515 cm
1) or absorptions of aspartate and
glutamate residues at 1584 and 1566 cm
1,
respectively, were observed in agreement with previous studies (45,
46).

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Fig. 2.
FT-IR spectra of native RNase A at a
temperature of 30 °C. The dotted curve
displays an FT-IR spectrum of RNase A (buffer subtracted), while the
solid curves were obtained applying "resolution enhancement
techniques" to the original data (curve
A, Fourier self-deconvolution; curve
B, second derivative spectrum). The amide I region of RNase
A spectra (1690-1620 cm 1) is dominated by
band components at 1631 and 1680 cm 1, both
assigned to antiparallel -pleated sheets. Other amide I band
components at 1665 cm 1 (unordered structure)
and 1651 cm 1 (assigned to -helical
structures) are indicated. Absorptions of amino acid side chains at
1515 cm 1 (tyrosine ring vibration) and near
1584 and 1566 cm 1 (-COO
vibrational modes of the aspartate and glutamate residues), although
less intense than the amide I band, can be used to obtain additional
structural information. AU, absorbance units.
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Spectroscopic parameters of the amino acid side chain absorption bands
can be utilized to derive structural information on the specific
microenvironment of these functional groups. For example, the frequency
of the tyrosine band at 1515 cm
1 was used to
monitor specifically the formation of tertiary contacts upon refolding
(29). Fig. 3A displays a
series of Fourier self-deconvolution infrared spectra (corrected for
buffer) obtained by a linear temperature gradient measurement. As
previously described for RNase A (29, 43), the appearance of a broad
and featureless amide I band suggests the lack of stable secondary
structure elements at temperatures above 70 °C.

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Fig. 3.
Thermal unfolding of RNase A. A, series of FT-IR spectra in the amide I' region of RNase A
(20 mg/ml) in D2O buffer (10 mM cacodylat) at
pH* 7.1 is shown. The thermal unfolding was studied under control of a
linear temperature increase from 20 to 80 °C with a constant heating
rate of 0.5 K/min. FT-IR spectra were acquired at every temperature K. A shows buffer-subtracted and Fourier self-deconvoluted
FT-IR spectra of RNase A, while the B contains the
corresponding difference spectra (see "Experimental
Procedures"). AU, absorbance units.
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A frequently used approach to illustrate temperature-induced spectral
changes is given in Fig. 3B, which shows a series of FT-IR
difference spectra. These difference spectra have been calculated according to Dx = A80 °C
Ax, where A80 °C is the
absorbance spectrum at 80 °C and Ax is an
absorbance spectrum at the temperature x. Two negative bands at 1631 and 1680 cm
1 reflect the
disappearance of antiparallel
-pleated sheet structures upon
temperature-induced unfolding. The broad band between 1651 and 1680 cm
1 (Fig. 3A) indicates the
thermally induced formation of unordered structures. These
spectroscopic changes induced by thermal unfolding of RNase A will be
compared quantitatively with the spectroscopic effects observed during
the oxidation of RNase A by hydrogen peroxide. For quantification of
the fraction of unfolded protein, the absorbance/temperature dependence
of the most prominent amide I' contour at 1631 cm
1 (low frequency
-band) was analyzed
(see Fig. 4). Obviously, three main
phases of this absorbance/temperature plot can be observed: a linear
low temperature region (below 53 °C), a sigmoidal transition region
(53-72 °C), and a second linear post-transitional high temperature
region above 72 °C. The midpoint (inflection point) of the protein
melting curve Tm, at which 50% of RNase A is
supposedly in the unfolded state, was determined by curve fitting to be
64.5 °C, in accordance with literature data (Backmann et
al. (35): 64.5 °C.; Reinstädler et al. (29):
63 °C).

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Fig. 4.
Folding/unfolding transition profile as
obtained from the absorbance change of the 1631 cm 1 band (antiparallel
-pleated sheet structure). The absorbance
values were obtained from buffer-subtracted FT-IR spectra of a heating
run from 20 to 80 °C applying a constant heating rate of 0.5 K/min.
Both the dependence in the low (<50 °C) and the high temperature
region (>72 °C) were fitted with a linear function and extrapolated
to a temperature of 30 °C. Assuming that RNase A is completely
unfolded at T > 72 °C and completely folded at
30 °C, one can equate the absorbance difference
A30 °C (equal to 0.073 absorbance units
(AU)) between both states to 100% loss of secondary
structure of the protein. The ratio
( A/Af)30 °C of the
temperature unfolding experiment is concentration-independent and was
therefore used for quantification of
H2O2-induced structural alterations of RNase A. Quantitative estimations of other structure-sensitive IR bands
(e.g. the tyrosine ring vibration band at 1515 cm 1) were carried out in the same way.
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Two linear fits of the high and low temperature region and the
extrapolation of both curves to the temperature of the protein degradation experiments of 30 °C allowed us to estimate the value of
absorbance difference
A30 °C. This value
indicates the absorbance difference between the completely folded and
the completely unfolded states of the protein at a temperature of 30 °C. Finally, to account for the protein concentration dependence, the
A30 °C value was divided by the
absorbance of the completely folded RNase A species
Af,30 °C (see Fig. 4). The relation
(
A/Af)30 °C was set to 100% and was found to be particularly useful in quantifying the
IR-spectroscopic protein denaturation features induced by H2O2. The procedure described can be applied
similarly to obtain quantitative information from other infrared marker
bands (e.g. from the tyrosine band at 1515 cm
1).
The spectroscopic features of the temperature-induced reversible
unfolding and the partial denaturation of RNase A induced by
H2O2 are illustrated in Fig.
5. The upper curve of Fig. 5
(curve a) shows a second derivative spectrum of
the native protein. The peak positions of the major bands (amide I',
amino acid side chain absorptions) are indicated. Spectrum
b was recorded during the heating cycle of the linear
temperature gradient experiment at a temperature of ~65 °C.
Obviously, the band contour has changed significantly due to the
temperature-induced partial melting of the protein. The second
derivative spectrum c was recorded under isothermic conditions after 48 h of incubation of RNase A with H2O2 at a concentration of 4 µmol/mg of
protein. A comparison of spectra b and
c clearly demonstrates that thermal unfolding and hydrogen
peroxide degradation lead to similar FT-IR spectroscopic changes,
characterized by the partial disappearance of several narrow amide I'
band components. These are the high and low frequency
-bands at 1680 cm
1 and 1631 cm
1
and the peak at 1651 cm
1, which was assigned
to
-helical structures. However, it should be pointed out that
spectra b and c were recorded at
different temperatures (30 and 65 °C), which may help explain why
they are not identical. Spectrum d of Fig. 5 was
recorded at 80 °C and displays the typical spectral features of
completely unfolded RNase A.

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Fig. 5.
Spectral similarities of temperature- and
hydrogen peroxide-induced structural alterations of RNase A. The
uppermost curve (a) shows a second derivative spectrum of
the native protein RNase A in the amide I' and the amino acid side
chain absorption region (after complete H/D exchange, T = 30 °C). The amide II band (near 1550 cm 1, typical for 1H protein
solutions, can be found in 2H solutions near 1450 cm 1 (amide II'-band, not shown).
Spectrum b was acquired near the melting point of
the protein (Tm = 64.5 °C), where ~50% of the
protein is unfolded. In contrast, spectrum c was
recorded after 48 h of incubation of a solution containing
initially 4 µmol of H2O2/mg of RNase A. Spectrum d was obtained at a temperature of
80 °C, where RNase A is expected to exist in a completely unfolded
state. The second derivative spectra a-d were
normalized utilizing the tyrosine ring vibration band at 1515 cm 1 as an internal standard. A comparison of
spectra b (temperature induced unfolding) and
c (oxidation) indicates a high degree of conformity of the
IR spectroscopic changes, particularly in the secondary
structure-sensitive amide I' region.
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Fig. 6 displays a series of FT-IR
difference spectra recorded during the oxidation of RNase A by hydrogen
peroxide. Fig. 6A shows IR difference spectra of RNase A in
the region of 1800-1400 cm
1 after an
incubation time of 48 h in 0 µmol of
H2O2/mg of protein (a, control), 0.5 µmol of H2O2/mg of protein (b), 1 µmol of H2O2/mg of protein (c) 2 µmol of H2O2/mg of protein (d),
and 4 µmol of H2O2/mg of protein
(e). Fig. 6 (B and C) displays a time
evolution of difference spectra, obtained for the 4 µmol of
H2O2/mg of protein samples. For both
experiments shown in Fig. 6, difference spectra were calculated
according to Dt = Alast
At, where At is the absorbance
spectrum at a given time t, and Alast
is the last absorbance spectrum of the FT-IR spectroscopic time series.
Again, negative bands indicate disappearance of respective structure
elements upon hydrogen peroxide-induced protein oxidation, and
vice versa. A comparison of the IR spectral differences
observed for the temperature-induced unfolding of RNase A (Fig. 3) and the hydrogen peroxide-mediated RNase A degradation (Fig. 6) suggests remarkable similarities in the structure change between both processes. The most notable spectral changes are observed in the amide I' region,
particularly at 1631 and 1680 cm
1 (low and
high frequency antiparallel
-pleated sheet structures). Another
interesting feature that coincides with variations in the amide I'
region is a subtle but highly reproducible frequency shift of the
tyrosine band at 1515.20 cm
1 of about

30 °C
0.5 cm
1 to
higher wave numbers. This behavior is interpreted in the literature as
a consequence of changes in the microenvironment of the aromatic tyrosine residues (36, 47). Upon unfolding of RNase A, the aromatic
tyrosine residues become more solvent-exposed, resulting in the
appearance of a new band at 1515.70 cm
1. This
band cannot be resolved spectroscopically from the original tyrosine
band at 1515.20 cm
1 but is observed via a
"band shift" to higher frequency. The "frequency shift" of the
tyrosine band near 1515 cm
1 is accompanied by
changes of the absorbance values of this band. Upon temperature-induced
unfolding of RNase A between 53 and 72 °C, the absorbance value of
the tyrosine band increases. This temperature-dependent
spectral effect is very small (cf. Fig. 3) compared with the
changes observed in the amide I region. Interestingly, the difference
spectra shown in Fig. 6, A-C, display a clear decrease of
absorbances of the tyrosine band with increasing hydrogen peroxide concentration or time, respectively. Furthermore, for the aspartate absorption band at 1584 cm
1, the decrease of
absorbance (at 4 µmol of hydrogen peroxide/mg of protein, 48-h
incubation time) was about twice as high as the corresponding effect
observed during complete unfolding of RNase A. Obviously, some specific
marker bands due to distinct amino acid residues may indicate covalent
modifications of amino side chains by hydrogen peroxide. In contrast,
changes in absorbance value of the amide I band contour indicate global
conformation changes.

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Fig. 6.
Concentration and time dependence of hydrogen
peroxide-induced alterations of RNase A structure. A
shows FT-IR difference spectra of RNase A, incubated with varying
H2O2 concentrations (T = 30 °C, t = 48 h). Difference spectra were
calculated according to Equation 1. Negative bands indicate
disappearance of structure elements upon RNase A oxidation.
H2O2/RNase A concentration ratios were as
follows: 0 (a, control), 0.5 (b), 1.0 (c), 2.0 (d), and 4.0 µmol/mg of protein
(e). B and C illustrate the time
dependence of the spectral changes of RNase A at 4 µmol of
H2O2/mg of protein concentration (pH* 7.1, tmax = 48 h). C, illustration
of the spectral changes below 1500 cm 1. A
small positive peak in the difference spectra at 1047 cm 1 ((R2)-S=O stretching
vibration?) is indicated by an arrow. The amplitude of this
difference peak is comparable with the intensity changes of the
tyrosine band at 1515 cm 1 (also marked).
AU, absorbance units.
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The difference spectra, given in Figs. 3B and 6B,
also exhibit significant variations in the spectral region from 1620 to 1580 cm
1. While the difference spectra
obtained upon reversible thermal unfolding of RNase A display no
significant changes in this spectral region (Fig. 3B), at
least one positive band at 1594 cm
1 is found
for the RNase A oxidation measurement series (Fig. 6B). This
band is assigned in the literature to an asymmetric carboxylate stretching vibration (
(-COO
)as). The
generation of additional carboxylate groups during protein oxidation
may be interpreted as a result of disruption of covalent bonds of amino
acid side chains. To some extent, however, these spectral features may
overlap with a so-called
-aggregation band near 1615 cm
1. The formation of protein aggregates is
widely observed for many proteins (48-51). A more detailed examination
of difference spectra in Fig. 6B reveals the simultaneous
appearance of an additional shoulder at 1714 cm
1 in the positive band contour around 1695 cm
1, which is not present in the difference
spectra of the unfolding experiments (Fig. 3B). Carbonyl
stretching bands can be expected around this wave number. Some authors
reported the generation of carbonyl groups during the oxidation of
amino acid side chains producing carbonyl derivatives (1, 52).
Fig. 6C shows the difference spectra of Fig. 6B
below 1500 cm
1. These difference spectra
display a small positive peak at 1047 cm
1
((R2)-S=O stretching vibration), which is indicated by an
arrow. The amplitude of this difference peak is comparable
with the intensity changes of the tyrosine band at 1515 cm
1 (also marked by an arrow). The
difference pattern in the spectral region 1180-1240
cm
1 results from the very strong
D2O deformation signal at 1210 cm
1.
Fig. 7 illustrates the
time-dependent structural changes of RNase A induced by
H2O2 treatment. These plots were derived using the absorbances of the low frequency
-band at 1631 cm
1 (Fig. 7A) and the absorbance
values of the tyrosine band at 1515 cm
1 (Fig.
7B). To estimate quantitatively the changes of distinct structural elements as a function of H2O2
treatment, the following model was used. From the temperature gradient
measurements, the value of the parameter
(
A/Af)30 °C is known. In the example of Fig. 4, this ratio indicates the maximal absorbance change of the low frequency antiparallel
-pleated sheet band at 1631 cm
1, which is a concentration-independent
ratio describing complete unfolding of the protein. According to
Equation 2, this ratio can be used to estimate the percentage of
hydrogen peroxide induced structural changes of RNase A.
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(Eq. 2)
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The denominator of this equation was calculated from spectral
parameters of the temperature profile measurements, and the numerator
was calculated from parameters of the hydrogen peroxide experiments.
P0 and Pt are any
structure-sensitive spectroscopic parameter like the absorbance at a
given frequency, band frequency, or the half-width at t = 0 or at a given time t, respectively. We used Equation 2
to compare semiquantitatively the structural changes of RNase A
oxidation with the structural changes detected during
temperature-induced unfolding. This approach allowed us to analyze
structural changes in RNase A using the infrared marker bands from the
amino acid side chains or secondary structure-sensitive components of
the amide I band.

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Fig. 7.
Time and concentration dependence of hydrogen
peroxide-induced structural changes of RNase A as seen by FT-IR
difference spectroscopy using the low frequency band of
antiparallel -pleated sheets at 1631 cm 1 as a secondary
structure-sensitive monitor (A) or a local infrared
marker (absorbance of the tyrosine ring vibration band near 1515 cm 1) (B). See
inset for the ratio of H2O2/RNase A
concentrations.
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Fig. 7 shows the time and concentration dependence of the absorbances
of two distinct spectral marker bands: the low frequency antiparallel
-pleated sheet band at 1631 cm
1, and the
tyrosine band at 1515 cm
1. Generally, the
higher the concentration of hydrogen peroxide and the longer the
incubation time, the larger were the structural alterations detected by
FT-IR spectroscopy. The plots of Fig. 7, A and B,
and the quantitative results of Table
I derived from these plots suggest
that substantial differences exists between various secondary
structure-sensitive marker bands (amide I band, absorbances at 1680, 1651, and 1631 cm
1) and IR bands of defined
amino acid residues (absorbance of the tyrosine and aspartate band at
1515 and 1584 cm
1). While the direction and
the time-dependent changes in the amide I region indicate
an "unfolding-like" behavior of the protein as a result of
H2O2 treatment, the information derived from
the amino acid side chain bands cannot be explained simply on the basis
of protein unfolding (see ordinate values of Fig.
7B). These interesting findings will be discussed below.
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Table I
Quantitative estimations of changes upon H2O2 treatment
of RNase A marker bands ( t = 48 h)
All data are given in percent relative to spectral alterations of RNase
A during complete thermal unfolding (see "Results"). While infrared
bands for secondary structure elements (e.g.
" -bands") or the tyrosine peak shift (tertiary contacts) monitor
the global unfolding process, other spectral markers like the
absorbance of Tyr or Asp bands probably reflect local events,
presumably covalent modifications of the amino acid side chains induced
by hydrogen peroxide.
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Oxidation of RNase A by Hydrogen Peroxide (120-h
Incubation)--
Armed with the knowledge that the oxidative damage of
RNase A may be accompanied by conformational and covalent
modifications, we undertook a series of experiments to detect subtle
spectral changes, especially of the amino acid side chain absorptions. Those measurements required a very high spectral signal/noise ratio and
instrument stability. Therefore, the sampling time for each individual
spectrum of these experiments was increased to 20 min (2200 scans), and
the time of data collection was increased to 120 h. The
experiments shown in Fig. 7 (48-h duration) were carried out at a
temperature of 30 °C. The protein concentration and the composition
of the buffer solutions were not changed. To analyze the smallest
spectroscopic effects detectable and to minimize unavoidable base line
shifts (a common problem of long time measurements), the spectroscopic
parameters were calculated exclusively from second derivative spectra
(see Fig. 8). High signal/noise ratio
(see Fig. 8, B or C), stability of the
experimental setup over 5 days (temperature, water vapor content,
etc.), and state-of-the-art data evaluation were essential
prerequisites to be able to obtain information from these types of
experiments.

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Fig. 8.
A comparison of spectral changes induced by
temperature increase of RNase A solutions (unfolding) and hydrogen
peroxide-induced structural alterations of RNase. All
spectroscopic parameters of this figure were calculated from
second derivative spectra. Left row, time dependence of band
parameters obtained on a hydrogen peroxide/RNase A incubation
experiment (I) and the respective control experiment
(II). The relation of intensity values at a given time
t and t = 0 (A and B)
or frequency values (C) were calculated and plotted as a
function of time. Curves in A were obtained, using the
absorbance information at 1631 cm 1 (low
frequency -pleated sheet band), while B and C
display the time dependence of the absorbance or frequency parameters
of the tyrosine band at 1515 cm 1,
respectively. Right, temperature profile measurements of
RNase A. D and E display the normalized second
derivative/temperature dependence at 1631 and 1515 cm 1, respectively, while F shows
the frequency/temperature dependence of the tyrosine band. A comparison
of the curves in the left and right rows
indicates that hydrogen peroxide-induced structural alterations of
RNase A are to some extent similar to spectral changes during the
unfolding of the protein. However, the structural information derived
particularly from infrared marker bands of the amino acid side chains
makes it evident that additional "local" events (e.g.
oxidation of aromatic residues) may take place (see
"Results").
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The interpretation of band parameters derived from second derivative
spectra is not as straightforward as for data obtained from the
original absorbance spectra. The calculation of
d(A)2/d2(
) values from a
simulated Lorentz band yields a negative band where the position of the
minimum of the second derivative band coincides with the frequency
value of the maximum of the original Lorentzian, while the ordinate
value at the minimum depends on the half-width and the absorbance value
of the Lorentz band. Therefore, one can utilize second derivative
intensities for comparative purposes in the same way as absorbances
from original spectra, provided that the half-widths of the bands are
constant and do not depend on temperature or other experimental
parameters. For the determination of band frequency, second derivatives
are even more precise than the original absorbance spectra.
In Fig. 8, the low frequency
-pleated sheet band absorbance at 1631 cm
1 (Fig. 8, A and D)
and the absorbance and the frequency values of the tyrosine band at
1515 cm
1 (Fig. 8, B, C,
E, and F) were evaluated from second derivative spectra. While the curves in the left column of
Fig. 8 (A-C) display the time dependence of the structural
alterations of RNase A induced by hydrogen peroxide, the plots of the
right column (D-F) show the
corresponding changes during temperature-induced unfolding of the
protein. The curves (I) of the left
panels display the fraction of structural change during
RNase A oxidation at 1 µmol of H2O2/mg of
protein, while the corresponding results of the control experiment
without H2O2 are given by curves
(II). The protein melting curves of Fig. 8, D and
E, were corrected for linear temperature effects and
normalized according to a method proposed by Heyn (53). The calculation
of folded/unfolded protein fractions in the hydrogen peroxide
measurement series was carried out using Equation 2, where the spectral
parameters Pi were obtained from second derivative spectra.
Curve I in Fig. 8A indicates a
significant decrease of the absorbances of the low frequency
-band
intensity at 1631 cm
1 in accordance with the
measurement series of Figs. 5-7. In general, the magnitude and the
direction of the time-dependent changes detected in the
amide I region coincide with those observed during thermal unfolding of
RNase A. In contrast, the spectral changes of the tyrosine band at 1515 cm
1 (induced by hydrogen peroxide or by
temperature variation) have an opposite direction. As indicated by the
FT-IR difference spectra of Fig. 6, A-C, the intensity of
the tyrosine band is decreasing as a function of hydrogen peroxide
concentration and incubation time, while it is increasing with
temperature-induced unfolding of the protein. Consequently, the
ordinate values of Fig. 8B are negative (see also Fig.
7B). Specifically, the absorbance changes in the amide I
region indicate a decrease of about 70% of secondary structure
elements after 120 h of incubation time at 1 µmol of H2O2/mg of protein, while the corresponding
tyrosine band absorbances changes are comparable in the magnitude but
opposed in direction. Interestingly, the absorbance time-dependence of
the "aspartate band" at 1584 cm
1 exhibits
much larger changes in the oxidation experiment compared with the
effects of the unfolding measurements (cf. Table I). But in
contrast to the behavior of the tyrosine band, the intensity of the
aspartate band at 1584 cm
1 is diminishing
upon unfolding of RNase A as well as upon oxidation. Thus, relative
absorbance changes calculated by Equation 2 were found to be positive
for the aspartate band at 1584 cm
1 and
negative for the tyrosine band at 1515 cm
1
(Table 1).
In analogy to the analysis of the band intensity of the
-structure
band at 1631 cm
1, the frequency analysis of
the tyrosine ring vibration band at 1515 cm
1
indicates similarity between temperature (Fig. 8F) and
hydrogen peroxide (Fig. 8C)-induced structural alterations
of RNase A. The temperature-induced unfolding is accompanied by a
tyrosine "peak shift" to higher wave numbers by about

30 °C
0.4 cm
1. A
similar effect was observed for the 120-h H2O2
incubation experiment, where a tyrosine peak shift of about 0.35 cm
1 indicates a global unfolding of about
70% of the RNase A molecules. Interestingly, this value is close to
that determined from the high frequency
-pleated sheet band at 1680 cm
1 (not shown) and from the low frequency
-pleated sheet band at 1631 cm
1
(cf. Fig. 8A). Presumably, the intensity of the
IR marker bands such as the high and the low frequency
-pleated
sheet band at 1680 and 1631 cm
1 and the
frequency of the tyrosine band monitor global folding/unfolding events
of the protein, while other spectral markers such as the intensities of
the tyrosine and the aspartate bands reflect more local
consequences of protein oxidation (chemical modifications).
Time Dependence of the Proteolytic Susceptibility of RNase
A--
As demonstrated by Fig. 1, the reaction with hydrogen peroxide
up to 4 µmol of H2O2/mg of RNase A leads to
an increase of proteolytic susceptibility of RNase A. Since it is known
that protein damage is not only a concentration-dependent
but also a time-dependent process, we also investigated the
time dependence of the proteolytic susceptibility of RNase A. In this
study, only the incubation time of RNase A with hydrogen peroxide was
varied, while the reaction time of the 20 S proteasome with the
oxidized RNase A was kept constant (2 h). In this way, we measured the proteolytic susceptibility of the substrate and not a maximal amount of
protein oxidation. As shown in Fig. 9, a
time-dependent increase in the proteolytic susceptibility
of the RNase A up to 48 h was detected. At all time points tested,
a clear dependence of proteolytic rates on the
H2O2 concentration was found.

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Fig. 9.
Concentration- and time-related increase in
degradation of hydrogen peroxide-treated RNase A by the isolated 20 S
proteasome. RNase A solutions were treated with the indicated
concentrations of hydrogen peroxide. After mixing and incubation for a
given time (see inset), the proteins were washed and diluted
from the initial concentration of 10 to 1 mg/ml and incubated afterward
for 2 h with the isolated 20 S proteasome. Proteolysis was
measured by analyzing the acid-soluble supernatant for free amines. The
data represent the mean of three independent experiments (S.D. less
then 10%). The experimental data clearly show an increase of the
concentration of free amines as a function of time and
H2O2 concentration.
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Correlation of Proteolytic Susceptibility and FT-IR Spectroscopic
Changes--
One aim of this study was to get new insights into the
molecular basis of the recognition process of mildly oxidized RNase A
by the 20 S proteasome. Assuming that distinct structure-sensitive IR
spectroscopic parameters of oxidized RNase A are related to the
proteolysis rates, we correlated hydrogen peroxide-induced changes of
specific infrared bands with the proteolytic susceptibility of RNase A
toward the 20 S proteasome.
Fig. 10 clearly shows that hydrogen
peroxide-induced structural reorganization of the protein coincides
with increased proteolysis rates of RNase A. For all infrared bands
analyzed in this study, i.e. for "global" as well as for
"local" parameters, a clear linear dependence between structural
changes and the proteolytic susceptibility was detected. Consequently,
this approach did not allow the identification of a specific
recognition site causing proteasome binding and subsequent protein
degradation. However, an improved experimental setup to increase the
sensitivity and the time resolution of the experiment may be helpful to
address this problem more adequately.

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Fig. 10.
Correlation of structural changes as seen by
FT-IR spectroscopy and the proteolytic susceptibility of RNase A. For hydrogen peroxide/RNase A ratios below 4 µmol/mg of protein, the
spectral changes of the following infrared marker bands were related to
the proteolytic susceptibility of RNase A to digestion by the 20 S
proteasome: absorbance values of the low frequency -pleated sheet
band at 1631 cm 1 (100% = complete unfolding;
A) frequency of the tyrosine band at 1515 cm 1 (A), and the absorbance of the
tyrosine band (B).
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DISCUSSION |
Covalent modifications of proteins by oxidative agents are thought
to play a key role in various physiological and pathological conditions
such as inflammation, ischemia reperfusion, or aging (for reviews see
Oliver et al. (54) and Stadtman (55)). It has been shown
that oxidative modification of proteins can be mediated by a number of
different systems including oxidases, ozone, hydrogen peroxide,
hypochloride, superoxide,
-irradiation, and metal-catalyzed
oxidation, to mention a few. As a result of protein oxidation, an
accumulation of enzymes with partially altered structure and function
is observed. These proteins exhibit changes in thermostability (41)
and, when mildly oxidized, show an increased susceptibility to
degradation by proteasome (1, 2, 5, 6, 15, 18), which is known
to be a part of a complex antioxidant repair and removal system. This
main intracellular proteolytic system for the degradation of
oxidatively damaged proteins exists in at least two forms, the ATP- and
ubiquitin-dependent 26 S form and an independent 20 S form.
The 20 S proteasome used in this study is a 700-kDa soluble proteinase
complex that is found in the cytosol and the nucleus of mammalian cells
(56). Proteolysis of oxidatively damaged proteins by the 20 S form of
the proteasome seems to be the major pathway (5, 6, 18). As observed by
a number of studies, proteolysis by the 20 S proteasome is most
efficient after treatment of the protein with moderate oxidative stress, whereas greater oxidative damage actually leads to decreased proteolytic susceptibility (15, 17, 57). It has been shown that the
formation of protein aggregates, by whatever mechanism, may contribute
to the decrease of proteolytic rate (41).
However, the molecular basis of the recognition process of mildly
oxidized proteins by the proteasome still remains questionable. Experimental evidence for a possible role of increased surface hydrophobicity (16, 20-22), the formation of dityrosines (57), the
conversion of methionine to methionine sulfoxide (21), and the increase
of reactive carbonyl content (21) were found. Nevertheless, an in
vitro study showing a direct relationship between structural changes of a model protein and proteolytic susceptibility to the 20 S
proteasome is still lacking. To get new insights into the recognition
process of mildly oxidized proteins by the 20 S proteasome, we combined
a structure-sensitive spectroscopic technique, FT-IR spectroscopy, with
measurements of the proteolytic susceptibility of RNase A toward the 20 S proteasome. We report here experimental evidence for
oxidation-induced conformational rearrangements of the secondary
structure of the model protein RNase A and, at the same time, for
covalent modifications of amino acid side chains such as tyrosine and
aspartate residues. These modifications could be correlated to the
proteasome-induced proteolysis rate of oxidatively damaged RNase A.
Oxidatively damaged RNase A can be degraded by the 20 S proteasome.
This degradation exhibits a typical biphasic dependence; at comparably
low concentrations of H2O2, the proteolytic
rate of the mildly oxidized protein is increased if the oxidant
concentration is increased. At concentrations above 4 µmol of
hydrogen peroxide/mg of protein, an inverse relation is observed
(cf. Fig. 1). These findings are in good agreement to
literature data for other model systems (6, 15, 58), which were a
starting point for the present study.
The remarkably high degree of similarity between the infrared
difference patterns, particularly in the amide I region, for oxidation-
and temperature-induced unfolding of RNase A prompted us to propose a
correlation between hydrogen peroxide-induced protein modifications and
protein unfolding. In fact, the similarity of spectral characteristics
observed for both processes (cf. spectra b and c in Fig. 5) support the hypothesis that
oxidative damage of proteins may result in global unfolding
rearrangements of the polypeptide. This is also corroborated by the
thermal and denaturant-induced unfolding of RNase A (29). The proposed
correlation is supported by two facts. First, a quantitative comparison
of RNase A oxidation using several spectroscopic parameters of the
amide I region (such as high and low frequency
-band or the band for
-helical structure) indicate a remarkably high degree of internal
consistence (Table I). Second, even when relatively high concentrations
of the oxidants were applied for time periods up to 120 h, the
changes of all secondary structure elements of the amide I region were
always smaller then the changes observed during the temperature-induced two-state transition of global unfolding. Thus, the results of the
protein oxidation experiments suggest that the hydrogen
peroxide-induced structural rearrangements of RNase A are processes
similar to global unfolding, involving all parts of the protein.
Protein backbone fragmentation may be a potential candidate producing unfolded fragments of RNase A. However, the results of mass
spectrometry demonstrate that oxidative unfolding of RNase A does not
perturb the protein sequence.
The detailed analysis of some amino acid side chain absorption bands
indicates distinct discrepancies that cannot be explained on the basis
of the simple model of protein unfolding. While the absorbance values
of the tyrosine band at 1515 cm
1 are
increasing during temperature-induced unfolding of RNase A, the
oxidation experiments exhibit a decrease of this band. Furthermore, for
the absorption band of the aspartate residues at 1584 cm
1 we found a decrease in intensity twice as
large compared with the corresponding changes observed during complete
unfolding. From the literature, it is known that side chains of
proteins are primary targets of oxidation (e.g. Ref. 57).
Therefore, it is possible that some of the tyrosine (and aspartate)
side chains of RNase A were oxidized by hydrogen peroxide. Tyrosine is
one amino acid residue that can readily undergo oxidation, forming a
number of reaction products such as dityrosines (57). RNase A contains
six tyrosine residues and, as the calculations of the ASA show, only
one of these side chains is initially not accessible by the solvent
(Tyr97). The remaining residues revealed relative
ASA values between 11 and 65% (Tyr25 and
Tyr65). Therefore, an oxidative modification of at least
some tyrosine residues seems likely.
Unlike the absorbances of the tyrosine band, the time dependence of the
tyrosine band frequency confirmed quantitatively the corresponding
structural alterations found in the amide I region (see Table 1). It is
expected that any covalent modification of the tyrosine ring will
change its spectral characteristics considerably. Thus, oxidation of
the tyrosine residues will necessarily be followed by a decrease of the
tyrosine band intensity at 1515 cm
1 (and
accordingly by the appearance of a number of new bands), whereas the
frequency of the tyrosine band should remain unchanged. Therefore, we
interpret frequency shifts of the tyrosine band during RNase A
oxidation as a consequence of changes in the microenvironment, in this
case due to the unfolding of the protein.
Previous literature demonstrated (25) that under nonphysiological high
concentrations of the oxidant, proteins may aggregate to large and
densely packed complexes that are not accessible to proteases.
Aggregates can be either formed by covalent cross-linking (dityrosines;
Ref. 57) or by intermolecular hydrophobic interactions (intramolecular
-sheets; Ref. 20). According to these well established facts, the
decrease of proteasome degradation activity at high
H2O2/protein concentration ratios
(cf. Fig. 1) was correlated to the formation of aggregates
of this protein. Previous IR studies have shown (48, 50, 51) that
densely packed intermolecular
-aggregates give rise to highly
characteristic infrared bands. Particularly, a so-called
"
-aggregation band" at 1615 cm
1 is
known to be typical for the presence of these aggregates. The FT-IR
measurements of the present study, however, were carried out at
comparably low H2O2/protein concentration
ratios, i.e. when protein aggregation is supposed to play
only an inferior role. It cannot be ruled out completely that the
difference spectra shown in Fig. 6B show small signs of an
aggregation band. However, the presence or absence of a minor
-aggregation band seems to be irrelevant for the interpretation of
the data of the present study.
Oxidative modification of amino acid side chains can severely reduce
the conformational stability of proteins. Stadtman and colleagues (52)
used an example of two-model proteins to demonstrate that during
exposure of amino acids to ozone. Primarily methionine and aromatic
amino acid residues were oxidized in the order Met > Trp > Tyr
His > Phe (52). Hence,
methionine residues appear to be the primary targets of oxidation. It
was furthermore proposed that methionine residues constitute an
effective antioxidant on the surface of proteins (14). Since the
oxidation product of methionine, methionine sulfoxide, can be recycled
by a catalytic system, methionine residues may act as oxidant
scavengers. Interestingly, for many proteins it was demonstrated that
protein conformation is little affected if solvent-accessible
methionines on the protein surface are oxidized (14, 59-61). These
studies support the hypothesis that surface exposed methionines
generally preserve the biological function of the protein.
The S-C stretching vibration of methionines
(R-CH2-S-CH3) can be expected in the spectral
region of 730-570 cm
1 (62). The experimental
setup used in this study (CaF2-windows) did not allow us to
monitor IR-spectroscopic changes in this low frequency region.
Nevertheless, the absorption band arising from the S=O stretching
vibration of methionine sulfoxide ((R2)-S=O) is known to
occur between 1060 and 1015 cm
1 (62). In
fact, at 1047 cm
1 a small and narrow positive
band (cf. Fig. 6C) was observed for the RNase A
oxidation experiments, while the control measurements did not indicate
any additional peak at these wave numbers. In RNase A, the four
methionine residues are poorly accessible by the oxidant. For the side
chain of Met29, an ASA value of 18% was calculated
(Met13, 7%; Met30, 0%; Met79,
9%). However, we demonstrated that large scale disorganization of the
protein conformation occurs upon H2O2
treatment, which implies that Met residues could have been oxidized
that were initially shielded.
The other aromatic amino acid side chains of RNase A such as Phe (there
is no Trp in RNase A) are also poorly accessible. For three Phe side
chains, ASA values between 0 and 8% were calculated. On the other
hand, the three His residues of RNase A, which are also known to
undergo oxidative modification, exhibit ASA values of >35%.
Therefore, we think that in native RNase A, the primary targets of
oxidation are tyrosine and histidine residues. This could be shown
convincingly by FT-IR difference spectroscopy at least for tyrosine residues.
A summarizing description of possible conformational and covalent
modifications of proteins during oxidative damage is given in Fig.
11. Areas A-D of Fig. 11
schematically illustrate how the concentration of the different RNase A
fractions may vary if the concentration of the oxidant is increased.
Initially, at comparably low oxidant concentration, some of the
solvent-accessible amino acid residues such as histidine, methionine,
or aromatic amino acid residues are modified (Fig. 11B). The
FT-IR spectroscopic results of this study clearly indicate that
tyrosine and aspartate amino acid residues are involved. Possibly, the
more side chains are oxidized by hydrogen peroxide, the more the
conformational stability of the protein is reduced and the higher the
probability of local or even global unfolding (Fig. 11C).
Unfolding causes the exposure of initially buried hydrophobic groups to
the solvent and accordingly to the oxidant. Thus, residues that were
initially not exposed to the solvent were made accessible to oxidation
by hydrogen peroxide and other secondary oxidants. As a result, the protein surface hydrophobicity, potentially one of the recognition signals for proteasome binding, is increased. However, with increasing concentrations of the unfolded protein, the probability of
intermolecular formation of protein aggregates that are not accessible
to proteasome digestion will increase as well (Fig. 11D).
Therefore, at nonphysiologically high concentrations of the oxidants,
the proteasome turnover is not further enhanced but will be reduced
instead. This situation is reflected in Fig. 11, where a difference
curve between the unfolded fraction of RNase A (C) and
aggregated forms of the protein (D) is displayed.
Interestingly, the shape of this difference curve matches the main
features of the proteasome turnover plot of Fig. 1. However, it cannot
be completely ruled out that products of the oxidative modification of
amino acid side chains, such as methionine sulfoxide, play a
significant role in the proteasome recognition process as well. Yet we
believe that hydrophobicity is the key signal for the 20 S proteasome
because of the initially low accessibility of the methionine residues.
However, additional experimental efforts are necessary to answer these
questions. FT-IR difference spectroscopy is one of the potentially
techniques for detecting oxidant-induced changes in protein structure
due to its very high sensitivity and specificity. Further increase of
the specificity of the method may be achieved by the use of isotopic
labeled compounds e.g. of methionines marked by
13C and 15N and/or the substitution of specific
amino side residues.

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Fig. 11.
Schematic illustration of the
H2O2 concentration-dependent
effects on RNase A structure. At low hydrogen peroxide
concentrations, solvent-exposed amino acid side chains of RNase A are
potentially targets of the oxidants (curve B). In
our model, with an increasing number of modified side chains, the
stability of the model protein is more and more reduced, leading
initially to local and finally to global unfolding (curve
C). The higher the concentration of unfolded RNase A
species, the higher the probability of protein aggregation
(curve D). Possibly, unfolded RNase A species are
recognized by the 20 S proteasome due their exposure of hydrophobic
surfaces (curves C and D), whereas
aggregates are not accessible. Please note that all processes are
concentration- and time-dependent.
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