Biphasic reductive unfolding of ribonuclease A is temperaturedependent Yong-Bin Yan1,2, Ri-Qing Zhang1,2and Hai-Meng Zhou3 1 NMR Laboratory, Department of Biological Sciences and Biotech
Trang 1Biphasic reductive unfolding of ribonuclease A is temperature
dependent
Yong-Bin Yan1,2, Ri-Qing Zhang1,2and Hai-Meng Zhou3
1
NMR Laboratory, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China;2State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China;3Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China
The kinetics of the reversible thermal unfolding, irreversible
thermal unfolding, and reductive unfolding processes of
bovine pancreatic ribonuclease A (RNase A) were
investi-gated in NaCl/Pi solutions Image parameters including
Shannon entropy, Hamming distance, mutual information
and correlation coefficient were used in the analysis of the
CD and 1D NMR spectra The irreversible thermal
unfolding transition of RNase A was not a cooperative
process, pretransitional structure changes occur before the
main thermal denaturation Different dithiothreitol
(dithio-threitolred) concentration dependencies were observed
between 303 and 313 K during denaturation induced by a small amount of reductive reagent The protein selectively follows a major unfolding kinetics pathway with the selec-tivity can be altered by temperature and reductive reagent concentration Two possible explanations of the selectivity mechanism were discussed
Keywords: image analysis; proton nuclear magnetic reson-ance; reductive unfolding; thermal unfolding; unfolding kinetics
Dynamic analysis of the unfolding and refolding pathways
and identification of the specific conformational changes
which form the individual intermediates involved in the
rate-limited pathway(s) can distinguish one pathway from
another and play fundamental importance for protein
folding [1,2] It is usually of considerable interest to estimate
the conformational changes of both the whole protein
tertiary structure and of specific sites observed by
spectro-scopic techniques in different redox systems and solvent
conditions Protein unfolding is highly pertinent to protein
folding [3] and is more controllable for more comprehensive
study by slowing the unfolding process carried out at
physiological pH and temperature [4] Such studies in turn
provide new insights into the functional properties and
mechanisms of proteins, which will lead to a more detailed
and more complete description of biological functions [5]
Bovine pancreatic ribonuclease A (RNase A; EC
3.1.27.5) contains 124 residues with four native disulfide
bonds (Cys26–Cys84, Cys40–Cys95, Cys58–Cys110, and
Cys65–Cys72) RNase A has played a crucial role as a model system in studies of protein structure, folding, stability, and chemistry [6] It folds and unfolds through multiple pathways, with the rate-limiting steps in the well-populated pathways involving the formation of distinct transition intermediates [1–3,7,8] The complexity of the multiple pathways means that different mechanisms may occur with different types of redox systems and different solvent conditions [9,10] Thus a comprehensive investiga-tion under different condiinvestiga-tions using different methods is essential to elucidate the protein folding and unfolding processes
Much effort has been devoted in recent years to understanding the mechanism and the main factors that control protein folding, and to developing approaches that allow researchers to investigate the multifarious aspects of protein folding and unfolding While the determination of the protein structural transitions that occur in the pathways
is at the heart of studies on unfolding and refolding processes [10], dynamic analysis of the different processes is necessary to evaluate the different pathways These studies will also clarify the key factor(s) controlling the processes and clarify the unfolding and refolding mechanism associ-ated with the redox properties and solvent conditions Therefore, we have used1H NMR spectra to investigate the unfolding dynamics of RNase A during denaturation by different concentrations of reductive reagent dithiothreitol (dithiothreitolred) at different temperatures Similar studies were carried out by Rothwarf and Scheraga [10] in which the temperature dependence of RNase A regeneration was studied with dithiothreitolox/dithiothreitolred and GSSG/ GSH systems Their results suggested that the regeneration process with the two types of redox reagents proceeded through different pathways with significantly different temperature dependencies Here we present a temperature
Correspondence to Y.-B Yan, Department of Biological Sciences
and Biotechnology, Tsinghua University, Beijing 100084, China.
E-mail: ybyan@mail.tsinghua.edu.cn or H.-M Zhou, Department of
Biological Sciences and Biotechnology, Tsinghua University,
Beijing 100084, China E-mail: zhm-dbs@mail.tinghua.edu.cn
Abbreviations: C, Correlation coefficient; des-[40–95], RNase A
lacking the 40–95 disulfide bond; des-[65–72], RNase A lacking the
65–72 disulfide bond; DSS, 2,2-dimethyl-2-sila-pentanesulfonate;
FID, free induction decay; GSH, reduced glutathione; GSSG,
oxidized glutathione; H, Shannon entropy; HD, Hamming distance;
MI, Shannon mutual information; RNase A, Bovine pancreatic
ribonuclease A.
(Received 23 June 2002, revised 3 September 2002,
accepted 11 September 2002)
Trang 2dependence study based on the same reductive reagent
(dithiothreitolred) but different reagent concentrations In
addition to discussing the temperature effect, the thermal
denaturation transition of RNase A and the effect of the
reductive reagent concentration, which have not been
previously associated with protein folding and unfolding,
will also be discussed A new approach of image analysis,
which is established by us recently [4,11], was used to
analyze the unfolding dynamics and also was introduced
into the analysis of the thermal transition study by CD and
1H NMR
M A T E R I A L S A N D M E T H O D S
Sample preparation
Highly purified lyophilized ribonuclease A (type XII-A, lot
110K7665) from bovine pancreas (RNase A) was
pur-chased from Sigma Chemical Co (St Louis, MO, USA)
and used without further purification Dithiothreitolredwas
also a product of Sigma Chemical Co All other reagents
were of the highest grade commercially available Samples
of the native RNase A were dissolved in 0.5 mL 100 mM
NaCl/Pi or triple-distilled water, pH 8.0 The protein
concentration was 14 mgÆmL)1(for NMR measurements)
or 0.3 mgÆmL)1 (for CD measurements) The NMR
samples contained 10% D2O to provide a signal for the
lock and the final pH was 7.6 which was measured with no
corrections for isotope effects
NMR Spectroscopy
All1H NMR experiments were carried out on a Bruker
AM 500 superconductor spectrometer (Bruker, Fa¨llanden,
Switzerland) at Tsinghua University The carrier frequency
was fixed at the center of the H2O/HOD resonance
frequency The chemical shifts of the spectra were referenced
to the most upfield resonance which in turn had been
calibrated against 2,2-dimethyl-2-sila-pentanesulfonate
(DSS) The 90° pulse width was 6.5 ls, the sweep width
was 8333 Hz and each FID had 400 scans or 240 scans with
16 K data points Two dummy scans were performed for
each FID with a recycle delay of 1.8 s or 1 s Solvent
suppression was carried out by presaturation at all times
except during acquisition For a given sample, the
unfold-ing experiments were carried out over a contiguous block
of time (about 10–72 h) without removing the sample from
the spectrometer The Fourier transform of the FID signal
was obtained without additional modification The phase
correction parameters were the same as for the first
spectrum
The irreversible thermal transition measurements was
carried out by increasing the temperature in one degree steps
from 303 to 333 K Approximately 5 min was allowed for
thermal equilibration at each measured temperature The
spectra were collected at 30 min intervals (including thermal
equilibration), with 400 scans per spectrum to get a better
signal to noise ratio
The experimental temperature for the reductive unfolding
was maintained at 303, 308 and 313 K The protein stability
was examined by maintaining the sample without
dithio-threitolredat 313 K for 48 h and no difference was found
between the original and end spectra (data not shown)
RNase A was unfolded with a dithiothreitolred concentra-tion of 10–100 mM(10–100-fold molar excess of the protein, see below) Spectra were collected every 20 min or 8 min after dithiothreitolredwas added Zero corresponds to the dithiothreitolredinjection time into the cold NMR tube To allow for temperature equilibrium and operational delay, the first spectra were obtained at 10 min RNase A was also reduced with a 100-fold molar excess of dithiothreitolredin NaCl/Piat 313 K for comparison of the reductive denatur-ation endpoint (a complete unfolding was obtained in 4 h with 100-fold molar excess of dithiothreitolred) [1] No aggregation was observed along the experiments
The data analysis was carried out by spectral image analysis method established recently [4,11] The spectrum parameters (Shannon entropy, H; Mutual Shannon Infor-mation, MI and Correlation coefficient, C) that describe the nature of each image (spectrum) and the correlation between different images were calculated using MATLAB software (MATLAB 5.2, The MathWorks, Inc., Natick,
MA, USA) by programs developed in-house The spectral window for the reductive unfolding analysis was 6.3– 10.0 p.p.m The thermal denaturation curves were repre-sented directly by these parameters, while the Hamming distance was calculated instead of the mutual information The reductive unfolding kinetics was analyzed by a linear expansion least-squares algorithm and graphically using a least mean squares fit procedure The rate constant errors were defined as the standard deviation
Thermal denaturation monitored by CD spectropolarimetry
CD measurements were performed on a Jasco J-715 spectropolarimeter equipped with a thermoelectrically con-trolled cell holder CD spectra of RNase A in NaCl/Piand
in triple-distilled water were measured in the far-UV range (195–245 nm) in 2 mm pathlength quartz cells For the thermal denaturation, the measurements were typically made at two degree intervals in the temperature range from
293 to 353 K, at a heating rate of 1 KÆmin)1 Spectra were scanned twice at a rate of 100 nmÆmin)1, a resolution of 0.5 nm, and a bandwidth of 1 nm
The thermal denaturation curves were obtained by measuring the ellipticity at 222 nm and analyzed with a nonlinear least-squares algorithm The image parameters (H, HD, C) were also calculated for the image analysis of the
CD spectra The calculating routines were the same as those for the NMR spectra Curve fits was obtained by the standard method using the Marqurdt-Levenberg routine [12] as provided in theORIGIN6.0 software (Microcal Inc., Northampton, MA, USA)
R E S U L T S
The thermal stability of proteins, especially enzymes, has long been a practical concern, because this is usually the factor that most limits their usefulness Moreover, tempera-ture and the relevant free energy change DG, usually influence the behavior of protein folding and unfolding Both the protein folding and unfolding processes are accelerated by increased temperature Protein conformation ensembles are undoubtedly affected by temperature, but can temperature affect the folding and unfolding mechanism of
Trang 3proteins due to the different conformation ensembles at
different temperatures This research investigates the
rela-tionship between the unfolding mechanism and the
con-formational change at different temperatures
Thermal denaturation measured by far-UV CD
The changes in the secondary structure of RNase A as a
function of temperature were followed by CD
measure-ments Figure 1A shows the reversible thermal transition
curves of RNase A in triple-distilled water and in NaCl/Pi
measured by far-UV CD spectra at 222 nm To compare
the results from NMR spectra, the spectral image
param-eters, Shannon entropy (H), correlation coefficient (C) and a
new parameter, Hamming distance (HD), were introduced
into the CD spectra analysis (mutual information was not
presented for large errors) Figure 1B,D show the thermal
transition curves described by the Shannon entropy and the
correlation coefficient HD is defined as
HD¼ 1
N
X abs h ki hkj
ð1Þ
where hkmeans the ellipticity at k nm, N means the number
of data points used in the calculation, i and j are the CD
spectra at temperatures i and j Here, we used i¼ 1 to
calculate the relative ellipticity change referenced to the first
CD spectra The relative ellipticity change rate can be
calculated by j¼ i + 1 (data not shown) It can be seen in
Fig 1C that the Hamming distance exactly reflects the
ellipticity change but with minimal errors relative to the
traditional method All these parameters present a
well-defined two-state process for RNase A thermal
denatura-tion, which is consistent with the multiprobe principle [13]
The thermodynamic properties obtained from the curves in
Fig 1 and given in Table 1 are quite consist with previous
studies [14] The data clearly indicates that RNase A in
NaCl/Piand in H2O have a similar transition properties but
those in NaCl/Piare more stable A small amount (5–10%)
of irreversible denaturation was observed reproducibly in
both solutions
Irreversible thermal denaturation measured by NMR The extent of irreversible denaturation of proteins has been determined previously to depend on the protein concentra-tion and the period of time that the protein soluconcentra-tion is incubated at the higher temperatures during the heating and cooling process [15] Therefore, the denaturation and renaturation times were usually chosen which minimize the thermal equilibration at each temperature However, a temperature appearing in the predenaturation part of a thermodynamic curve does not mean stabilization for relatively long-term investigations at this temperature Thus,
a thermal denaturation study of a high concentration protein used in reductive unfolding studies will be useful to determine the initial conformational ensembles of the investigation Here we present a study of irreversible unfolding using image analysis of the NMR spectra to
Fig 1 Thermal denaturation profiles of RNase A in H 2 O (s) and in NaCl/P i (h) measured by far-UV CD spectra presented by (A) ellipticity at 222 nm; (B) Shannon entropy; (C) Hamming distance; (D) correlation coefficient.
Table 1 Thermodynamic properties of RNase A The errors were calculated as the standard deviations of the curve fits.
T m (K)
DH 0 (T m ) (kJÆmol)1)
DS 0 (T m ) (eu)
CD in H 2 O
CD in NaCl/P i
NMR in NaCl/P i
Trang 4investigate the relationship between the protein initial
conformational ensembles and the reductive unfolding
mechanism for different temperatures
The dynamics of the thermal transition can be analyzed
by observing specific resonance’s split phenomena, chemical
shift changes and the integral areas The resonance at
0.4 p.p.m was resolved as CH3 of Val63 Its
thermody-namic curve is shown in Fig 2 as a typical two-state process
If the protein shows a cooperative two-state transition
during the thermal unfolding process, the thermodynamic
curves obtained by different parameters should give similar
results The image parameters (H, MI, C, HD) calculated to
characterize the1H NMR spectra of the thermal unfolding
process of RNase A are presented in Fig 3 As described
previously [4, 11], Shannon entropy values reflect the
entropy of the spectra, which is the sum probability of
the peak intensity in a certain region In other words, the
H-values reflect the dispersion properties of the resonance in
selected regions Mutual Shannon information reflects the correlation of the Shannon entropy between the corres-ponding data of two spectra The MI value increases as the degree of correlation increases MI reflects how many unchangeable parts occur in the spectra The correlation coefficient reflects the correlation between two spectra using their covariance A larger value of C indicates more correlation In Fig 3, the significant increase of the values above 323 K in the downfield region was due to the protein aggregation and bad resolution Three of these parameters have similar curves between the downfield and upfield region if the aggregation parts are ignored The HD values have nearly equal curves, while the Shannon entropy values have rather different curves between the downfield and upfield regions which may be due to the H-values of the downfield region is more affected by the change of microenvironment No clear explanation can be given now for the differences obtained from H One explanation may be that H cannot exactly reflect the main changes during thermal denaturation A second explanation may be that there are some significant conformational differences that were only be observed by H However, there were some conformational differences between 303 and 313 K, and the thermal transition curves characterized by C and HD were flat between 310 and 316 K, which suggests a relative thermal stabilization The transition between 310 and 323 K characterized by MI, C, and HD shows a similar two state transition to Fig 3 The Tmvalues obtained from the MI, C and HD curves are presented in Table 1 Though the thermal transition is irreversible when the protein is heated
to 323 K, it is nearly reversible ( 95%) in the temperature range of 301–319 K (data not shown)
Reductive denaturation The reductive denaturation of 1 mMRNase A induced by 10–100 mM dithiothreitolred at 303, 308 and 313 K was investigated by image analysis of the 1D NMR spectra as
Fig 2 Integral area of CH3(Val63) during irreversible thermal
dena-turation of RNase A in 0.5 mL 100 m M NaCl/P i buffer.
Fig 3 Time course of Shannon entropy (A),
mutual information (B), correlation coefficient
(C), and Hamming distance (D) values of1H
NMR spectra during irreversible thermal
denaturation of RNase A (s) Upfield region
and (h) downfield region.
Trang 5performed in a previous paper [4] As relatively low
dithiothreitolred concentrations (only 10–100-fold molar
excess of the protein) and relatively high temperatures were
used, no intermediates peaks were observed from the 1D
NMR spectra or 2D NOESY NMR spectra (data not
shown) The image parameters (Shannon entropy, mutual
information, and correlation coefficient) were calculated to
characterize the unfolding process of RNase A induced by
dithiothreitolred The Hamming distance is not presented
because the curve is similar to those of the other parameters
All the results were fit best by a model of two consecutive
first-order reactions, with rate constants listed in Table 2 A
comparison of the rate constants from MI for different
dithiothreitolred concentrations and different temperatures
is presented in Figs 4 and 5 (Similar curves can be obtained
from other parameters)
As can be seen in Fig 4A,B, the rate constants increased
while increasing temperature, as described in a previous
paper [4] The rate constants of the fast process (kf) have
similar increases with temperature at the two
dithiothrei-tolred concentrations It should be noted that the rate
constants of the slow process (ks) increased dramatically
when the temperature was increased from 303 to 313 K for
a dithiothreitolredconcentration of 30 mM, while no such
large increase occurred for a dithiothreitolredconcentration
of 50 mM Figure 5A,B present the relationships between
the rate constants and the dithiothreitolredconcentration at
different temperatures The dithiothreitolredconcentration
affected the fast and slow process unfolding rate constants
in a complex way The kfvalues increased similarly when the
dithiothreitolredconcentration was increased from 30 mMto
50 mM at different temperatures As the dithiothreitolred
concentration was increased from 30 mMto 50 mM, the ks
values increased at 303 K, but decreased dramatically at 308
and 313 K
D I S C U S S I O N
It has been proposed that the regeneration of RNase A
follows different pathways in the GSSG/GSH and
dithio-threitolox/dithiothreitolredsystems [9] Li et al also found
that the reductive unfolding of RNase A took a parallel
biphasic pathway for unfolding induced by dithiothreitolred
[3] Recent studies demonstrated that the oxidative folding
of RNase A has a multiple pathway mechanism [7,8,16–18] Although the oxidative regeneration and the reductive cleavage of the disulfide bond were suggested to be kinetically the same [3], des-[65–72] RNase A is thought
to be the most highly populated intermediate in the unfolding pathways [19] However, the unfolding and
Table 2 Rate constants for changes in Shannon entropy, mutual information and correlation coefficient observed in the reductive denaturation of 1 m M
RNase A in NaCl/P i , at 313 K,308 K and 303 K.
Temperature
(K)
[dithiothreitolred] (m M )
k H (10 4 min)1) a k MI (10 4 min)1) a k C (10 4 min)1) a
a
Rate constants of Shannon entropy, mutual information and correlation coefficient, respectively.bThe errors were of the order of 20–30% determined by repeating experiments.
Fig 4 Comparison of the fast and slow process rate constants, k f (A) and k s (B), from mutual information as a function of temperature reduced
by 30 m M and 50 m M dithiothreitolred.
Trang 6folding processes, especially at relatively high temperatures
[20], of the protein are usually dominated by a major
pathway related to a major rate-determining intermediate in
similar redox systems For a single pathway, the biphasic
process can be expressed as
N!k1
I!k2
where N, I, and U mean the native state, transition
intermediate, and unfolding state, respectively The
observed image parameter rate constants, kfand ks, can
be expressed by a1k1[N][dithiothreitolred], a2k2
[I][dithiothre-itolred], where a1 and a2 are defined as the link constants
between the image parameter rates and the actual rates If
the unfolding process of RNase A was dominated by a
major pathway, the observed rate constants should increase
as the dithiothreitolred concentration increased Thus the
results shown in Fig 5 cannot be explained by a single or
major pathway mechanism although similar redox systems
were used The dithiothreitolred concentration and the
temperature have a relatively simple effect on the rate
constants of the fast process (Figs 4A and 5A), but have a
complex effect on the rate constants of the slow process
(Figs 4B and 5B) As there are multiple unfolding pathways
with multiple three disulfide intermediates [7,8,16–18], these
intermediates seem have different sensitivities to
dithiothre-itolredand thus may exhibit different kinetics from NMR
spectra Therefore, a change in dithiothreitolred concentra-tion will change the relative populaconcentra-tion of these intermedi-ates, which will be reflected by the image analysis of the NMR spectra These results suggest that the external solvent conditions affect the properties of the unfolding rate-limiting intermediates Thus different unfolding inter-mediates may dominate under different solvent conditions
A possible mechanism for the results in Figs 4 and 5 is shown in Fig 6 The protein presents a selective behavior of unfolding process under different conditions The selectivity
of the major unfolding pathway may be altered by the temperature and the dithiothreitolredconcentration, though the mechanism for this selectivity is not clear now The effects of pH and phosphate concentration were also studied, and it was found that ions only affect the rate of protein unfolding but have no effect on the pathways (data not shown) N313 and N303 mean the different conformational ensembles at 313 and 303 K (Fig 6) The free energy change for folding at any temperature DG(T), can be obtained using the modified Gibbs-Helmholtz equation [21],
DGðTÞ ¼ DHmð1 T=TmÞ
þ DCp½ðT TmÞ T lnðT=TmÞ ð3Þ Here DCp¼ 1.15 (0.08 kcalÆK)1Æmol)1as recently obtained
by Pace et al [22] Using the parameters listed in Table 1, the DG(T) change between 313 and 303 K was calculated to
be 6.6 ± 0.4 kJÆmol)1 (1.6 ± 0.1 kcalÆmol)1) Though there is no other evidence that the conformation of RNase A at 313 K is stabilized by a thermal transition state, the different features of the NMR spectra at 303 and
320 K suggest different molecular chemical environments Such microenvironmental changes, like the effects induced
by ions [13,20,22–24], may result in changes of the protein behavior It has been proposed that the nonzero surface exposure of Cys40 and Cys65 causes the 40–95 and 65–72 disulfides in RNase A to be the first to break [3] Thus one possible explanation of the mechanism in Fig 6 might be that the conformational ensembles at 313 K, a relatively high energy state, may result in little difference between the cystine (or the amino-acid residues around cystine) surface exposures This may also explain the complexity of the rate constant changes between 15 and 25°C observed by Li
Fig 6 Possible reductive unfolding pathways of RNase A at 303 and
313 K in NaCl/P i , pH 8.0 N 313 and N 303 represent the different con-formational assemblies at 303 and 313 K I 1 and I 2 represent two dis-tinct unfolding rate-determining intermediates, while U represents the unfolded state of RNase A.
Fig 5 Comparison of the fast and slow process rate constants, k f (A)
and k s (B), from mutual information as a function of dithiothreitolred
concentration reduced under three temperatures.
Trang 7et al [3] The major parts of intermediates I1 and I2 are
likely to be the three-disulfide-intact species, though they are
not characterized here Thus the selectivity of the breakage
of the first disulfide may be altered by the different cystine
surface exposures induced by temperature and
dithiothre-itolredconcentration
Another possible explanation of the selectivity
mechan-ism dependence on temperature and reductive reagent
concentration in Fig 6 may be obtained from the studies of
the folding intermediate properties The solution structure
and the thermodynamics of the analogs of the major and
minor rate-determining three-disulfide folding intermediates
have been characterized in recent years [25–27] The
thermodynamic parameters for these analogs are
summar-ized in Table 3 All the Tmvalues of these analogs are lower
than 313 K ( 40 °C) at pH 4.6 The major intermediate,
des-[65–72] RNase A, was more stable than the minor
intermediate, [C40A, C95A] RNase A The thermal
unfolding of des-[65–72] RNase A began at about 303 K
and ended at about 318 K [25], while thermal unfolding of
[C40A, C95A] RNase A began at about 298 K and ended
at about 313 K at pH 4.6 Considering the pH effect on Tm,
the Tmvalues are expected to well above 313 K at neutral
pH [28] The dramatic increase of ks from 303 to 313 K
(Fig 4B) may result from such intermediate
thermody-namic properties The close-to-unfolding state at 313 K of
the three-disulfide intermediates may drive the slow process
to the unfolded state The selectivity mechanism may
therefore arise from the different thermodynamic properties
between the two intermediates At 313 K, des-[40–95]
RNase A was closer to the midpoint of the thermal
unfolding transition than des-[65–72] RNase A Such a
difference accompanied by the increased concentration of
the reductive reagent may lead the (major) unfolding
process along different pathways The thermodynamic
properties may also resulted in the disulfide reshuffling at
such a relatively high temperature of 313 K, though few
populated intermediates were observed during the unfolding
process (see Results)
Recently Sogbein et al [29] reported that pH has a
pronounced influence on the kinetic mechanism of
myo-globin unfolding Their results show that myomyo-globin unfolds
through a short-lived intermediates only at acidic pH, with
no intermediates observed for basic conditions Our results
suggest that the protein kinetic unfolding mechanism might
depend on external solvent conditions such as temperature
Sogbein et al hypothesized that the observed pH
depend-ence of the protein unfolding mechanism could be related to
the pH dependence of heme solubility [29] From the
temperature and reductant concentration dependence of the
protein unfolding mechanism presented in this paper, we
can further hypothesize that the observed difference in the protein unfolding behavior could be related to different initial conformational ensembles for different external solvent conditions Furthermore, the protein unfolding behavior could be related to different conformational stabilities which could be demonstrated by different free energy change for different external conditions It should be noted that such the free energy change is not distributed equally among every amino-acid residue of a well-folded protein Thermal denaturation studies can help elucidate the structural stabilities of proteins Traditionally such studies used a simple two-state mechanism of thermally induced transitions in small, compact globular proteins, which are thought to act as single stage systems Many groups have identified the multiple steps involved in thermal unfolding pathways [30,31] A theoretical model was presented recently to study the stepwise thermal unfolding of globular proteins using the stabilizing or destabilizing characteristics
of amino-acid residues in protein crystals [32] All these results show that in RNase A, the a helix of the shell residues around 16–22 unfolds in the temperature range 303–318 K, while the b sheet segment 106–118 is relatively stable thermally Our experiments also confirmed that structural changes occur in RNase A before the main thermal denaturation transition (Fig 2) The pretransition structural changes occurring in a temperature range of
Table 3 Thermodynamic properties of the analogs of the rate-determining three-disulfide folding intermediates.
a Data from Laity et al [28] b Data from Shimotakahara et al [26] c Data from Talluri et al [25] d Data from Laity et al [27].
Fig 7 Effect of external solvent conditions on the kinetic mechanism of protein unfolding N T(a) and N T(b) represent the initial conformational assemblies under different solvent conditions I 1 and I 2 represent two distinct unfolding intermediates or different energy states of one intermediate U T(a) and U T(b) represent the different unfolding assem-bles under different solvent conditions #1 and #2 represent the fast and slow transition states.
Trang 8303–318 K may result in significant structural changes of
RNase A between 303 and 313 K though the tertiary
structure is still maintained as was confirmed by CD
measurements The influence of different initial
conforma-tional ensembles on the selectivity of the protein unfolding
kinetics is shown in Fig 7 The different pathways show
how the external solvent conditions such as temperature
affects the kinetic mechanism of protein unfolding If the
external effect is significant, such as the pH effect on
myoglobin unfolding, the protein selectively follows a
different unfolding kinetics pathway If the external effect
is very small, the protein follows a simpler pathway (where
I1and I2are the same rate-limiting intermediates)
In conclusion, the irreversible thermal unfolding
trans-ition of RNase A is not a cooperative process,
pretransi-tional structure changes occur before the main thermal
denaturation The different initial conformational
ensem-bles at 303 and 313 K may lead to the different
dependen-cies on the reductive reagent concentration of the biphasic
pathway reductive denaturation The protein selects a
preferred one from several major pathways with the
selectivity altered by temperature and reductive reagent
concentration The two possible explanations of the
selec-tivity mechanism described here need to be clarified by more
detailed investigations
A C K N O W L E D G E M E N T S
This investigation was supported by the National Key Basic Research
Special Funds, P R China, No G1999075607, the National Key
Science and Technology Item, P R China, no 96-900-09-03, the 985
Funds of Tsinghua University, P R China and THSJZ of Tsinghua
University, P R China The authors also thank Dr Bo Jiang, Dr Sen
Li, Mrs Xue-Chun Luo, Mrs Xiao-Lan Ding at Tsinghua University,
P R China, and Dr Guang-Zhong Tu at the Institute of
Microchem-istry, P R China for expert technical assistance.
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