(Received for publication, April 20, 1995; and in revised form, September 18, 1995)
From the
Ovotransferrin N lobe contains six intrachain disulfides
(SS-I/Cys-Cys
;
SS-II/Cys
-Cys
;
SS-III/Cys
-Cys
;
SS-IV/Cys
-Cys
;
SS-V/Cys
-Cys
;
SS-VI/Cys
-Cys
) in a single
polypeptide chain of 332 amino acid residues. Upon the protein
disulfide reduction with dithiothreitol under nondenaturing conditions,
the intermediate species with four, three, and two disulfides were
generated. The partially disulfide-reduced intermediates were isolated,
and the localization of intact disulfides in the intermediates was
determined by an indirect end-labeling method. This method included the S-cyanocysteine-specific protein fragmentation, followed by
gel electrophoresis and the immunochemical visualization of the C
terminus-intact fragments using antiserum raised against a non-cysteine
C-terminal fragment (Ser
-Arg
).
Results clearly showed that first SS-IV and SS-V, second SS-III, and
then SS-VI are cleaved. No reduction was observed for SS-I and SS-II
under the employed reducing conditions. The conclusion was confirmed by
peptide mapping analyses for the same disulfide intermediates using
reverse phase high performance liquid chromatography. Transverse urea
gradient gel electrophoresis and visible absorption spectra revealed
that the four-disulfide intermediate, but not the three- or
two-disulfide intermediate, retains essentially the same iron-binding
function as the native protein. By far-UV CD analyses, the residual
native conformation of the partially disulfide-reduced intermediates
was found to decrease with increased number of the reduced disulfides.
Implications of the partially disulfide-reduced intermediates for the
disulfide-reductive unfolding pathway in ovotransferrin N lobe are
discussed.
The redox folding/unfolding system of a disulfide protein is a
useful model for the investigation of protein folding mechanism,
because all disulfide intermediates may be trapped in stable forms
(Creighton, 1986). In bovine pancreatic trypsin inhibitor of the most
well documented example, the predominant folding/unfolding pathway
includes intrachain disulfide rearrangements that inevitably require
the formation of non-native disulfide intermediates (Creighton, 1977;
Creighton and Goldenberg, 1984; Weissman and Kim, 1992). A similar
non-straightforward regeneration/reduction pathway has been shown for
many small single-domain proteins, such as lysozyme (Acharya and
Taniuchi, 1980), ribonuclease A (Rothwarf and Scheraga, 1993),
ribonuclease T1 (Pace and Creighton, 1986), -lactalbumin (Ewbank
and Creighton, 1993a), and hirudin (Chatrenet and Chang, 1993). To
quantitatively elucidate the kinetic pathways, the structural
characteristics of partially disulfide-bonded intermediates have been
extensively studied with these small single-domain proteins (Pace et al., 1988; Radford et al., 1991; Darby et
al., 1992; Staley and Kim, 1992; Ewbank and Creighton, 1993b; van
Mierlo et al., 1994; Talluri et al., 1994).
As a
useful model for the multidomain protein, we have been interested in
the folding/unfolding problem of ovotransferrin N lobe. Ovotransferrin
consists of a single polypeptide chain with a molecular mass of about
80 kDa, which is folded to give N and C lobes; each of the lobes has
one specific iron-binding site (Aisen and Listowsky, 1980). Either lobe
can be isolated after mild proteolysis (Oe et al., 1988), and
the x-ray crystallographic structure of the N lobe has recently been
established (Dewan et al., 1993). As shown in Fig. 1,
the N lobe of ovotransferrin consists of a single polypeptide chain of
332 amino acid residues, which is folded into two domains (domains 1
and 2); Domain 1 contains two disulfide bonds in the N-terminal
motif
(SS-I/Cys
-Cys
;
SS-II/Cys
-Cys
), and domain 2 contains
three disulfides in the kringle bridges
(SS-III/Cys
-Cys
;
SS-IV/Cys
-Cys
;
SS-V/Cys
-Cys
) and one disulfide in
the C-terminal loop of this domain
(SS-VI/Cys
-Cys
). The structural and
functional renaturation of the isolated N lobe has been shown to be
attained by a two-step procedure, in which the denatured, reduced
proteins are initially transformed into a partially folded, reduced
state and are then reoxidized into the native, disulfide-bonded form
(Hirose et al., 1989; Hirose and Yamashita, 1991; Yamashita
and Hirose, 1993). The structural and functional characteristics of the
disulfide intermediates involved in the redox folding/unfolding
pathway, however, have still not been determined.
Figure 1:
Schematic view of
the native structure of ovotransferrin N lobe. The figure was drawn
based on the x-ray crystallographic data of iron-loaded ovotransferrin
N lobe (Dewan et al., 1993) using the program MolScript
(Kraulis, 1991). The upper and lower halves correspond to domains 1 and 2, respectively. N and C represent the N-terminal and C-terminal ends, respectively. Closed spheres are sulfur atoms that are involved in SS-I
(Cys-Cys
), SS-II
(Cys
-Cys
), SS-III
(Cys
-Cys
), SS-IV
(Cys
-Cys
), SS-V
(Cys
-Cys
), and SS-VI
(Cys
-Cys
).
One of the major
difficulties on studying the redox folding/unfolding pathway of a
complex protein is related to the analytical approach for the disulfide
localization in the partially disulfide-bonded intermediates. The
established method of the peptide mapping procedure that includes
extensive protein digestion and subsequent identification of disulfide
peptides is highly complex and time-consuming for the analysis of a
large protein having many disulfides. A previous chemical fragmentation
approach that includes specific polypeptide cleavages in S-cyanocysteine sites and subsequent SDS-PAGE ()has
been shown to be useful for the simple and sensitive analysis of the
localization of half-cystines (Jacobson et al., 1973;
Subramanian, 1980; Walser et al., 1981; Mikami and Morita,
1983; Nefsky and Bretscher, 1989). Practical application of the
technique, however, has been limited to proteins with a small number of
half-cystines.
In the present study, we developed an indirect end-labeling procedure that enables, in combination with the S-cyanocysteine-specific fragmentation technique, the localization of sulfhydryls and disulfides in a complex protein with many disulfides. The reliability of the indirect end-labeling procedure was confirmed in such a way that with respect to the partially disulfide-reduced intermediates of ovotransferrin N lobe, the sulfhydryl and disulfide localizations determined by this procedure were essentially the same as those determined by the established peptide mapping analysis. The structural and functional characteristics of the partially disulfide-reduced intermediates were also investigated using different analyses. The implications of the disulfide intermediates for the disulfide-reduction pathway are discussed on the basis of their structural characteristics.
Figure 2:
Strategy for determining the
localization of sulfhydryls and disulfides by indirect end-labeling
approach. In Panel A, the localizations of the six native
disulfide bonds and the C fragment
(Ser-Arg
) that was employed for the
probe antiserum are shown. Panel B represents the strategy for
determining the sulfhydryl and disulfide localizations in
ovotransferrin N lobe. Step 1 includes disulfide reduction with DTT
under nondenaturing conditions, alkylation of the generated sulfhydryls
with IAA yielding alkylated cysteines (A-), and then
isolation of disulfide intermediates. The figure shows the case for the
hypothetical two-disulfide intermediate having SS-III. Step 2 is the
cyanylation procedure, in which the intact disulfides are fully reduced
by DTT in the presence of urea. The newly generated sulfhydryls are
modified with DTNB and subsequently S-cyanylated (CN-). In step 3, the protein is fragmented by S-cyanocysteine-specific cleavage at pH 9.0, electrophoresed
on a SDS-polyacrylamide gel, and visualized by the Western blotting
technique using the antiserum specific for the C fragment. In the
standard run, the native protein is analyzed in the same way except
that step 1 is skipped. By the S-cyanocysteine
specific-fragmentation, 91 different polypeptide species should be
generated. However, only the 13 species (12 fragments of F1-F12 and one intact molecule, Int) that
retain the C-terminal segment of the intact protein should be detected
by the Western blotting analysis. On the basis of the molecular size of
the fragments, the half-cystine sites can be directly mapped from the C
terminus of the intact N lobe. In the sample run, only the intact
protein and the fragments that come from the cleavages at the
disulfide-involved half-cystine sites in a disulfide intermediate
should be detected. The fragments that are detected in the standard
run, but not in the sample run, correspond to those coming from the
cleavages at the free sulfhydryls in the disulfide intermediate. The
term of ``indirect end labeling'' was employed after Wu(1980)
who mapped the nuclease-sensitive sites in nuclear chromatin DNA as
probed with a cloned radiolabeled DNA
segment.
In step 1, protein disulfides were reduced under nondenaturing
conditions by incubating the isolated ovotransferrin N lobe at 0.2
mg/ml and 6 °C with 10 or 30 mM DTT for various times in
buffer A (0.1 M Tris-HCl buffer, pH 8.2, 0.1 mM Na-EDTA). The buffer was degassed at reduced pressure and
equilibrated under a N atmosphere prior to the reduction
experiments. Immediately after initiating the protein reduction, sample
vials were placed under a stream of N
and then sealed with
a screw cap. The protein was alkylated with 0.1 M IAA at 6
°C for 5 min and then at 37 °C for 5 min. The number of intact
disulfide bonds was determined by the PAGE technique as described
elsewhere (Hirose et al., 1988). For the isolation of
[3S-S] and [4S-S], the native protein
was reduced with 10 mM DTT for 50 min in a total volume of 30
ml and alkylated with IAA in the same way. The sample was concentrated
using a concentrator (Amicon, Centriprep-30), passed through a Sephadex
column (NAP-10, Pharmacia Biotech Inc.) equilibrated with buffer B (50
mM sodium acetate buffer, pH 5.5, containing 8 M urea), and applied to an ion-exchange column (Mono S HR 5/5,
Pharmacia) equilibrated with buffer B. Proteins were eluted by a linear
20-ml gradient of 0.05 to 0.9 M sodium acetate buffer, pH 5.5,
in the presence of 8 M urea; peak fractions as monitored by
absorbance at 280 nm were collected and analyzed for the number of
disulfides by the same PAGE technique. Protein samples containing
[3S-S] and [4S-S] were applied to a
reverse-phase column (Cosmosil 5C18 AR-300, 4.6
150 mm), eluted
by a linear 20 ml gradient of 30 to 60% acetonitrile in 0.1%
trifluoroacetic acid, lyophilized, and stored at -20 °C until
use for further analyses. [2S-S] was isolated in the
same way after incubating the native half-molecule with 30 mM DTT for 5 h.
In step 2, the cysteine-specific cyanylation was carried out essentially as described by Nefsky and Bretscher(1989). Briefly, the isolated disulfide intermediates were fully reduced at 1.0 mg/ml by incubation with 3 mM DTT at 37 °C for 20 min in buffer A containing 9 M urea in a total volume of 0.1 ml. The sample was mixed with 0.5 volume of 30 mM DTNB dissolved in 0.1 M sodium phosphate buffer, pH 7.5, incubated for 15 min at 25 °C, and then passed through a Sephadex G-25 column (NAP-5, Pharmacia) equilibrated with 0.1 M Tris-HCl buffer, pH 7.5. The modified protein was applied to a reverse phase column (Cosmosil 5C18-AR-300) equilibrated with 0.1% trifluoroacetic acid, 30% acetonitrile, eluted by a linear 30 to 60% gradient of acetonitrile, and lyophilized. The protein modified with DTNB was dissolved in 50 µl of cleavage buffer (0.1 M sodium borate, 0.5 M glycylglycine, 8 M urea, pH 9.0) and mixed with 0.5 µl of 0.1 M KCN.
In step 3, the S-cyanylated
protein was fragmented by incubation at 40 °C. At different times,
10 µl of the sample was withdrawn and the fragmentation was
terminated by addition of one volume of 0.2 M DTT. The sample
was analyzed by SDS-PAGE using the standard buffer system as described
by Laemmli (1970). Polyacrylamide gels (13 13
0.075 cm)
consisted of 15% acrylamide and 0.08% bisacrylamide. Protein fragments
were electroblotted onto a polyvinylidene difluoride membrane and
detected by an immunochemical procedure described by Sambrook et
al. (1989), using the antiserum raised against the C fragment
(Ser
-Arg
), anti-IgG alkaline
phosphatase conjugate, nitro blue tetrazolium, and
5-bromo-4-chloro-3-indolyl phosphate.
One nmol (36 µg) of the native
protein or either of the isolated disulfide intermediates was dissolved
in 35 µl of the tray buffer, mixed with 2 µl (0.5 nmol iron) of
0.25 mM iron-nitrilotriacetate (1:1 mixture of FeCl and the chelating agent), so that half of protein molecule can be
saturated with the metal. The sample was mixed with 0.33 volume of the
tray buffer containing 50% glycerol and applied to the top of the gel.
Electrophoresis was carried out at 4 °C and a constant voltage of
100 V for 2 h. The gel was stained with Coomassie Blue.
Figure 3: Disulfide reduction of the native protein under nondenaturing conditions. The native protein was incubated at 0.2 mg/ml with 30 mM DTT in buffer A at 6 °C for various times: lane 2, 0 min; lane 3, 5 min; lane 4, 15 min; lane 5, 30 min; lane 6, 50 min; lane 7, 1.5 h; lane 8, 3 h; lane 9, 5 h; lane 10, 8 h; lane 11, 24 h. The disulfide-reduced protein was alkylated with IAA, fully reduced with DTT, again alkylated with iodoacetamide, and then electrophoresed on an acid-urea gel as described in the text. In lanes 1 and 12, the standard protein with different numbers of disulfide bonds was electrophoresed. The numbers on the right side represent the numbers of disulfide bonds in a protein molecule.
To isolate the partially disulfide-reduced intermediates, the native protein was reduced with 10 mM DTT for 50 min, alkylated with iodoacetic acid, and then fractionated by ion-exchange column chromatography. As shown in Fig. 4, the protein was mainly separated into three peaks labeled 1, 2, and 3. Analyses for the number of disulfides by the PAGE technique revealed that the peaks 1, 2, and 3 correspond, respectively, to [3S-S], [4S-S], and the native six-disulfide protein. For the isolation of [2S-S], the native protein was reduced by 30 mM DTT for 5 h. On the same ion-exchange column chromatography, [2S-S] was eluted as a single peak with a retention time of 26.7 min (data not shown). The peaks corresponding to [2S-S], [3S-S], and [4S-S] were collected and employed for further analyses.
Figure 4: Isolation of partially disulfide-reduced intermediates. The native protein was reduced at 0.2 mg/ml with 10 mM DTT in buffer A at 6 °C for 50 min in a total volume of 30 ml. Disulfide-reduced proteins were alkylated with IAA, concentrated, passed through a Sephadex column, applied to an ion-exchange column, and eluted as described in the text. The same PAGE analysis as in Fig. 3revealed that the peaks 1, 2, and 3 correspond to [3S-S], [4S-S], and the six-disulfide native protein, respectively.
Figure 5: Indirect end-labeling visualization for the six-disulfide protein. In Panel A, the six intrachain disulfides in the native ovotransferrin N lobe were all reduced with DTT in the presence of 9 M urea. The protein was modified with DTNB and then cyanylated. In Panel B, the native protein was processed in the same way as in Panel A, except that disulfide-reduced protein was alkylated with IAA prior to the modification with DTNB. The protein was subjected to fragmentation at pH 9.0 for 0 min (lane 1), 5 min (lane 2), 10 min (lane 3), 20 min (lane 4), 3 h (lane 5), or 20 h (lane 6). The samples were analyzed by SDS-PAGE, electroblotted onto a membrane, and visualized immunochemically using the C fragment-specific antiserum as described in the text. In Panel C, to detect all fragments in one lane, the same volumes of 10- and 20-min fragmentation samples in Panel A were mixed and analyzed in the same way. Int represents the intact protein, and F1-F12 represent the fragments that retain the C-terminal segment of the intact protein. Roman numerals represent the corresponding native disulfide bonds.
To localize the intact disulfide bonds in ovotransferrin N lobe, the isolated disulfide intermediates of [4S-S], [3S-S], and [2S-S] were analyzed by the indirect end-labeling method as the sample run in Fig. 2B. Fig. 6clearly shows that, for either [4S-S], [3S-S], or [2S-S], the number of the detected fragments was exactly consistent with the one expected from the number of the intact disulfide bonds. The number of the fragment bands was eight for [4S-S]; the fragment bands corresponding to SS-I, SS-II, SS-III, and SS-VI were clearly detected, but no other fragments corresponding to SS-IV and SS-V were produced (Fig. 6C). For [3S-S], only the six fragments that corresponded to SS-I, SS-II, and SS-VI were detected (Fig. 6B). When [2S-S] was analyzed, only the four fragment bands corresponding to SS-I and SS-II were detected. These results were consistent with the idea that the disulfide cleavage occurs first at SS-IV and SS-V, second at SS-III, and then at SS-VI. Both SS-I and SS-II are resistant to DTT under the employed reducing conditions.
Figure 6: Disulfide localization for the partially disulfide-reduced intermediates. The isolated disulfide intermediates of [2S-S] (Panel A), [3S-S] (Panel B), and [4S-S] (Panel C), in which free sulfhydryls were all alkylated with IAA, were fully reduced with DTT in the presence of 9 M urea. The sample was modified with DTNB, cyanylated, and fragmented for 0 min (lane 1), 5 min (lane 2), 10 min (lane 3), 20 min (lane 4), 3 h (lane 5), or 20 h (lane 6). SDS-PAGE, electroblot onto a membrane, and immunochemical detection using the C fragment-specific antiserum were carried out as described in the text. Roman numerals on the right side represent the corresponding native disulfide bonds.
According to our peptide mapping procedure (Yamashita and Hirose, 1993), the extensive tryptic digestion of the native half-molecule yields, on reverse-phase HPLC, nine disulfide peaks denoted A-I. By the rechromatography, peaks C and I, but not the other peaks, are separated into two peaks: peak C, into peaks C-1 and C-2, and peak I, into peaks I-1 and I-2. The peaks, A, B, C-1, C-2, D, E, F, G, H, I-1, and I-2, respectively, correspond to SS-V, SS-VI, SS-II, SS-III, SS-II, SS-III, SS-IV, SS-III, SS-VI, SS-VI, and SS-I.
The isolated disulfide intermediates of [3S-S] and [2S-S] along with the native protein control were analyzed by the same peptide mapping procedure. We reconfirmed by analyzing amino acid sequences and amino acid compositions that the 11 disulfide peptide peaks were all produced from the native protein control. For [3S-S], however, the disulfide peptides (A, C-2, E, F, and G) corresponding to SS-III, IV, and V were missing, while the occurrence of all the other disulfide peptides (B, C-1, D, H, I-1, and I-2) corresponding to SS-I, SS-II, and SS-VI was confirmed. For [2S-S], the occurrence of the peptides (C-1, D, and I-2) corresponding to SS-I and SS-II was confirmed, but no other disulfide peptides were detected. All disulfide peptides from [3S-S] and [2S-S] were analyzed for amino acid compositions, and peptide recoveries were compared between the partially disulfide-reduced proteins and the native protein control. As summarized in Table 1, the recoveries of SS-I and SS-II from [3S-S] were more than 93% of those from the native protein, although the recovery of SS-VI showed a slightly lower value of 72.8%. The recoveries of SS-I and SS-II from [2S-S] were also greater than 94% of those of the native protein control. These data from the peptide mapping analyses along with previous results (Yamashita and Hirose, 1993) led us to the same conclusion as the one obtained by the indirect end-labeling approach: the disulfide reduction in ovotransferrin N lobe occurs first at SS-IV and SS-V, second at SS-III, and then at SS-VI. Both SS-I and SS-II are highly resistant against reduction with DTT.
Figure 7: Transverse urea gradient PAGE of the native and partially disulfide-reduced intermediates. The native protein (Panel A), [4S-S] (Panel B), [3S-S] (Panel C), and [2S-S] (Panel D) was incubated with iron-nitrilotriacetate complex at an iron to protein molar ratio of 0.5, electrophoresed from top (cathode) to bottom (anode) on transverse linear 0 to 8 M urea gradient gels at 4 °C, and stained with Coomassie Blue as described in the text. For each protein, the fraction of unfolded form, Fu, is indicated on the right side scale.
When [4S-S] was analyzed in the same way, the protein was separated into two distinct bands (Fig. 7B); one band displayed urea-induced denaturation transition, and the other showed no denaturation in urea concentrations from 0 to 8 M. Protein species corresponding to the urea-resistant band can be again accounted for by the iron-bound form. The urea concentration for the midpoint of the denaturation was the same 4.0 M for the iron-free form.
In contrast to the native protein and [4S-S], either [3S-S] or [2S-S] that had been incubated with iron at metal to protein molar ratio of 0.5 showed a single band, suggesting the absence of iron-binding capacity. Both [3S-S] and [2S-S] showed some conformational transition by urea; the urea concentration giving the midpoint of the denaturation was 3.1 M for [3S-S] and 2.6 M for [2S-S] (Fig. 7, C and D).
Figure 8: Far-UV CD spectra of the native and partially disulfide-reduced intermediates. The native protein (dashed and dotted lines), [4S-S] (short-dashed line), [3S-S] (solid line), and [2S-S] (long-dashed line) were analyzed for far-UV CD spectrum at 6 °C in buffer A in the presence (Panel B) and absence (Panel A) of iron as described in the text. For the dotted line, the native protein was incubated with 8 M urea in buffer A and analyzed for the CD spectrum in the same way.
As shown in Fig. 8B, [3S-S] and [2S-S] appeared, also in the presence of iron, to contain significantly lower secondary structures than the native protein. When the spectrum data were compared between the presence and absence of iron, the CD profiles were almost indistinguishable for these protein species. This may be related to the absence of iron-binding capacity in either [3S-S] or [2S-S] (see Fig. 7, C and D). The disulfide intermediate, [4S-S], however, displayed significantly different CD spectra in the presence and absence of iron; the CD spectrum of this intermediate was very similar to that of the native protein in the presence of iron. This disulfide intermediate may, therefore, undergo some conformational change upon the iron binding in such a way that partially unfolded state is transformed into the native-like conformation.
Figure 9: Visible absorption spectra of the native and partially disulfide-reduced intermediates. The native protein (solid line), [4S-S] (dashed line), and [3S-S] (dashed and dotted line) were incubated with iron and analyzed for visible absorption spectra as described in the text.
The chemical fragmentation method that includes specific polypeptide cleavages in S-cyanocysteine sites and subsequent SDS-PAGE has been shown to be potentially useful for the simple and sensitive analysis of the localization of half-cystines in a protein (Jacobson et al., 1973; Subramanian, 1980; Walser et al., 1981; Mikami and Morita, 1983). The method has, however, yielded only indirect evidence about the localization without the use of a specific end-labeling procedure. Matsudaira et al.(1985) first attempted to localize the half-cysteine sites in villin by the immunoblot analysis of the S-cyanocysteine cleavage products using antiserum specific for the terminal sequence peptides. However, it has not been clear because of the use of a protein with unknown chemical structure whether their approach works as a general method for localizing half-cystine sites in a protein; the S-cyanocysteine cleavage data have shown the presence of 6 half-cystines in villin, while the data from the radiolabeled alkylation and from optical determinations using DTNB have shown the occurrence of 2.6-3.9 half-cystines. Furthermore, their procedure has not included the way to differentiate cystine and cysteine sites. The data in our present report demonstrate that the indirect end labeling reliably works for the cystine and cysteine localizations in a complex protein, such as ovotransferrin N lobe with 12 half-cystines, providing a promising example for its general application for a variety of sulfhydryl and disulfide proteins.
The indirect end-labeling
method has some advantages over previous ``landmark mapping''
(Nefsky and Bretscher, 1989), in which the carbon atom of CN is introduced, upon the S-cyanocysteine-specific fragmentation reaction, into the N
termini of all the newly generated fragments. First, the number of the
fragment bands detected in SDS-PAGE by a nonspecific staining should be (p + 1)(p + 2)/2 (91 bands for
ovotransferrin N lobe) for a protein having p of the number of
half-cystines, while that detected by autoradiography should be p(p + 1)/2 (78 bands for the N lobe). The
difference in these numbers, p + 1, corresponds to the
number of the bands that are assigned to the fragments retaining the N
terminus of the original protein. This complex procedure may hamper the
clear mapping of S-cyanocysteine sites for a protein having
many disulfides. In the indirect end-labeling method, however, only (p + 1) bands that retain the N or C terminus of intact
protein should be directly detected. Second, cellular protein folding
is one of the most important subjects in modern bioscience. A disulfide
protein in which all disulfide intermediates may be trapped in stable
forms (Creighton, 1986) is a useful model for investigating in vivo protein folding. The potential usefulness for such a nonpurified
protein system appears to be an alternative advantage of the indirect
end-labeling approach, since this technique includes specific protein
visualization with high sensitivity after gel electrophoresis.
Data
from the optical and PAGE analyses of the disulfide intermediates
provide important information about structural and functional roles of
the disulfide bonds in ovotransferrin N lobe. One of the most striking
feature is that [4S-S] still retains the iron-binding
function ( Fig. 7and Fig. 9). The observation
unambiguously proves that SS-IV
(Cys-Cys
) and SS-V
(Cys
-Cys
) that are both strictly
conserved in N and C lobes of ovotransferrin, lactoferrin, and serum
transferrin (Williams, 1982) are not essential for the iron-binding
function. Another interesting observation for [4S-S] is
the lower mobility, in a low urea concentration range (from 0 to about
2.5 M urea) on the transverse urea gradient PAGE, for the
iron-free form than for the iron-loaded form of [4S-S] (Fig. 7B). This may be explained as follows. As
observed with the far-UV CD spectrum, [4S-S] may
undergo some conformational change upon the iron binding in such a way
that partially unfolded state is transformed into the native-like
conformation (Fig. 8). The lower mobility of the iron-free form
can, therefore, be accounted for, at least in part, by the partially
unfolded conformation. In addition, the iron-dependent conformational
change might be closely related to the amino acid residues that are
involved in the disulfide loop of SS-III
(Cys
-Cys
); in the absence of iron,
the loop might be more exposed to protein environment in
[4S-S] than in the native protein, since
[4S-S] lacks both SS-IV
(Cys
-Cys
) and SS-V
(Cys
-Cys
). The loop, however, should
be less exposed to protein environment, since it includes an
iron-coordinating ligand (Tyr
) and the synergistic
anion-binding ligand (Arg
) that are both localized in the
interior of the interdomain cleft in the iron-loaded form (Dewan et
al., 1993). The disulfide loop contains 12 basic amino acid
residues (Arg
, His
, and Lys
) and 10
acidic residues (Glu
, Asp
, and
carboxymethyl-Cys
) (Jeltsch et al., 1987). These
would confer increased positive charges on the accessible surface of
the iron-free form of [4S-S], thereby reducing the
electrophoretic mobility toward anode.
The results from the indirect
end-labeling analysis clearly shows that the number of the detected
fragments is exactly consistent with the one expected from the number
of the intact disulfide bonds for either [4S-S],
[3S-S], or [2S-S] (Fig. 6).
Furthermore, the disulfide peptides are recovered in high yields (Table 1) from either [3S-S] or
[2S-S] that shows a single peak on the ion-exchange
column chromatography (Fig. 3) and no non-native disulfide
peptide has been detected on the peptide mapping analyses of
[4S-S], [3S-S], and
[2S-S]. ()These data strongly suggest that
the isolated disulfide intermediate of either [4S-S],
[3S-S], or [2S-S] consists of a single
molecular form that specifically lacks the corresponding disulfides.
A question arises as to the implication of these disulfide
intermediates for the redox folding/unfolding pathway of ovotransferrin
N lobe. In the oxidative folding pathway of bovine pancreatic trypsin
inhibitor, the kinetically preferred mechanism involves intramolecular
disulfide rearrangements (Creighton, 1977; Creighton and Goldenberg,
1984; Weissman and Kim, 1992), while the reductive unfolding mechanism
includes the parallel pathways of the direct reduction and
rearrangement pathway (Mendoza et al., 1994). As a technical
problem on detecting the correct disulfide intermediates, it has been
pointed out that the rate for sulfhydryl alkylation is sometimes too
slow to quench rapid sulfhydryl/disulfide interconversions (Rothwarf
and Scheraga, 1991; Weissman and Kim, 1991). The following
observations, however, make it very unlikely that the partially
disulfide-reduced intermediates of the ovotransferrin N lobe are
artificial ones that are formed during slow alkylation with 0.1 M IAA employed in the present report. We observe by the peptide
mapping analysis that [4S-S] and
[3S-S], which are isolated from the disulfide
intermediates quenched at an increased concentration of 0.9 M IAA, possess the same disulfide structures as the ones
characterized in the present report. All of the disulfide
intermediates of [4S-S], [3S-S], and
[2S-S] have some stable and partially folded
conformations, and their residual native conformations are decreased
with the increased number of the reduced disulfides ( Fig. 7and Fig. 8). We therefore conclude that either
[4S-S], [3S-S], or
[2S-S] is implicated as a major stable
disulfide-intermediate for the reductive unfolding pathway of
ovotransferrin N lobe. The pathway for the disulfide reduction from the
native form to [4S-S], however, is still not clear,
since no five-disulfide intermediate is detected in the disulfide
intermediates quenched at either 0.1 M IAA (Fig. 3) or
0.9 M IAA.
According to the x-ray crystallographic
structure (Dewan et al., 1993), the sulfur atoms of SS-V are
somehow exposed to solvent, while all sulfur atoms of the other
disulfides are almost completely buried in the protein molecule. The
SS-V, therefore, may be the disulfide that first cleaved by DTT. In an
oxidatively refolded form of ovotransferrin N lobe, small amounts of
non-native disulfides (Cys
-Cys
and
Cys
-Cys
) that correspond to the
mispairings of SS-IV and SS-V have been detected (Yamashita and Hirose,
1993), probably reflecting the fact that the
C
-C
distances between Cys
and Cys
and between Cys
and
Cys
are only about 5 Å in the native structure
(Dewan et al., 1993). It is, however, not clear whether or not
such non-native disulfide intermediates are involved in the pathway for
the second reduction of SS-IV. The question of direct reduction or
rearrangement reduction remains until the kinetic pathway for the redox
folding/unfolding of the ovotransferrin N lobe is established.