From the National Center for Cell Science, Ganeshkhind, Pune-411007, India
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ABSTRACT |
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The physical state of two model mutants of
The activity of It has been observed that the carboxyl-terminal end of The extension mutant was constructed with an aim to see whether or not
All bacterial strains employed in this study were obtained from
commercial sources. Ultrapure bovine serum albumin, S-Sepharose, and
8-anilino-1-naphthalene-sulfonate (ANS) were obtained from Sigma.
Protein estimations were carried out with the Bradford reagent
(Bio-Rad) using ultrapure lipid-free bovine serum albumin as the
standard. Hemolysis assays were carried out with freshly drawn blood
from New Zealand White rabbits of the local animal facility. All other
chemicals used were of analytical grade.
Cloning of Purification of Purification of CDM--
E. coli BL21(DE3) cells
harboring the CDM plasmid were grown at 37 °C and induced at 0.4 A600 with 0.2 mM
isopropyl-1-thio- Purification of CEM--
Purification of the soluble protein was
carried out employing similar protocol reported by us earlier for
Limited Proteolysis--
The proteins (native and refolded as
applicable) were subjected to digestion with Proteinase K at 25 °C
by keeping the substrate:enzyme ratio at 50:1. At appropriate time
points, the enzyme was inactivated by the addition of 5× Laemmli
sample buffer and boiled at 100 °C for 5 min. The samples were
analyzed by 14% SDS-PAGE.
Hemolysis Assays--
The lysis of rRBCs was measured by adding
100 µl (7 µg/ml) of protein to 100 µl of K-PBSA (150 mM NaCl, 20 mM KH2PO4,
pH 7.4, containing 1 mg/ml bovine serum albumin) in well number 1, and a 100-µl aliquot was taken for 2-fold serial dilution to 12 wells. An
equal volume of 2% rRBCs was added to the wells and left at 25 °C
for 1 h. At the end of the incubation period, the well number exhibiting 50% lysis was recorded visually. For CDM, the lysis was
recorded after 24 h. Unless specifically mentioned, all hemolytic assays were performed in 96-well microtiter plates.
Quantitative Hemolysis Assay--
The toxins (2 µg) were added
to 1 ml of K-PBSA and a 500-µl aliquot was subjected to 2-fold serial
dilutions with the same buffer. To the serially diluted samples an
equal volume (500 µl) of 1% rRBCs was added and incubated at
25 °C for 30 min. The samples were then centrifuged, and the
absorbance at 545 nm (due to hemoglobin release) was plotted against
toxin concentration. For the mixed hemolysis assays with Fluorescence Measurements--
Fluorescence measurements were
carried out in a Perkin-Elmer LS-50B spectrofluorometer. The protein
samples (30 µg/ml in 10 mM MOPS, pH 7.0) were excited at
295 nm, and the slit widths were 5 nm for both excitation and emission.
All fluorescence spectra are corrected for buffer contribution and are
an average of at least eight scans. For the pH-induced unfolding
studies,
For ANS binding studies, a stock solution of ANS (5 mM) was
prepared in methanol. Binding of ANS to CD Studies--
CD spectra were recorded on a Jobin Yvon
spectropolarimeter, which was calibrated with
D-10-camphorsulfonic acid. The proteins were diluted to a
concentration of 0.1 mg/ml in 10 mM sodium acetate, pH 5.2, buffer. The far (190-250 nm) and near (250-320 nm) UV spectra were
recorded in 1-mm and 5-cm path length quartz cuvettes, respectively.
The spectra are an average of four scans, with subtraction of
appropriate blanks.
Coupled In Vitro Transcription and Translation--
Supercoiled
plasmid DNA was used for in vitro transcription and
translation in an Escherichia coli T7-S30 extract in the
presence of rifampicin and complete amino acid mix as reported earlier (4). A 5-µl aliquot was withdrawn at various time intervals from the
initiation of translation. The 5-µl aliquot was added to a 96-well
plate containing 200 µl of 0.5% rRBCs. The decrease in absorbance at
595 nm due to hemolysis was monitored at regular time intervals.
Purification and Characterization of
We have carried out limited proteolysis of the three proteins in
solution with Proteinase K. Limited proteolysis is a sensitive probe
for analyzing the conformation of proteins (11). Polypeptides that are
devoid of tertiary structure have been observed to be very sensitive to
proteolysis (12, 13). Proteinase K cleaves Hemolytic Activity--
The hemolytic activity of the three
proteins was compared by a quantitative assay. As seen from Fig.
2, the CDM exhibited no lysis at all in
the time course of the experiment, whereas the CEM showed efficient
lysis. However, it is about 5-fold weaker than Unfolding and Refolding of Fluorescence Studies--
The normalized fluorescence emission
spectra of native and denatured states of
The fluorescence emission maximum as a function of pH for Probing the Hydrophobic Regions of CD Studies--
The secondary structure of
The near UV-CD spectra of In the present study, we have designed a carboxyl-terminal
deletion and a carboxyl-terminal extension mutant of Our efforts to isolate the CDM have met with partial success, since it
appears to be unstable. The protein is almost exclusively found in
inclusion bodies, unlike recombinant The CDM has a tendency to aggregate both in vitro and
in vivo (as seen by the extensive inclusion body formation).
Studies have shown that inclusion bodies form due to aggregation of
partially folded intermediates (23). Hence, the occurrence of CDM in
inclusion bodies suggests that the mutant protein was unable to achieve the final folded conformation in vivo. Limited proteolysis
experiments have revealed that the CDM gets completely digested unlike
The fluorescence emission of the CDM lies in between the native and
denatured states of The far UV-CD spectrum of CDM shows that it possesses nearly
native-like secondary structure. However, its tertiary structure as
analyzed by near UV-CD is greatly diminished. The near UV-CD spectrum
indicates that most of the phenylalanine and tryptophan of CDM are
in a mobile, symmetric environment but some tertiary contacts are
present in the environment of some of the tyrosines. A cursory glance
at the distribution of the aromatic residues along the All of these observations strongly suggest that significant
perturbations to the The non--hemolysin (
HL),
HL(1-289), a carboxyl-terminal deletion
mutant (CDM), and
HL(1-331), a carboxyl-terminal extension mutant
(CEM), were examined in detail to identify the role of the carboxyl
terminus in the folding and function of native
HL. Denatured
HL
can be refolded efficiently with nearly total recovery of its activity
upon restoration of nondenaturing conditions. Various biophysical and
biochemical studies on the three proteins have revealed the importance
of an intact carboxyl terminus in the folding of
HL. The CDM
exhibits a marked increase in susceptibility to proteases as compared
with
HL.
HL and CEM exhibit similar fluorescence emission maxima, and that of the CDM is red-shifted by 9 nm, which indicates a greater
solvent exposure of the tryptophan residues of the CDM. In addition,
the CDM binds 8-anilino-1-naphthalene sulfonic acid (ANS) and increases
its fluorescence intensity significantly unlike
HL and CEM, which
show marginal binding. The circular dichroism studies point that the
CDM possesses significant secondary structure, but its tertiary
structure is greatly diminished as compared with
HL. These data show
that the CDM has several of the features that characterize a molten
globule state. Experiments with freshly translated mutants, using
coupled in vitro transcription and translation, have
further supported our observations that deletion at the carboxyl terminus leads to major structural perturbations in the water-soluble form of
HL. The studies demonstrate a critical role of the carboxyl terminus of
HL in attaining the native folded state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Hemolysin (
HL)1
of Staphylococcus aureus has attracted lot of attention from
structural biologists and biotechnologists for its potential
applications in biotechnology and therapeutics (1). It is a 293-amino
acid polypeptide that binds target cells as a monomer, and the
cell-bound monomers undergo lateral diffusion to form a transmembrane
heptameric pore. The heptamer is a rigid mushroom-like structure,
resistant to SDS and temperatures up to 80 °C (2). The water-soluble
monomer undergoes a series of conformational changes to form the
heptameric pore on the membrane. The amino acid residues 110-148,
termed the stem domain, penetrate the membrane bilayer to access the
interior of a target cell for the functional pore formation.
HL was earlier shown to be critically dependent on
an intact amino and carboxyl termini (3). Recent studies from our
laboratory have shown that deletion of four amino acids at the amino
terminus of
HL leads to delayed pore opening. Although this mutant
(
HL(5-293)) could undergo the oligomerization process as fast as
native
HL, the conformational changes that lead to the opening of
the pore were retarded (4). Fluorescence spectroscopic studies carried
out by Valeva et al. (5) have also arrived at similar
conclusions regarding the role of the amino terminus. Previous studies
have shown that deletion of three, five, or eight amino acids at the
carboxyl-terminal end of
HL impairs its oligomerization and pore
formation abilities. The carboxyl-terminal deletion mutants, however,
have been shown to bind to rabbit red blood cells (rRBCs), where they
remain in a cell-bound monomer form (3). In addition, the
carboxyl-terminal deletion mutants are very inefficient in forming
hetrooligomers with full-length
HL. All of the studies conducted so
far have attributed the role of the amino terminus for pore opening and
the carboxyl terminus for the initial oligomerization process of
HL.
However, the reasons for the inefficient oligomerization of the
carboxyl-terminal deletion mutants are not yet clear.
HL becomes
more exposed to the solvent in the oligomeric state than in the monomer
form in solution, as revealed by IASD modification of single cysteine
mutants (IASD is a membrane-impermeant reagent that covalently modifies
surface-accessible cysteine residues in proteins (6, 7)). This
observation was supported by the crystal structure of the fully
assembled
HL pore, which shows that the carboxyl terminus is well
exposed to solvent. Thus, the carboxyl terminus does not appear to be
critically involved in interprotomer interactions (3). Hence, a
deletion of as few as three carboxyl-terminal residues ought not to
have any drastic effect on the oligomerization process. Another
possibility is that the carboxyl-terminal deletion is hampering a
process prior to the oligomerization step, which might occur in the
water-soluble monomer or at the membrane-bound monomer stage.
Therefore, in order to have a better understanding of the role of the
carboxyl terminus in the structure and function of
HL, we have aimed
to examine the properties of a carboxyl-terminal deletion and an extension mutant.
HL can accept a polypeptide at the carboxyl terminus and still carry
out its folding and function. For this purpose, we have added a nearly
neutral, nonaromatic amino acid-rich sequence to the carboxyl terminus
of
HL. In this paper, the results from biochemical and biophysical
studies on the above proteins have revealed that the carboxyl terminus
of
HL stabilizes the native structure of
HL. In the absence of
the carboxyl terminus,
HL is unable to acquire its water-soluble,
fully folded, native form. This defective folding caused by truncation
is responsible for the loss of function of
HL.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
HL, CDM, and CEM--
The cloning of
HL was
described in detail by Vandana et al. (4). The PCR
amplification of CDM was achieved by advancing the stop codon present
in
HL at 294 to Glu290 by a downstream primer that
contains a HindIII site. (The upstream primer is the same as
that used for
HL.) The resultant PCR product was digested with
NcoI and HindIII and ligated to the pT7 vector described earlier (4). The CEM was constructed in two stages by PCR
amplification of the
HL gene using an upstream primer that contains
an EcoRI site at Asn293 and the downstream
primer (with HindIII site) of
HL reported earlier (4).
The upstream primer eliminates the stop codon present in the
HL
gene. The primers containing the EcoRI and HindIII sites were first joined by PCR using
HL template.
The CEM in final form was obtained by re-PCR of
HL template using a
T7 promoter primer and the EcoRI and HindIII
joined product obtained in the first stage. The resultant PCR product
was digested with NcoI and EcoRI to remove the
3'-untranslated region of
HL. This double-digested PCR product was
ligated to the parent pT7 vector between NcoI and
EcoRI. As a result of removal of the stop codon of
HL,
the translation proceeds beyond the EcoRI site and incorporates the following 38-amino acid stretch from the vector backbone:
294SSSVDKLEYSIVSPKSELDPAANKARKEAELAAATAEQ331.
HL--
HL was purified from S. aureus wood 46 (ATCC 10832) as reported earlier (8). Briefly, a
2% mid-log phase inoculum of S. aureus was added to 1 liter
of tryptic soy broth, and the culture was grown for 18 h at
37 °C. The cells were removed by centrifugation, and the supernatant
was brought to 80% saturation with ammonium sulfate. The mixture was
left overnight at 4 °C with mild stirring. The precipitate was
collected by centrifugation and dialyzed against 10 mM
sodium acetate, pH 5.2, buffer at 4 °C for 48 h with at least
eight changes. The dialysate was clarified and loaded on an S-Sepharose
column pre-equilibrated with 10 mM sodium acetate buffer,
pH 5.2. The bound
HL was eluted with a step gradient, and the yield
of
HL was typically 3-4 mg/liter. The protein was >95% pure as
judged by SDS-PAGE (9).
-D-galactopyranoside. After 4 h of
induction, the cells were harvested by centrifugation at 4000 × g for 20 min, resuspended in buffer A (50 mM
Tris-HCl, pH 8.0, containing 5 mM EDTA), and treated with
0.2 mg/ml lysozyme at 20 °C for 25 min. The cells were passed once
at 1000 p.s.i. through a French press followed by 5-min
sonication. The resultant cell lysate was centrifuged at 6000 × g for 10 min. The inclusion body pellet was washed three
times with 1 M urea and 0.5% Triton X-100 in buffer A,
rinsed with buffer A, and stored at
4 °C until further use. The
inclusion bodies were suspended in 8 M urea in buffer B (10 mM sodium acetate, pH 5.2), diluted 2-fold with buffer B,
and centrifuged at 13,000 × g. The supernatant was
passed through an S-Sepharose column pre-equilibrated with 4 M urea in buffer B. The mutant protein eluted with 200 mM NaCl and 4 M urea in buffer B. The protein
was renatured either by dialysis against buffer B at 4 °C or by
simple dilution as desired for specific experiments, and hereafter this
protein is referred to as renatured. The renatured protein was found to
be >95% pure as judged by SDS-PAGE.
HL(5-293) (4).
HL and CEM,
known quantities of toxins were mixed, and the assay was carried out as
outlined above.
HL Unfolding and Refolding--
A stock solution of
HL
(1.75 mg/ml) was diluted with freshly prepared 10 M urea or
8 M guanidine HCl to a final concentration of 0.350 mg/ml
HL in 8 M urea and 6 M guanidine HCl,
respectively. For acid denaturation studies,
HL was incubated in 50 mM citrate-phosphate buffer at pH 2.5, 3.5, and 7.0 at a
concentration of 350 µg/ml. After incubation at 25 °C for
different periods of time in the above described conditions, samples
were withdrawn and diluted 50 times with various buffers (given in
Tables I and II), and a hemolysis assay was performed immediately.
HL and CDM were incubated in 50 mM citric
acid/Na2HPO4 buffer of pH values ranging from
2.5 to 7.0 for 20 min before measuring the emission spectrum. The
buffer solutions were prepared by suitably mixing solutions of 50 mM citric acid and 50 mM
Na2HPO4 in order to obtain the desired pH.
HL, CDM, and CEM was carried
out in cuvettes containing 30 µg/ml protein in 10 mM
MOPS, pH 7.00, and 50 µM ANS. ANS fluorescence was
obtained in the range of 410-580 nm with the excitation wavelength
fixed at 390 nm using the same slit widths mentioned above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
HL, CDM, and CEM--
All
of the toxins employed for the present study were constructed under the
control of the T7 promoter. The relative sizes of the polypeptides are
in agreement with the cloning strategy (Fig.
1A). Unlike recombinant
HL
and CEM, the soluble form of CDM could not be obtained due to extensive
inclusion body formation (>95%). Since inclusion body formation is
often due to temperature-sensitive denaturation of the protein and can
be avoided at lower culture growth temperatures (10), the CDM culture
was grown at 30 °C. However, this had no effect on the extent of
inclusion body formation. Hence, we have purified CDM by solubilizing
its inclusion bodies as described under "Materials and Methods."
The purity of all three proteins was routinely assessed by SDS-PAGE and
was found to be >98% as shown in Fig. 1A.
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Fig. 1.
A, SDS-PAGE of purified HL, CDM, and
CEM. The full-length
HL and its mutants were isolated as described
under "Materials and Methods" and electrophoresed on 12% SDS-PAGE.
Lane 1, 2 µg of CDM; lane
2, 2 µg of
HL; lane 3, 2 µg of
CEM. B, proteolysis of
HL and CDM. The purified proteins
were subjected to limited proteolysis with Proteinase K as mentioned
under "Materials and Methods" and electrophoresed on 15% SDS-PAGE.
Lane 1,
HL at 0 min; lane
2,
HL at 2 min; lane 3,
HL at 15 min; lane 4,
HL at 60 min; lane
5, CDM at 0 min; lane 6, CDM at 2 min;
lane 7, CDM at 15 min; lane
8, CDM at 60 min. The two halves are marked with an
arrow.
HL monomer in solution
between residues 131 and 136 of the polypeptide chain, yielding an
approximate two halves of
HL. Such a cleavage does not occur for the
membrane-bound forms of monomer and oligomer because these residues get
occluded in the membrane (14). As seen in Fig. 1B, the
refolded CDM was completely digested by Proteinase K in minutes,
whereas
HL gave the expected two halves, which were resistant toward
further protease attack. This enhanced susceptibility of CDM towards
protease suggests that its structure is not as rigid as that of
HL.
HL and increasing the
amount of CEM 5 times in the assay gave an identical curve like that of
HL.
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Fig. 2.
Quantitative hemolysis assays of
HL, CDM, and CEM. The toxins were added to 1 ml of K-PBSA and subjected to 2-fold serial dilutions. An equal volume
of rRBCs was added to get a final concentration of 0.5% and 1 µg/ml
of toxin in the first dilution. After incubation at 25 °C for 30 min, the absorbance at 545 nm of the centrifuged sample was measured.
,
,
, and
,
HL, CDM, CEM, and CEM (5 times the amount of
other toxins in each tube), respectively.
HL--
The denaturation and
renaturation of
HL were carried out by employing a wide spectrum of
denaturing conditions as described under "Materials and Methods."
It is clear from Table I that >95% of
hemolytic activity of
HL can be recovered upon restoration of
nondenaturing conditions. However, when
HL was incubated at pH 3.5 at room temperature, the recovery of hemolytic activity decreases with
longer incubation times. At pH 3.5, the carboxyl-terminal portion of
HL is said to undergo a transition to a molten globule-like state
with exposed hydrophobic regions (15). The apparent reason for the loss
of activity at pH 3.5 could be due to aggregation of the partially
unfolded intermediate. However, total recovery of activity was achieved
when the incubation was carried out at 4 °C, which could be either
due to slow denaturation or due to a significant decrease in
hydrophobic interactions at the lower temperature.
Recovery of hemolytic activity of HL after its denaturation and
renaturation
HL, CDM, and CEM are shown
in Fig. 3, and the emission maxima
obtained are 336, 345, and 336 nm, respectively. It is interesting to
note that the emission maximum of CDM exhibited a 9-nm red shift with
respect to native
HL, indicating a change in the polarity of
environment of tryptophans due to solvent exposure (16). The emission
maxima for all three toxins were further red-shifted in presence of 8 M urea to 352.5 nm. In contrast to CDM, the fluorescence
spectrum of CEM was identical to that of
HL, indicating that the
extra 38 residues at the C-terminal end of
HL did not have any
influence on its folding and function. The residues that were deleted
in the case of CDM are
Glu290-Met291-Thr292-Asn293,
and the residues extended in the case of CEM do not contain any
tryptophans. Hence,
HL, CDM, and CEM contain an equal number of
aromatic residues in their primary sequence, and the differences in
their fluorescence spectra clearly reflect the degree of compactness of
the individual toxins.
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Fig. 3.
Fluorescence spectra of
HL, CDM, and CEM in the presence and absence of 8 M urea. The fluorescence spectra of the respective
toxins (30 µg/ml) in 10 mM MOPS, pH 7.0, were recorded in
quartz cuvettes with excitation fixed at 295 nm and 5-nm slit widths
for both excitation and emission. The spectra are corrected for buffer.
Solid line,
HL; dotted
line, CDM; short dashed
line, CEM. Very short
dashed line, dashed line and
dot, and long dashed line are
HL, CDM, and
CEM, respectively, in 8 M urea. Spectra for CDM were
obtained by renaturation of urea-solubilized inclusion bodies by
dilution to the appropriate concentration followed by clarification by
high speed centrifugation. AU, arbitrary units.
HL and CDM
is depicted in Fig. 4. The curve obtained
for
HL is in agreement with previous results (15). The CDM in 8 M urea was either 1) diluted with an appropriate buffer to
the desired pH (final urea concentration was kept at about 80 mM) or 2) renatured by dialysis, and the pH of the
dialysate was adjusted to the desired value. The curves obtained by
both of the approaches do not overlap (as one would expect) even after
incubation for 48 h, in that the case 2 CDM appears to be more
resistant to acid denaturation compared with the case 1 CDM. This
hysteresis could be due to formation of soluble aggregates among the
renatured CDM molecules, because it has been observed that aggregation
commonly interferes with the correct equilibrium, giving rise to such
hysteresis (17). This possibility was examined by glutaraldehyde
cross-linking of the renatured CDM, and high molecular weight forms of
CDM were observed in contrast to
HL (data not shown).
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Fig. 4.
Effect of pH on the fluorescence
emission HL and CDM. The toxins were
incubated in 50 mM citrate-phosphate buffer at various pH
values, and their fluorescence emission maxima were recorded as
described in the legend to Fig. 3.
,
HL.
and
represent
CDM refolded and denatured, respectively.
HL, CDM, and CEM--
ANS
has been extensively used to characterize the hydrophobic pockets of
proteins and enzymes for understanding their folding and function (18).
The fluorescence emission of ANS is known to increase when the dye
binds to hydrophobic regions of proteins that are normally absent in
totally unfolded states and rarely present in native states (19). While
the fluorescence of ANS marginally increased in the presence of
HL
and CEM, the increase in case of CDM was rather dramatic, as shown in
Fig. 5. In addition, the emission maximum
of ANS had blue-shifted from 513 nm in buffer to 483.5 nm upon binding
to CDM. On the other hand, the denatured states of the three proteins
did not bind any ANS (data not shown). This result indicates that CDM
has hydrophobic regions exposed to the solvent, unlike
HL and CEM.
It is interesting to note that the CEM did not show any concomitant
increase in fluorescence intensity of ANS. These observations reflect
the compactness of native
HL and the role of the carboxyl terminus
in maintaining such a compact structure.
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Fig. 5.
Binding of ANS to
HL, CDM. and CEM. Binding of ANS to
HL,
CDM, and CEM was carried out in cuvettes containing 30 µg/ml toxin
and 50 µM ANS. The excitation was at 390 nm, and the
emission range recorded was 410-580 nm with 5-nm slit widths.
Long dashed line, buffer containing
ANS. Solid line, dotted
line, and short dashed
line,
HL, CDM, and CEM, respectively, in the presence of
50 µM ANS. AU, arbitrary units.
HL and CDM was
examined by far UV-CD spectroscopy. The CD spectrum of CDM shows a
significant content of secondary structure and is characteristic of a
predominantly
-sheet protein, as is the case for
HL. However,
comparison with the
HL spectrum suggests some minor conformational
differences between the two species (Fig.
6A). The spectrum of
HL
shows a minimum at 214.5 nm and is consistent with earlier reports (14, 15, 20). In case of CDM, the minimum was red-shifted to 218.5 nm. This
might result from a change in the polarity of the environment of
-sheets, since it is well known that the
-sheet is very sensitive to a change in environment conditions (21).
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Fig. 6.
Far (A) and near
(B) UV-CD spectra of HL and
CDM. The far and near UV-CD spectra were taken in 1-mm and 5-cm
path length cuvettes, respectively, for
HL (dashed line)
and CDM (solid line) in 10 mM sodium acetate, pH
5.2, and a concentration of 0.1 mg/ml. The spectra are an average of
four scans and are corrected for buffer.
HL and CDM are shown in Fig.
6B. The
HL spectrum is consistent with the previously
published reports. Comparison of the two spectra shows a drastic
difference in the tertiary structure of the two proteins. In case of
CDM, the negative peaks at 265 and 295 nm, which correspond to the
vibrionic regions of phenylalanine and tryptophan, respectively (22),
are totally absent. This indicates that most of the phenylalanine and
tryptophan are in a more mobile environment. However, CDM still
possesses the positive peak at 280 nm, whose ellipticity is about 30%
of the corresponding peak in the
HL spectrum. This reveals that some
rigid tertiary contacts are present in CDM, particularly around a
fraction of the tyrosine residues, but the overall spectrum reflects a
significant disorder in the tertiary structure of CDM.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
HL in order to
investigate the contribution of the carboxyl-terminal residues of the
protein to its structural organization and function.
HL and CEM, which can be
isolated in soluble, active form. A variety of attempts to purify the
CDM by ion exchange and gel filtration techniques have led to
precipitation of the protein in the column. Hence, a wide spectrum of
conditions was employed in an attempt to stabilize the CDM, and the
conditions suitable for spectroscopic studies are low ionic strength
and pH 5.0-7.0. The CEM, on the other hand, can be purified like
HL. The hemolytic data presented in Table I show that
HL can be
unfolded and refolded to its native form in a variety of conditions.
Refolding is extremely efficient, with almost total recovery of
activity. Hemolysis studies carried out with the mutants showed that
the CDM is very weakly lytic, which is in agreement with prior studies
with other C-terminal deletion mutants (3). On the other hand, the CEM,
which has 38 extra amino acids, was able to lyse the rRBCs efficiently
(Fig. 2).
HL, which exhibited its typical "two halves" pattern. This
pronounced susceptibility of CDM to proteolytic digestion suggests a
more relaxed structure in which many proteolytic sites that are
otherwise hidden in
HL are getting exposed. All these observations
suggest that the CDM possesses a non-
HL like structure.
HL. This red shift observed for the CDM
indicates that its tryptophan residues are more exposed to the solvent
as compared with
HL. This was further corroborated with binding
studies with ANS, a dye widely used to probe the molten globule states
of proteins. The fluorescence intensity of ANS increased by about
10-fold upon binding to CDM but showed negligible increase in the
presence of
HL and the denatured states of the two proteins. The
increase is accompanied by a blue shift of the emission maximum by 29.5 nm, which is an indication of specific binding. These results indicate
the presence of exposed hydrophobic regions in CDM, which are
sequestered in native
HL.
HL
polypeptide chain shows that seven of the eight tryptophan residues are
in the carboxyl-terminal half of
HL, and eight of the 14 tyrosine
residues are located on the N-terminal half. This gives rise to the
possibility that the tertiary contacts may be localized on the first
half of the polypeptide chain.
HL structure had occurred in the absence of the
carboxyl terminus. Interestingly, the additional amino acids at the
carboxyl terminus (as in CEM) did not have any influence either on the
structure or the function of native
HL. The CEM exhibited all of the
characteristics of
HL. Proteolytic digestion revealed an
HL-like
pattern, and the protein was able to oligomerize and cause the lysis of
rRBCs. It has the same fluorescence emission maximum as
HL, which
suggests that the tryptophans of both of the proteins are in a similar
environment. Like
HL, it does not have any of its hydrophobic
regions exposed to the solvent, as demonstrated by the negligible ANS
binding. All of these points illustrate that it is possible to build
residues at the carboxyl terminus of
HL without inhibiting its
folding or function.
HL like structure of the CDM can be interpreted as its being
unable to "attain/maintain" the native,
HL-like conformation, and for that the carboxyl terminus is very crucial. We have tested for
such a possibility by in vitro transcription and
translation, whereby the activity of the CDM was assessed as it emerged
from the ribosome during in vitro translation. The hemolytic
activity of
HL, CDM, and CEM was examined as a function of
initiation of translation. It is clear from Fig.
7 that at constant rate of transcription
and translation,
HL begins to show lysis within minutes after
initiation of translation, whereas the CDM fails to show any lysis. On
the other hand, the CEM lyses rRBCs efficiently but marginally slower
when compared with
HL. This shows that freshly synthesized
HL
folds rapidly to attain its native, active structure, but the CDM is
unable to do so although it lacks just four amino acids. This suggests
that the CDM is probably stuck in a partly folded inactive state.
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Fig. 7.
Monitoring the hemolytic activity of
HL, CDM, and CEM during translation. Coupled
in vitro transcription and translation was carried out as
mentioned earlier (3). Typically, an aliquot of translation mix was
withdrawn and added to 0.5% rRBCs, and the decrease in light
scattering of rRBCs at 595 nm due to lysis was monitored at regular
time intervals.
,
, and
represent
HL, CDM, and CEM,
respectively at 3.5 min after initiation of translation.
,
, and
represent
HL, CDM, and CEM, respectively, at 6.5 min after
initiation of translation. No detectable lysis was observed before 3 min.
The physical state of the carboxyl-terminal deletion mutants of HL
is intriguing, since these mutants cannot participate in interprotomer
interactions to form oligomers on rRBCs. They can however, bind the
rRBCs and remain in the form of a cell-bound monomer (3). This is not
surprising, because partially folded states of proteins are shown to
bind membranes with high affinity (24). The crystal structure of
HL
reveals that the N-terminal latch makes extensive contacts with the
inner
-sheet of an adjacent protomer. Yet, when as many as 22 residues are deleted from the N terminus,
HL is still able to form
oligomers (although yet to be fully characterized). This is because in
a heptamer, each protomer participates in about 120 salt bridges and
hydrogen bonds and 850 van der Waals contacts (2). In the absence of a
few of these interactions, as in the case of an N-terminal deletion mutant, the net force of the remaining interactions is enough to drive
the process to the oligomeric state. However, this does not seem to
happen in the case of carboxyl-terminal deletion mutants, and they are
unable to carry out such fruitful interactions either among themselves
(i.e. formation of homooligomer) or with
HL (i.e. formation of hetrooligomer). Our data are clearly able
to reveal the reason behind the inefficient oligomerization of
carboxyl-terminal deletion mutants. The present data unequivocally
prove that the CDM possess a partially folded nonnative conformation
with greatly diminished tertiary structure. Such a species could still
bind rRBCs through nonspecific hydrophobic interactions but does not possess the requisite motifs for carrying out interprotomer
interactions. As a result, it cannot form either homooligomers with
itself or hetrooligomers with
HL. We have also examined the lysis
efficiency of the hetrooligomers of
HL and CEM by mixing a fixed
amount of
HL with increasing amounts of CEM. As seen in Table
II, the percentage of lysis increases
with increasing amounts of the CEM, which shows that the extended
carboxyl terminus does not interfere with the assembly of
HL
(i.e. during formation of functional pore). This is in
contrast to the amino-terminal deletion mutant
HL(5-293), which
retards the lysis of
HL as reported by us earlier (4). This again
supports our conclusion that the carboxyl terminus is not very critical
for the oligomerization process but instead is involved in the folding
of the monomer form.
|
The molten globule states of proteins have been attributed with four
distinct features, viz. compactness, a near native secondary structure, loss of tertiary structure, and exposure of hydrophobic regions (25). High affinity of CDM for ANS reveals that it has hydrophobic regions exposed to the solvent. In addition, the CDM exhibited the presence of substantial secondary structure accompanied by a significant loss of tertiary structure. We could not, however, determine the compactness of the CDM due to its tendency to interact with gel filtration matrices. All these observations suggest that in
the absence of the carboxyl terminus, the physical state of HL may
lie close to a molten globule-like state. Furthermore, the observations
presented here strongly suggest that
HL is able to fold to its
native form only after its complete synthesis, and/or the CDM is not
able to "latch on" to native
HL-like structure because of the
absence of the carboxyl terminus.
In summary, for the first time, the role of the carboxyl terminus of
HL has been delineated in greater detail. While the amino terminus
is important for the functional pore formation step, the carboxyl
terminus is crucial for correct folding and maintenance of the
water-soluble monomer form of
HL. Removal of just four residues from
the carboxyl terminus makes the protein unable to proceed beyond a
molten globule-like state. This information would be valuable to
understand how membrane binding toxins like
HL carry out their
folding and function. Since single amino acid substitutions at the
carboxyl terminus do not affect the function, the length
(i.e. backbone) of the protein appears to be vital instead
of the actual sequence at the carboxyl terminus. In addition, we have
shown that the addition of an extra sequence at the carboxyl terminus
does not affect the folding or function of
HL. Such molecules could
form the basis for construction of new molecules in the future.
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ACKNOWLEDGEMENTS |
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We thank Prof. A. Surolia, Dr. G. C. Mishra, and Dr. Amit Ghosh for encouragement.
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FOOTNOTES |
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* This work was supported by the Department of Science and Technology. This is a L198-01 communication from the National Center for Cell Sciences.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Council of Scientific and Industrial Research
senior research fellowship.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed: National Center for Cell Sciences, University of Pune Campus, Ganeshkhind, Pune 411 007, India. Fax: 91-20-372259 or 91-20-377756; E-mail: infonccs{at}giaspn01.vsnl.net.in.
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ABBREVIATIONS |
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The abbreviations used are:
HL,
-hemolysin(1-293) full-length protein;
CDM,
-hemolysin(1-289);
CEM,
-hemolysin(1-331) with 38-amino acid sequence added to
HL;
ANS, 8-anilino-1-naphthalene sulfonic acid;
UV-CD, ultraviolet circular
dichroism;
rRBCs rabbit red blood cells, PAGE, polyacrylamide gel
electrophoresis;
IASD, 4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonic acid;
MOPS
3-(N-morpholino)propanesulfonic acid, PCR, polymerase chain
reaction.
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REFERENCES |
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