From the Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
A central question in biological chemistry is the
minimal structural requirement of a protein that would determine its
specificity and activity, the underlying basis being the importance of
the entire structural element of a protein with regards to its activity vis à vis the overall integrity and
stability of the protein. Although there are many reports on the
characterization of protein folding/unfolding intermediates, with
considerable secondary structural elements but substantial loss of
tertiary structure, none of them have been reported to show any
activity toward their respective ligands. This may be a result of the
conditions under which such intermediates have been isolated or due to
the importance of specific structural elements for the activity. In
this paper we report such an intermediate in the unfolding of peanut
agglutinin that seems to retain, to a considerable degree, its
carbohydrate binding specificity and activity. This result has
significant implications on the molten globule state during the folding
pathway(s) of proteins in general and the quaternary association in
legume lectins in particular, where precise subunit topology is
required for their biologic activities.
Despite their preponderance in biologic systems, studies on the
folding pathways of oligomeric proteins are less common and information
regarding the same, meager. The folding process for oligomeric proteins
is more complex as the acquisition of the quaternary structure entails
both, the intramolecular refolding of the individual polypeptide chains
and the simultaneous intermolecular interactions between the various
subunits. Hence, elucidation of the hierarchy of events occurring
during the denaturation of an oligomeric protein provides important
means to delineate such a process.
Legume lectins, a class of highly homologous, carbohydrate-binding
proteins exhibit the same jelly roll tertiary structural fold but
differ considerably in their ligand specificity and quaternary structures (1, 3). They can hence be considered to be "natural mutants" of quaternary structures. However, apart from their
agglutinating activity, the role of oligomerization in lectins is not
clearly understood (1-11). Peanut (Arachis hypogea)
agglutinin (PNA),1 a
homotetrameric nonglycosylated protein, violates an important principle
of quaternary association in globular proteins, a unique case of a
tetramer without a 4-fold or 222 symmetry (10, 11). Unlike most other
legume lectins, which dissociate and unfold as single entities
(12-14), we describe here its chaotrope (guanidine hydrochloride,
GdnHCl) induced unfolding that consists of a substantially unfolded
monomeric species, as an intermediate, which retains its carbohydrate
specificity despite a considerable loss of tertiary structure.
Existence of such a molten globule-like structure with retention of
binding activity is perhaps unprecedented so far. Moreover, the
occurrence of such a species for PNA suggests that the monomers of
legume lectins are competent to bind sugars and oligomerization appears
to impart them stability and necessary spatial disposition of sugar
binding sites for manifestation of their respective biologic activities.
Materials--
GdnHCl was a product of Amersham. All other
chemicals used were of the highest purity available from Sigma. PNA
purified as described previously, on SDS-polyacrylamide gel
electrophoresis, showed a single band of Mr
27,000 (15). The protein concentration was determined by its specific
absorbance A280 nm1% = 7.7 (16). Stocks of 8 M GdnHCl were freshly prepared in
appropriate buffers, filtered through 0.45-µm filters, and their
concentrations determined by refractive index measurements (17). The
buffers used were: 20 mM malate for pH 3, 50 mM
acetate for pH 4 and 5, 50 mM dimethylglutarate for pH 6, and 50 mM phosphate for pH 7.4.
Unfolding Studies--
Protein samples (10 µM)
were incubated for 8-10 h at the desired temperature and denaturant
solutions before measurements, to ensure equilibrium. Unfolding as a
function of GdnHCl concentration was monitored by fluorescence
spectroscopy, near- and far-UV circular dichroism. Intrinsic tryptophan
fluorescence of protein samples (10 µM) was monitored in
a 1-cm quartz cell in the 300-400 nm region, when excited at 280 nm,
in a Jasco-FP777 spectrofluorimeter connected to a circulating water
bath. Excitation and emission band passes of 3 nm were used. Far-UV CD
(200-250 nm) and near-UV CD (250-300 nm) was followed in a 1-mm and a
10-mm path length cell, respectively, on a Jasco-J500A
spectropolarimeter connected to a circulating water bath. The spectra
were collected with a slit width of 1 nm, response time of 8 s,
and a scan speed of 10 nm s Gel Filtration--
Samples (10 µM PNA in 200 µl) incubated for 8 h with denaturant were injected onto a
SuperdexR75 HR 10/30 column (1 × 30 cm) connected to
a Pharmacia FPLC system preequilibrated and eluted with the 50 mM phosphate buffer (pH 7.4) and the required denaturant
concentration at a flow rate of 0.5 ml/min. Samples were monitored by
their absorbance at 280 nm.
Isothermal Titration Calorimetry--
Isothermal titration
calorimetric experiments were performed as described previously, on an
Omega titration calorimeter (Microcal Inc.) (12, 13, 18). Aliquots of
the ligand solution at 10-20 × the binding site concentration
were added via a 250-µl rotating stirrer-syringe to the solution cell
containing 1.34 ml of the 0.6-1.2 mM protein.
Chaotrope-induced Unfolding Profiles of PNA Display Biphasic
Nature--
The simple two-state assumption for denaturant-induced
unfolding of protein may not apply for many oligomeric proteins as, in
addition to the interactions within the same polypeptide chain, those
between the subunits may make distinct contributions to its overall
conformational stability. Consequently, although unfolded oligomers are
unlikely to exist, the occurrence of native-like compact monomeric
states is not excluded (19). In such instances, the overall unfolding
reaction may be more appropriately described as An The Chaotrope Intermediate Is Monomeric in Nature--
That the
first transition could well be associated with the dissociation of
tetramer to monomers was examined by size exclusion chromatography
(Fig. 1B). Whereas the native PNA and that in the presence
of 0.6 M GdnHCl (which correspond to the pretransition base-line region of the denaturation profile) eluted as a tetramer (retention time: 18.4 min), the completely denatured form of the protein (in 5.2 M GdnHCl) eluted earlier (at 13 min), which
is consistent with the retention time expected for a completely
unfolded random polypeptide of a PNA monomer. However, at 1.8 M GdnHCl (the intermediate region of the melting curve) it
elutes at 38.5 min, the position where a monomer of 25 kDa is expected
to emerge. Also, under no circumstances was a dimer, in the entire
region of the denaturation curve of the protein, observed. Taken
together, these data show that the intermediate species is monomeric
and that the unfolding of PNA proceeds through a compact monomeric form
to a completely unfolded polypeptide chain. At pH 3.0 PNA is known to
exist mostly as a dimeric species (21), consistent with its elution (28 min) in FPLC (not shown). The unfolding of the dimer also involves a
monomer as the intermediate. Thus PNA dimer also unfolds through the
monomeric form (Fig. 1A).
The Intermediate Has "Molten Globule"-like
Characteristics--
The nature of the intermediate was further
examined by comparing its near- and far- UV CD spectra with the native
and the fully denatured forms and by its ability to bind to the
hydrophobic indicator ANS. As can be seen from Fig.
2, A and B, while
its tertiary structure is lost dramatically (~70%), the secondary structure is retained significantly. Thus the unfolding of PNA by
chaotropes leads to the formation of a stable intermediate species,
with most of its secondary structure intact but substantial loss of
tertiary structure. Such intermediate species with similar spectral
losses have been observed for a number of proteins, such as
Binding of the hydrophobic fluorescent probe, ANS, to proteins occurs
upon the exposure of hydrophobic clusters during the unfolding process.
We observed that ANS binds neither to the native nor the fully
denatured states of PNA, but it does so very strikingly to the
intermediate (Fig. 3A).
Obviously, there are large clusters of solvent exposed hydrophobic
regions that are unveiled in the intermediate and that are impinged by
molecules of ANS, with a resultant increase in its emission intensity
and a blue shift of its emission maximum. The intermediate is thus a
molten globule-like state (26-30). Moreover, as shown in Fig.
3B, the maximum of fluorescence intensity of ANS occurs at
progressively lower temperatures as the pH is lowered. Thus the
intermediate occurs with greater propensity at more acidic pH which is
not surprising as these conditions would promote deoligomerization of
PNA (30).
The Molten Globule-like Intermediate Retains Its Carbohydrate
Binding Activity--
Finally, the FPLC-purified monomeric
intermediate in 2 M GdnHCl, which was confirmed to retain
its molten globule-like characteristics at the concentrations used
for titrations, bound to the sugar ligands almost akin to its native counterpart (Fig. 4 and Table I). Their binding constant was nearly
80% that of native PNA. The stoichiometry (n) of
interaction of the intermediate was comparable with that of the native
PNA, both being close to one. It thus seems that the molten
globule-like intermediate retains, to a significant extent, the sugar
binding capability of the protein.
The unfolding process of peanut agglutinin studied by
GdnHCl-induced denaturation was found to be completely reversible. The profiles were biphasic in nature when monitored by all the three spectroscopic probes, suggesting a non-two-state unfolding process. The
intermediate in the unfolding of PNA was further characterized spectroscopically (fluorescence, near- and far-UV-CD), by gel filtration and by titration calorimetry, to determine the extent of its
activity. The intermediate has about 80% of its secondary structural
elements intact, yet has considerably decreased tertiary structure
(nearly 70% loss of tertiary structure). It seems to be fairly compact
in nature (its gel filtration elution volume corresponds to that of the
monomer of PNA), and yet highly solvent exposed, as observed by its
strong ability to bind ANS. This species may hence be described as a
molten globule-like intermediate, as similar properties have been
observed for the molten globule states of a number of proteins. For
example, in the case acid isolated molten globule of Legume lectins exist as oligomers, the nature of which has important
functional implications (1, 2). Studies by Brewer and co-workers (3,
6), for example, have shown that various lectins form well defined and
characteristic lattices when complexed with multivalent
oligosaccharides that may underlie their effects on cells such as
mitogenesis. From our studies, it appears that lectin monomers may have
all the necessary features compatible with carbohydrate binding and
that oligomerization essentially appears to endow them with additional
stability and the requisite topology of binding sites for manifesting
their biologic activity.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
1. Each data point was an
average of eight accumulations. 8-Anilino-1-naphthalene sulfonate (ANS)
emission spectra were recorded in the range of 400-500 nm, of samples
excited at 380 nm at the desired temperature using slit widths of 5 nm.
The changes in ANS fluorescence induced by the conformational changes
in PNA were followed by measuring the intensity at 470 nm at constant
concentrations of PNA (10 µM) and ANS (50 µM). The temperature inside spectrometer cells were
monitored using a CIE digital thermometer.
RESULTS
nA
nU rather than An
nU, where the unfolding may start with the folded oligomers
(An), end with unfolded monomers (U), but have a significantly
populated monomeric state. Such a non-two-state transition may display
biphasic denaturation curves and/or nonsuperimposable transitions when the reaction is followed by different spectroscopic probes (19, 20).
The GdnHCl-induced unfolding of PNA followed by fluorescence (at 321 nm), far- (222 nm) and near- (270 nm) UV CD and shows a very distinct
biphasic nature (Fig. 1A).
These unfolding transitions were found to be completely reversible. In
the case of the fluorescence and the near-UV CD transition, this
intermediate species is populated predominantly in the 1.0-2.0
M GdnHCl range. In the far-UV CD transition, the
intermediate appears in 1.4-2.2 M GdnHCl range. Additionally, the melting curves obtained by the different
spectroscopic probes do not superimpose (Fig. 1A,
inset). Thus, additional intermediates during the unfolding
pathway of PNA may indeed occur, but we were unable to observe them as
they are not populated significantly.
View larger version (24K):
[in a new window]
Fig. 1.
GdnHCl-induced unfolding of PNA at
25 °C. A, protein (10 µM) at the
required GdnHCl concentration was incubated for 8-10 h, and its
fluorescence at 321 nm was followed as a function of GdnHCl
concentration at pH 3.0 ( ), pH 5.0 (
), and pH 7.4 (
).
Inset shows nonsuperimposition of PNA (10 µm) melting
curves obtained by fluorescence (
), near-UV (
), and far-UV CD
(
), monitored at 270 and 222 nm, respectively. B, gel
filtration of PNA at 25 °C in "0" M (1), 0.6 M (2), 1.6 M (3), 1.8 M (4), and
5.2 M (5) GdnHCl in 50 mM sodium phosphate
buffer (pH 7.4). Native PNA elutes at 18.5 ± 0.5 min, which in
1.6 and 1.8 M GdnHCl concentration elutes at 38.5 ± 0.5 min. Fully denatured (5.2 M GdnHCl) form elutes at
13 ± 0.4 min. C, PNA, in the absence of GdnHCl, under
the experimental conditions used (0.5-10 µM protein)
always exists as a tetramer.
-lactalbumin, cytochrome c, apomyoglobin, etc.
(20-29).
View larger version (23K):
[in a new window]
Fig. 2.
CD spectrum of the native PNA and its
intermediates. Far-UV (A) and near-UV (B) CD
spectra of PNA at 0 M (spectrum 1),
0.8 M (spectrum 2), 1.6 M
(spectrum 3), 1.8 M (spectrum 4), 5.2 M (spectrum 5), and 6 M (6) GdnHCl
concentration at 25 °C. At 1.6 and 1.8 M GdnHCl, PNA
retains significant secondary structure, but loses much of the tertiary
structures.
View larger version (20K):
[in a new window]
Fig. 3.
ANS binding to PNA (A) ANS
binding as a function of GdnHCl concentration. A,
inset shows fluorescence emission spectra of ANS in PNA
solutions at 0 M ( ---
), 0.8 (· · · ·), 1.8 M (---), and 5.2 M (
) GdnHCl.
B, ANS binding to PNA at pH 3.0 (
), 4.0 (
), 5.0 (
),
6.0 (
), and 7.4 (
) as a function of temperature. ANS binding was
monitored by 380 nm excitation and 470 nm emission.
View larger version (16K):
[in a new window]
Fig. 4.
An ITC profile of PNA binding to
lactose (10 mM) at 25 °C.
A, binding of native PNA (ntPNA) at 0.7 mM
monomer concentration, Mr 27,000. B,
FPLC-purified monomeric fraction (mPNA) (0.7 mM) in 2 M GdnHCl. The Kb values for ntPNA
and mPNA were 1990.0 and 1620.0 M 1,
respectively.
Carbohydrate binding abilities of native PNA (ntPNA) and the folding
intermediate (mPNA) to various sugars
DISCUSSION
-lactalbumin,
there is nearly a 75% loss of tertiary structure, while the secondary
structure is almost intact in addition to being highly compact in
nature. Likewise, in cytochrome c this state is accompanied
by a loss of approximately 23-30% of the secondary structure (20, 21,
23-26). Yet, unusually, the PNA intermediate, despite the reduced
tertiary structure, retains its carbohydrate binding activity to a
considerable degree. It thus seems that, at least for PNA, the
architecture of the binding site in the intermediate seems sufficiently
intact, necessary for its ligand recognition. These data, hence, not
only provide information on the folding pathway of peanut agglutinin
tetramer, which may be correlated with its three-dimensional structure, but also provide novel insights on the architecture of a functional monomer of a legume lectin. It is thus pertinent, at this point, to
describe briefly the unusual features of its quaternary structure. Unlike any other legume lectin, PNA is best described as a dimer of two
back to back dimers each 2-fold symmetric (10, 11). Although the two
dimers, viz. subunits 1 and 4 and 2 and 3 in the tetramer,
are also related by a dyad, the different dyads neither intersect nor
are they mutually perpendicular. Consequently, the molecule has an open
quaternary arrangement. The interactions between the two dimers involve
two distinct kinds of interfaces. One is a side by side antiparallel
alignment of the flat six-stranded back
-sheets, between subunits 1 and 2, like the highly extended "canonical" dimeric interface in
legume lectins such as concanavalin A (ConA), pea lectins, etc.
However, the two sheets at this interface do not come close enough as
in ConA, pea lectin, etc. Instead the association between the two back
-sheets from subunits 1 and 2 are stabilized by six water bridges.
The other interface, viz. between subunits 3 and 4, an
incidental consequence of the presence of the two back to back (1-4 and
2-3) and a side by side (1-2) interfaces, has not been observed in any
other tetrameric protein so far. The back to back association between
the two flat back
-sheets across the subunits 1-4 and subunits 2-3 of PNA tetramer appears to be intrinsically less stable as compared
with the canonical dimeric interfaces of the legume lectins. The 1-2 interface in peanut agglutinin, which is common to 1-4 and 2-3 dimers,
does not link up to give a contiguous 12-strand sheet, as it does so in
ConA, pea lectins, etc., hence it is unable to confer stability to the
extent observed in the latter class of lectins and with a slight
chaotrope perturbation, dissociates into monomers readily. The
resultant exposure of the flat six-stranded back
-sheet may lead to
the display of extensive hydrophobic patches that is recognized by ANS,
although the exposure of a recently described additional hydrophobic
region in PNA is not ruled out altogether (11). This monomeric state
has considerably reduced tertiary structure and yet binds the ligand,
indicating that the regions of the two sheets supporting the four
carbohydrate binding loops as well as the loops themselves retain the
geometric features of the combining site like that in the native protein.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Departments of Science and Technology (DST) and Biotechnology (DBT), Government of India (to A. S.).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.
Research Associate under the Umbrella Program of the Department of Biotechnology.
§ Research Associate supported in the above DST grant.
¶ To whom correspondence should be addressed: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India. Tel.: 91-80-3092389; Fax: 91-80-3348535 (or 3341683); E-mail: surolia{at}mbu.iisc.ernet.in.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PNA, peanut agglutinin; GdnHCl, guanidine hydrochloride; ANS, 8-anilino-1-naphthalene sulfonate; FPLC, fast protein liquid chromatography; ConA, concanavalin A; ntPNA, native PNA; mPNA, monomeric PNA; ITC, isothermal titration calorimeter.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|