Islam Khan Division of Biochemical Sciences, National Chemical Laboratory, Pune, India Unfolding, inactivation and dissociation of the lectin from Artocarpus hirsutaseeds were studied by
Trang 1Artocarpus hirsuta lectin
Differential modes of chemical and thermal denaturation
Sushama M Gaikwad, Madhura M Gurjar* and M Islam Khan
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India
Unfolding, inactivation and dissociation of the lectin from
Artocarpus hirsutaseeds were studied by chemical (guanidine
hydrochloride, GdnHCl) and thermal denaturation
Con-formational transitions were monitored by intrinsic
fluor-escence and circular dichroism The gradual red shift in the
emission maxima of the native protein from 335 to 356 nm,
change in the ellipticity at 218 nm and simultaneous decrease
in the sugar binding activity were observed with increasing
concentration of GdnHCl in the pH range between 4.0 and
9.0 The unfolding and inactivation by GdnHCl were
par-tially reversible Gel filtration of the lectin in presence of
1–6MGdnHCl showed that the protein dissociates rever-sibly into partially unfolded dimer and then irreverrever-sibly into unfolded inactive monomer Thermal denaturation was irreversible The lectin loses activity rapidly above 45°C The exposure of hydrophobic patches, distorted secondary structure and formation of insoluble aggregates of the thermally inactivated protein probably leads to the irre-versible denaturation
Keywords: Artocarpus lectin; denaturation; intrinsic fluores-cence; unfolding; aggregation
Proteins that bind carbohydrates specifically and reversibly
are termed as lectins They occur ubiquitously in nature and
have diverse role in plants, animals and microbes The
recognition of carbohydrate moieties by lectins has
import-ant applications in a number of biological processes such as
cell–cell interactions, signal transduction, and cell growth
and differentiation [1] a-Galactoside specific lectin present
in the seeds of Artocarpus hirsuta [2–4], is a homotetrameric
protein with molecular mass of 60 000 Da and high
specificity for methyl a-D-galactopyranoside (Me a-gal)
The folding pathways of oligomeric proteins involve both
intramolecular and intermolecular interactions The
dena-turation of pea and peanut lectins, both oligomeric proteins,
has been studied in great detail [5–7] In this paper we show
the progressive unfolding and inactivation of the lectin in
presence of GdnHCl and heat, and refolding and
reactiva-tion under renaturing condireactiva-tions
M A T E R I A L S A N D M E T H O D S
Materials
GdnHCl was a product of Sigma Chemical Co All other
chemicals were of the highest purity available The lectin
from A hirsuta was purified as described previously [2]
Stocks of 7MGdnHCl were freshly prepared in appropriate buffers and filtered through 0.45-lm filter Buffers used were acetate for pH 4.0, citrate/phosphate for pH 5.0 and 6.0, phosphate for pH 7.0 and Tris/HCl for pH 8.0 and 9.0 (all 100 mM)
Fluorescence studies Protein samples (1.5 lM) were equilibrated for 4 h at the desired denaturant concentration at 30°C in the pH range
of 4.0–9.0 Unfolding as a function of GdnHCl concentra-tion was monitored by intrinsic tryptophan fluorescence emission in a 1-cm quartz cell in the 300–400 nm region, when excited at 280 nm, in a Perkin-Elmer LS 50B spectrofluorimeter with attached circulating water bath Excitation and emission band passes of 5 nm were used Activity of the sample was measured at the same time Unfolding as a function of temperature was carried out
by incubating the protein samples (1.5 lM) in duplicates at the temperature from 30–70°C for 15 min One of the duplicates was used to record the spectra and activity at the respective temperature The other sample was brought to
35°C, centrifuged to remove any particulate matter, spectra were recorded and activity was estimated
Circular dichroism studies Far UV CD (210–250 nm) spectra of the protein samples (15 lM) treated with different concentrations GdnHCl in respective buffers of pH 4.0–9.0 and incubated for 14 h were recorded in a 1-mm path length cell, on a Jasco J715 spectropolarimeter connected to a circulating water bath For thermal denaturation studies, the protein sample was incubated at various temperatures for 10 min and the spectra were recorded The spectra were collected with response time of 4 s, and scan speed of 100 nmÆs)1 Each data point was an average of three accumulations
Correspondence to S M Gaikwad, Division of Biochemical Sciences,
National Chemical Laboratory, Pune, 411008, India.
Fax: + 91 20 5884032, Tel.: + 91 20 5893034,
E-mail: gaikwad@ems.ncl.res.in
Abbreviations: GdnHCl, guanidine hydrochloride; Me a-gal, methyl
a- D -galactopyranoside; ANS, 1-anilino-8-naphthalene sulfonate.
*Present address: University of Medicine and Dentistry New Jersey,
Robert Wood Johnson Medical School, Division of Biochemistry
(Research tower), Piscataway, New Jersey 08854, USA.
(Received 21 December 2001, accepted 14 January 2002)
Trang 2Hydrophobic dye binding studies
8-Anilino-1-naphthalene sulfonate (ANS) emission spectra
were recorded in the range of 400–500 nm with excitation at
375 nm using slit widths of 5 nm The changes in the ANS
fluorescence induced by the binding to the lectin were
followed by recording the spectra at constant concentration
of protein (1 lM) and ANS (50 lM), in different
concentra-tions of GdnHCl (1–5M)
Determination of the lectin activity
Sugar binding activity of the samples was measured by the
enhancement in the intrinsic fluorescence of the protein at
335 nm after addition of the nonfluorescent ligand Me a-gal
(4 mM) at saturating concentration Increase in the
fluores-cence of the native lectin after adding Me a-gal was taken as
100% activity [3]
Light scattering studies
Rayleigh light scattering experiments were carried out with
the spectrofluorimeter to follow protein aggregation during
GdnHCl and thermal denaturation Both excitation and
emission wavelengths were set at 400 nm and the time
dependent change in scattering intensity was followed
Renaturation studies
Two-hundred microliters aliquot was removed from the
samples treated with different concentrations of GdnHCl
(3.0–5.0M) for 4 h at 30°C at pH 7.0 and diluted 10 times
with 100 mMbuffer of pH 7.0 After 30 min, the
fluores-cence spectra and activity of the original (treated with
GdnHCl) as well as diluted samples were recorded Protein
sample without GdnHCl treated under identical conditions
was taken as control
The renaturation of thermally denatured protein was
followed by cooling the heated samples to 35°C, removing
any particulate matter by centrifuging, and then recording
the fluorescence spectra and the activity
Gel filtration studies
Lectin samples (10 lMin 100 lL) were incubated for 14 h
with GdnHCl (1.0–6.0M) and injected onto Protein Pak
300SW HPLC column (7.8· 300 mm) connected to a
Waters HPLC system preequilibrated and eluted with
different concentrations of GdnHCl (1.0–6.0M) in
100 mM buffer of pH 7.0 at a flow rate of 0.5 mLÆmin)1
Elution was monitored by absorbance at 280 nm The
standard molecular mass markers run in the presence of
buffer were, BSA (66 kDa), ovalbumin (45 kDa), carbonic
anhydrase (29 kDa) and cytochrome c (14.5 kDa)
R E S U L T S A N D D I S C U S S I O N
Unfolding studies
The fluorescence emission spectrum of the native lectin
showed a maximum at 335 nm which characterizes
non-polar environment of the tryptophan residues On
dena-turation of the lectin with increasing concentration of
GdnHCl (1–5M) although fluorescence intensity at 335 nm does not change much, the fluorescence at 356 nm increases significantly thus changing the emission maxi-mum from 335 to 356 nm (Fig 1A) indicating that due to unfolding, most of the tryptophans in the protein are getting exposed to the solvent The ratio of fluorescence intensities at 335 and 356 F(335/356) (Fig 1B) decreases from 1.35 to 0.82 Similar trend of denaturation with GdnHCl is observed in the pH range of 5.0–8.0, while it is more drastic at pH 4.0 and 9.0 The concentration of GdnHCl required for 50% unfolding of the protein between the pH range 6.0–8.0 is higher (3.2M) than at
pH 4.0 and pH 9.0 (2.2M)
The lectin showed a typical far UV CD spectrum observed for proteins with high proportion of b sheet content, with minimum at 218 nm [2] The relative percentage of structural elements calculated using CDPro software package for analyzing protein CD spectra was
a helix 2%, b sheet 44%, turns 23% and random coil 30% for native protein The CD spectra of the GdnHCl treated protein when analysed using the above programs did not show any significant change in the different structural elements compared to the native protein, while there was visible difference in the respective CD spectra This was probably due to the incompatibility of the data with the programmes used Because GdnHCl was interfering the CD spectra below 210 nm, data in the range of 210–250 nm could be collected The negative ellipticity of the protein at 218 nm increases in 1–2 M GdnHCl and then decreases at higher concentration (Fig 1C) The change in the structure at 1–2M GdnHCl is concomitant with loss of activity and therefore cannot be a stable
Fig 1 GdnHCl-induced unfolding of A hirsuta lectin at 30 °C Protein (1.5 l M ) at the required GdnHCl concentration was incubated for 4 h and the fluorescence emission spectra were recorded between 300 and
400 nm with the excitation wavelength of 280 nm (A), shift in the emission max (B), ratio F(335/356) (C), mean residue ellipticity at
218 nm in far UV region and (D) activity of the GdnHCl treated protein The symbols used for all the figures are pH 4.0 (.), pH 5.0 (d), pH 6.0 (m), pH 7.0 (,), pH 8.0 (s) and pH 9.0 (n).
Trang 3conformation When Rayleigh light scattering studies of
the samples were carried out, the sample incubated at 1M
GdnHCl at 30°C showed lower light scattering intensity
than the native protein At 2.0 M GdnHCl, there was
lower light scattering than that with 1.0M and at still
higher concentrations of GdnHCl, there was no light
scattering at all (data not shown) Thus, the increase in
the negative ellipticity at 218 nm at low concentrations
of GdnHCl could be due to the solubilization of the
aggregates in the protein There is substantial loss in
the secondary structure of the lectin as indicated by the
decrease in the negative ellipticity at 218 nm with
increasing concentration of GdnHCl Similar trend was
observed for denaturation between pH 5.0–8.0, while the
rate of unfolding was faster at pH 4.0 and 9.0
The inactivation of the lectin was proportional to the
concentration of GdnHCl (Fig 1D) The maximum
enhancement in the intrinsic fluorescence of the lectin due
to the binding of sugar, Me a-gal, taken as measure of
100% activity of the lectin [3] determined at different pH
was different The percentage decrease in the enhancement,
i.e activity with increasing concentration of GdnHCl,
however, was equivalent in the pH range of 4.0–9.0 and
the loss in the activity of the lectin is concomitant with the
unfolding of the protein At 3MGdnHCl, more than 50%
activity was lost with 60% decrease in the ratio (F335/356)
and 25–35% shift in the emission maximum
Refolding of the protein
Renaturation or refolding of the protein was measured as
the extent of reappearance of the original spectra (F 335/
356) and recovery of the sugar binding activity After
dilution of the reaction mixture containing lectin and
GdnHCl (10 times), partial reactivation of the lectin was
observed The lectin treated with 3, 4, and 5M GdnHCl
had 45, 13 and 7% activity, which increased to 75, 37,
and 23%, respectively (Table 1) on renaturation
Refold-ing of the protein was indicated by substantial increase in
the F(335/356) ratio GdnHCl probably unfolds the
protein in such a way that substantial interactions are
reformed after removal of the denaturant, leading to the
significant reformation of the structure and regaining of
activity
Gel filtration studies The native protein gets dissociated first into dimer (Mr
30 000) and then into monomer (Mr14 000) with increasing concentration of GdnHCl (Fig 2) At 3–4M GdnHCl, a single peak at 10.4 min appears that seems arise from the totally denatured monomer Complete dissociation of the tetramer does not take place even at 6MGdnHCl The protein components corresponding to the peaks 1, 2, 3 and 0 were analysed separately for sugar binding activity Peak 1 was found to be the folded and active fraction of the total population of the lectin molecules treated with GdnHCl Peak 2 is partially unfolded form of the lectin with traces of activity Peak 3 is unfolded, inactive monomer Peak 0 is the totally denatured monomer similar to that observed in case
of peanut lectin [6] The dissociation of the native protein in presence of GdnHCl into dimer is reversible, that into monomer is irreversible as observed by rechromatography
of the individual peaks on gel filtration column under renaturing conditions (data not shown) Based on the dissociation pattern, the following scheme can be written:
T() D () M () M*
where T is tetramer, D is dimer, M is monomer and M* is totally denatured monomer
The monomer seems to be unstable and the conforma-tional stability of the oligomer seems to be contributed wholly by the quaternary interactions In case of peanut lectin, folded monomer is obtained after dissociation of the protein [7] and the molten globule-like state of the monomer was detected during its unfolding [6], both of which retain the sugar binding activity
Thermal denaturation The A hirsuta lectin loses sugar binding activity and starts precipitating above 45°C The fluorescence emission spec-trum broadens, but the emission maxima does not shift from 335 to 356 even at 70°C where almost total inactivation of the lectin takes place The decrease in the ratio F(335/356) observed for thermally denatured protein, from 1.36 (native) to 1.03 (70°C, 15 min) (Table 1) was less than that observed with GdnHCl denaturation (at pH 7.0), 1.35 (native) to 0.82 (5MGdnHCl) (Fig 1B)
Table 1 Effect of treatment GdnHCl and thermal denaturation and renaturation on A hirsuta lectin The samples treated with GdnHCl were diluted
10 times with 100 m M phosphate buffer, pH 7.0, the spectra were recorded and activity was estimated as described in Materials and methods The lectin samples incubated at respective temperatures were cooled to 35 °C, spectra were recorded and activity was estimated.
Treatment
Activity (%) F 335/356
On denaturation On Renaturation On denaturation On Renaturation Lectin + GdnHCl (0 M ) 100 100 1.35 1.35
Lectin Þ 35 °C,15 min 100 100 1.36 1.36
Trang 4On thermal denaturation, the protein forms insoluble
aggregates before total unfolding and loses its sugar
binding activity When the temperature of the samples
incubated from 45 to 70°C was brought down slowly to
35°C, the activity was not restored and no refolding was
observed as there is decrease in the F(335/356) ratio
(Table 1) indicating that the thermal denaturation is
irreversible Because the protein starts aggregating on
thermal denaturation, Rayleigh light scattering studies were
carried out The protein shows higher light scattering
intensity at 45°C (Fig 3A) which goes on increasing with
further increase in the temperature
ANS binding studies Binding of ANS to the proteins occurs upon the exposure of hydrophobic clusters during the unfolding process ANS does not bind to the native or the denatured states of the
A hirsutalectin but binds at the intermediate stage (at 2M
GdnHCl), showing increase in the fluorescence intensity, indicating temporary exposure of the hydrophobic patches
of the protein during unfolding (Fig 3B) The possibility of the occurrence of the molten globule during unfolding of
A hirsutalectin as observed in the peanut lectin [6], was ruled out because a significant amount of the tertiary and secondary structure was intact The ANS binding to the protein samples exposed to 50, 60, and 70°C was more than those incubated at 30 and 40°C (Fig 3C) indicating the exposure of hydrophobic patches are due to thermal denaturation The tendency of the protein to aggregate increases as the hydrophobic patches get exposed due to thermal denaturation The CD spectra of the protein exposed at 45–70°C for 10 min shows progressive loss in the secondary structure (Fig 3D)
There seem to be two different modes of denaturation of the A hirsuta lectin with GdnHCl and heat The former unfolds and inactivates the protein, allowing it to fold back and reactivate to certain extent after removal of the reagent Thermal denaturation leads to unfolding and simultaneous formation of insoluble aggregates and is therefore irrevers-ible Different modes of folding and unfolding observed under different conditions could be due to the unusual
Fig 2 Gel filtration of A hirsuta lectin in presence of GdnHCl in
100 m M potassium phosphate buffer (pH 7.0) Molarity of GdnHCl is
indicated on the figure M r values of the standards used were as
fol-lows, 1, BSA 66 kDa, 2, ovalbumin, 45 kDa, 3, carbonic anhydrase,
29 kDa and 4, cytochrome c, 14.5 kDa.
Fig 3 Rayleigh light scattering (A) and ANS fluorescence (B,C) studies
of A hirsuta lectin (A) The lectin (1.5 l M ) was incubated at different temperatures for 10 min and the light scattering was monitored by setting kex ¼ kem ¼ 400 nm 1, 50 m M buffer of pH 7.0, 2, 30 °C, 3,
35 °C, 4, 40 °C and 5, 45 °C (B) Change in ANS fluorescence in the presence of A hirsuta lectin and GdnHCl The fluorescence emission spectra of the lectin (2.0 l M ) in the presence of ANS (50 l M ) (kex, 375 nm) Numbers on the curves indicate the molarity of GdnHCl (C) Change in ANS fluorescence in the presence of the
A hirsuta lectin at various temperatures The spectra were taken as described in (B) protein samples treated at, 1, 30 °C, 2, 40 °C, 3, 50 °C,
4, 60 °C, and 5, 70 °C (D) Near UV CD spectra of A hirsuta lectin (15 l M ), lectin exposed at 1, 35 °C, 2, 45 °C, 3, 50 °C, 4, 55 °C, 5,
60 °C, 6, 65 °C and 7, 70 °C for 15 min.
Trang 5folding and association of subunits of the lectin as compared
to other plant lectins [5,6]
R E F E R E N C E S
1 Lis, H & Sharon, N (1991) lectin–carbohydrate interactions Curr.
Opin Struct Biol 1, 741–749.
2 Gurjar, M.M., Khan, M.I & Gaikwad, S.M (1998) a-Galactoside
binding lectin from Artocarpus hirsuta: characterization of the
sugar specificity and binding site Biochim Biophys Acta 1381,
256–264.
3 Gaikwad, S.M., Gurjar, M.M & Khan, M.I (1998) Fluorimetric
studies on saccharide binding to the basic lectin from Artocarpus
hirsuta Biochem Mol Biol Int 46, 1–9.
4 Rao, K.N., Gurjar, M.M., Gaikwad, S.M., Khan, M.I & Suresh, C.G (1999) Crystallization and preliminary X-ray studies
of the basic lectin from the seeds of Artocarpus hirsuta Acta Crystallo D 55, 1204–1205.
5 Ahmed, N., Srinivas, V.R., Reddy, G.B & Surolia, A (1998) Thermodynamic characterization of the conformational stability
of the homodimeric protein, Pea lectin Biochemistry 37, 16765– 16772.
6 Reddy, G.B., Srinivas, V.R., Ahmed, N & Surolia, A (1999) Molten globule like state of peanut lectin monomer retains its carbohydrate specificity J Biol Chem 274, 4500–4503.
7 Reddy, G.B., Bharadwaj, S & Surolia, A (1999) Thermal stability and mode of oligomerization of the tetrameric Peanut agglutinin:
a different scanning calorimetric study Biochemistry 38, 4464–4470.