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Protein & Peptide Letters, 2014, 21, 0000-0000 1

A Comparative Study of Unfolding of Erythrina cristagalli Lectin in

Chemical Denaturant and Fluoroalcohols

Debasish Sen and Dipak K. Mandal*

Department of Chemistry, Presidency University, 86/1 College Street, Kolkata 700 073, India

Abstract: The unfolding of dimeric Erythrina cristagalli lectin (ECL) has been investigated and compared under different
denaturing conditions in presence of chemical denaturant, guanidine hydrochloride (GdnHCl) and fluoroalcohols,
trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP). The GdnHCl-induced unfolding exhibits three-state mechanism
involving structured intermediate that corresponds to tertiary monomer. The intermediate has been characterized by 8-
anilino-1-naphthalenesulfonate (ANS) binding, which shows ~ 30 fold increase in ANS fluorescence and selective chemi-
cal modification with N-bromosuccinimide when Trp 45 and Trp 207 are possibly oxidized. The results are supported by
red edge excitation shift (10 nm), acrylamide quenching and phosphorescence studies which give a (0,0) band at 412.6
nm. TFE and HFIP show differing roles and characteristics of ECL unfolding. In TFE, but not in HFIP, a molten globule-
like monomeric intermediate is formed, being characterized by ANS binding and concentration dependent studies. TFE-
and HFIP-induced secondary structure changes of ECL, as monitored by far-UV CD, show that conversion of -sheet to

-helix occurs at lower HFIP concentration compared to TFE perturbation, helical content reaching to 65 % in 80 % HFIP
and 53 % in 90 % TFE. Temperature-dependent studies reveal that induced helix entails reduced thermal stability. FTIR
results show partial -sheet to
-helix conversion but with quantitative yield. The tryptophan environment of TFE- and
HFIP-induced states is dissimilar involving oxidation of four and three tryptophans respectively, and also differs from the
fully unfolded state in GdnHCl when all five tryptophans undergo oxidation. The results offer insights into the unfolding
problem of ECL.
Keywords: Chaotrope, conformational change, Erythrina cristagalli lectin, fluoroalcohol, oligomeric protein, unfolding.

INTRODUCTION
The unfolding of a protein can be induced by solvent
denaturation using chaotropic agent such as guanidine hy-
drochloride (GdnHCl) or fluoroalcohols like 2,2,2-trifluoro-
ethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP).
A major difficulty in experimental investigations of protein
unfolding reaction is the reliable identification and structural
description of the intermediate state(s), which would reveal
the unfolding sequence. GdnHCl-induced denaturation may
involve folded or partially folded intermediates with native-
like secondary structure [1-4] whereas fluoroalcohols lead to
structural states with nonnative secondary conformations [5-
8]. It has been proposed that fluoroalcohols destabilizes the
hydrophobic cores of protein because of its nonpolar charac-
ter while it induces helix formation by minimizing exposure
of peptide backbone [9]. In comparison to monomeric pro-
teins, the unfolding problem of oligomeric proteins is more
complex as it requires the disruption of additional molecular
interactions since both inter- and intrasubunit interactions
contribute to their overall structure and stability.
An important class of oligomeric proteins comprises lect-
ins that bind carbohydrate selectively, and are involved in
various biological recognition processes [10]. Among lectin
*Address correspondence to this author at the Department of Chemistry,
Presidency University, Kolkata, India; Tel: +91 33 2241 3893;
E-mails: dm_pcchem@yahoo.co.in; dm.pcchem@gmail.com
families, legume lectins have attracted much interest for pro-
tein folding studies because they exhibit the same tertiary
structural fold but differ considerably in their quaternary
structures [11,12]. Erythrina cristagalli lectin (ECL) is a
galactose-specific legume lectin, and exists as a dimer. X-ray
structure of ECL [13] shows that the ECL fold consists of
two stacked antiparallel -sheets (Fig. 1A), as seen for other
legume lectins. However, unlike canonical dimeric structure
for several legume lectins, for example, concanavalin A [11],
ECL forms non-canonical dimer in which two monomers are
arranged back-to-back in “handshake” fashion (Fig. 1B). The
dimer interface is characterized by mostly hydrophobic in-
teractions between the two monomers.
Solvent denaturation studies of many legume lectins have
been shown to be either a simple two-state process or a
multi-state process involving structured or partially folded
intermediates [14-18]. We have earlier shown the distin-
guishing features of soybean agglutinin and concanavalin A
in different structural states in their unfolding pathways
[19,20]. Recently, we have demonstrated a unique molten
globule fragment chain of pea lectin in GdnHCl-induced
unfolding [21] and presented the differential structural char-
acteristics of the molten globule intermediate formed in the
unfolding reaction of peanut agglutinin in urea and GdnHCl
[22]. Very recently, we have examined the effects of TFE on
the perturbation of secondary structure of soybean agglutinin
for quaternary and tertiary forms of the lectin [23]. Here, we

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2 Protein & Peptide Letters, 2014, Vol. 21, No. 1
Figure 1. A) Ribbon representation of three-dimensional structure
of ECL monomer (PDB entry 1UZY) in which five tryptophans are
indicated in ball-and-stick. B) Ribbon representation of ECL dimer
with a non-canonical interface resulting from back-to-back associa-
tion of two monomers.
have investigated and compared unfolding sequence of ECL
induced by the chaotropic agent (GdnHCl) and fluoroalco-
hols (TFE and HFIP) using fluorescence, phosphorescence,
chemical modification, far-UV CD and FTIR. The results
provide important insight into the folding problem of ECL
perturbed by chemical denaturant and alcohols.
MATERIALS AND METHODS
Materials. Erythrina crystagalli lectin (ECL), 8-anilino-
1-naphthalenesulfonate (ANS), guanidine hydrochloride
(GdnHCl), acrylamide, deuterium oxide (D2O) , 2, 2, 2-
trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol
(HFIP) were obtained from Sigma. The concentration of
ECL was determined spectrophotometrically at 280 nm from
its specific extinction coefficient, A1 %, 1 cm = 15.3 [24] , and
molarity was expressed in respect of monomer (Mr = 28000).
The concentration of ANS was measured spectrophotometri-
cally [25] using its molar extinction coefficient,
= 5,000 M-
1
cm-1 at 350 nm. GdnHCl concentration was determined by
refractive index measurements [26]. Other reagents were of
analytical grade. Double distilled water was used in all ex-
periments.
Sen and Mandal
Protein unfolding. The unfolding experiments in
GdnHCl were carried out in PBS (10 mM sodium phosphate
buffer containing 0.15 M NaCl), pH 7.2 by treating ECL
with varying concentrations of GdnHCl at 37 oC for 24 h.
For unfolding in fluoroalcohols, the protein samples in dif-
ferent concentrations of TFE / HFIP in PBS were incubated
at room temperature for 4 h. The unfolding reactions were
monitored by steady state intrinsic (tryptophan) as well as
extrinsic (ANS) fluorescence.
Size-exclusion chromatography. The size-exclusion
chromatography experiments were carried out using a
Superose-12 10/300 GL column attached to a Waters HPLC
system. An aliquot of 200 μL of a protein sample (20 μM)
prepared by incubation with required concentration of
GdnHCl in PBS as described above was injected into the
column. The column was preequilibrated with the sample
buffer. Flow rate was 0.5 mL/min. The eluent was detected
on-line by Waters 2489 UV-Visible detector at 280 nm.
Calibration of the column was made with the following
marker proteins: chicken egg ovalbumin (45 kDa), carbonic
anhydrase (29 kDa) and soybean trypsin inhibitor (20.1
kDa).
Ultraviolet absorption and fluorescence measure-
ments. Ultraviolet absorption was measured in Hitachi U-
4100 double-beam spectrophotometer. Steady-state fluores-
cence was measured in Hitachi F-7000 spectrofluorometer.
Sigma cuvette (2 mL) with path length of 1 cm was used. For
tryptophan fluorescence, excitation of samples was done at
280 nm and emission scanned from 300 to 400 nm with exci-
tation and emission band pass of 5 nm each. Scan speed was
60 nm / min. Corrected spectra were obtained after subtrac-
tion of appropriate blanks without ECL.
ANS binding experiments were performed at pH 7.2 with
native ECL and different protein samples in GdnHCl or TFE
/ HFIP. In a typical experiment, ECL sample (4
M) was
incubated with 100
M ANS for 5 min before excitation at
370 nm and emissions were scanned in the region of 400-600
nm. Appropriate blanks were subtracted to obtain the cor-
rected spectra.
Fluorescence quenching measurement. Acrylamide
quenching of tryptophan fluorescence was measured after
serial addition of small aliquots of freshly prepared acryla-
mide solution (2 M) to a sample of ECL in PBS taken in a
cuvette. After thorough mixing, the mixture was kept in the
dark for 10 min in the sample compartment. The excitation
wavelength was 295 nm. The fluorescence intensity was
monitored at the respective emission maximum for each
sample. The fluorescence intensities obtained were corrected
for dilution. Quenching data were analyzed by fitting to the
Stern-Volmer equation [27]:
F0 / F = 1 + KSV [Q]
where F0 and F are the fluorescence intensities in the ab-
sence and presence of the quencher, respectively, [Q] is the
molar quencher concentration and KSV is the Stern-Volmer
quenching constant.
Phosphorescence measurement at 77K. Measurement
of phosphorescence was performed in Hitachi F-7000 spec-
trofluorometer which was equipped with necessary accesso-
ries. The spectra were recorded using ECL samples without
Unfolding of Erythrina Cristagalli Lectin
or with GdnHCl or TFE at 77 K in a Dewar system using a
quartz tube (o.d. 5 mm). Samples prepared in 40% ethylene
glycol in appropriate buffer were frozen in liquid nitrogen.
Excitation wavelength was set at 280 nm. Excitation and
emission band pass was 10 nm and 1.0 nm respectively. The
phosphorescence spectra were free from any polarization
artifacts and found to be reproducible.
CD measurement. CD measurements were performed in
far-UV region (190-260 nm) on J-815 spectropolarimeter
(Jasco, Japan) which was equipped with a temperature con-
troller (Peltier type). ECL samples were prepared in PBS, pH
7.2 containing requisite quantities of GdnHCl or TFE /
HFIP. All the spectra were recorded in a cell of 1 mm path
length. The scan speed was 50 nm / min and response time
was 2 s. To eliminate signal noise, the spectra were averaged
over at least five scans.
FTIR measurement. FTIR spectra were obtained on a
FT/IR-680 plus spectrometer (Jasco, Japan) at 4 cm-1 resolu-
tion. The spectra were averaged over 512 scans. The sample
for native ECL was prepared by dissolving the protein in
D2O containing 0.15 M NaCl which was kept for 15 min.
The native ECL sample was placed in a sealed cell
consisting of two ZnSe windows and a Teflon spacer with
path length of 100 μm. In presence of TFE, the experiments
were carried out in a similar fashion when requisite amounts
of TFE-OD were added to ECL solution. In all cases appro-
priate blanks were subtracted and spectra in the range of
1700-1600 cm-1 were examined.
Chemical modification. Tryptophan oxidation with NBS
[28] was carried out at room temperature with native ECL
and other ECL samples in GdnHCl or TFE / HFIP. Aliquots
of NBS (2 M) were added to ECL samples in 20 mM sodium
acetate buffer, pH 5 containing 0.15 M NaCl taken in a 1-cm
path length cuvette and kept for 5 min. The protein concen-
tration was 500 μg / mL. The progress of reaction was moni-
tored by increase in absorbance at 250 nm due to formation
of oxindolealanine and decrease in absorbance at 280 nm due
to tryptophan modification. The absorbance recorded at 280
nm was corrected for dilution. The number of tryptophans
modified was estimated according to Spande and Witkop
[28].
RESULTS AND DISCUSSION
Native ECL exists as a dimer. The unfolding of ECL with
concomitant structural changes, as induced by the chemical
denaturant (GdnHCl), and by fluoroalcohols (TFE and HFIP)
have been investigated using fluorescence, low temperature
phosphorescence, selective chemical modification, far-UV
CD and FTIR.
GdnHCl-induced Unfolding of ECL: Detection and
Characterization of an Intermediate
Intrinsic fluorescence and ANS binding studies. Na-
tive ECL exhibits an emission maximum at 328±1 nm,
which undergoes gradual red-shift with increase in GdnHCl
concentration, and ultimately levels off at 351±1 nm in
3 M
GdnHCl indicating Trp exposure to aqueous environment
and complete unfolding of the protein. Fig. 2A shows the
Protein & Peptide Letters, 2014, Vol. 21, No. 1 3
denaturation curve in terms of the relative decrease of
fluorescence intensity at 320 nm as a function of GdnHCl
concentration. As shown, two distinct transitions have been
observed involving an intermediate around 1.5 M GdnHCl.
Free ANS is feebly fluorescent in aqueous solution, but
its spectrum is blue shifted (520470 nm) with large
increase of intensity when it binds to hydrophobic surfaces
of proteins. Usually the hydrophobic core of native proteins
is well protected from the solvent by the rigid packing of
tertiary structure. Thus ANS has a low affinity for the native
as well as fully denatured state [29,30]. However, the ability
of ANS to bind to intermediate state with hydrophobic
exposure provides a suitable method for the detection of
intermediate in protein unfolding reaction. Fig. 2B shows the
ANS fluorescent spectra of ECL in 0M, 1.6 M and 6 M
GdnHCl along with a plot of ANS fluorescence intensity at
470 nm as a function of GdnHCl concentration (inset). The
fluorescence intensity at 470 nm for the ANS-ECL dimer
complex is small, but a large increase (~30 fold) in ANS
fluorescence intensity occurs for the species populated in 1.6
M GdnHCl. The ANS fluorescence for ECL in
3 M
GdnHCl is negligible. The results clearly suggest and pro-
vide further evidence for the existence of an intermediate in
the unfolding of ECL. Since ECL is a noncovalently
associated dimer, one possibility is that the intermediate
detected through ANS binding is a dissociated monomer
with exposure of hydrophobic surface. The size-exclusion
chromatography on Superose-12 10/300 GL column of ECL
in 1.6 M GdnHCl gave a single peak which eluted later than
native ECL (Fig. 3). From the molecular weight calibration
curve, the protein peak in 1.6 M GdnHCl was found to ap-
pear at a position of molecular mass of 28 kDa that corre-
sponds to the lectin monomer. The result is also corroborated
by theoretical calculation from the x-ray structure of ECL
[13]. The monomer-monomer interface area has been deter-
mined using the program ProFace [31]. Upon dissociation,
there occurs an exposure of 762 Å2 surface area per subunit,
of which 530 Å2 corresponds to hydrophobic patches which
bind to ANS causing a blue shift of its emission maximum
with dramatic enhancement of fluorescence intensity.
Far-UV CD studies. Far-UV CD senses secondary struc-
ture of a protein, and is commonly used to monitor the melt-
ing of secondary structure during protein unfolding. The
secondary structure of ECL is predominantly
-sheet (Fig.
1). The far-UV CD spectra of ECL in 0 M, 1.6 M and 6 M
GdnHCl is shown in (Fig. 4). Native ECL shows a character-
istic negative maximum around 225 nm for an atypical -
sheet structure. This -sheet band shape has been found to be
retained in the intermediate monomer in 1.6 M GdnHCl ex-
hibiting the characteristics of a folded species. However, for
the unfolded species in 6 M GdnHCl, the secondary -
structure has been completely lost.
Chemical modification of tryptophan residues with N-
bromosuccinimide (NBS). Tryptophan environment in
proteins can be probed by selective oxidation with NBS. The
oxidation reaction converts the indole side chain of
tryptophan to oxindole, and thus causes a loss of absorbance
at 280 nm [28]. ECL has five tryptophan residues per
subunit. Table 1 shows that two tryptophans are oxidized for
4 Protein & Peptide Letters, 2014, Vol. 21, No. 1 Sen and Mandal

Figure 3. Size-exclusion profiles of ECL on Superose-12 10/300

GL column attached to a Waters HPLC system in native state (a)
and 1.6 M GdnHCl (b) at pH 7.2. Inset: Molecular weight calibra-
tion curve: (from left to right): chicken egg ovalbumin (45 kDa),
Figure 2. A) Denaturation curve of ECL measured in terms of the carbonic anhydrase (29 kDa) and soybean trypsin inhibitor (20.1
relative change of fluorescence intensity at 320 nm as a function of kDa). The elution volume of unfolding intermediate is shown by an
GdnHCl concentration. Protein concentration was 2
M. The spec- arrow. See Materials and Methods for details.
tra were corrected for the buffers by subtracting the blank in each
case. Excitation wavelength, 280 nm; excitation and emission band
pass, 5 nm each; scan rate 60 nm/min. B) Steady-state ANS fluo-
rescence spectra at pH 7.2 of ECL in native state (a), 1.6 M
GdnHCl (b), and 6 M GdnHCl (c). Protein concentration was 4
M
and ANS was 100
M in each case. Excitation wavelength, 370
nm; excitation and emission band pass, 5 nm each; scan rate, 120
nm/min. (Inset) ANS fluorescence intensity of ECL at 470 nm as a
function of GdnHCl concentration.

both native dimer and intermediate monomer whereas all

five tryptophans are susceptible to oxidation in the unfolded
state. The results are consistent with the crystal structure of
ECL, even though the reaction involves structural states of
the protein in solution. In general, NBS oxidizes exposed
indole side chains in folded proteins. The accessible surface
area (ASA) of each tryptophan residue in the ECL dimer and
monomer was calculated using NACCESS [32], and the re-
sults are shown in Table 1. It is seen that all five tryptophans
(Trp 45, Trp 60, Trp 135, Trp 207 and Trp 231) have identi-
cal ASA values in the dimeric and monomeric forms. Of five Figure 4. Far-UV CD spectra of ECL at pH 7.2 at 20 oC in native
tryptophans, Trp 45 and Trp 207 have large ASA values state (a), 1.6 M GdnHCl (b), and 6 M GdnHCl (c). Protein concen-
(78.27 and 109.05 Å2 respectively), and are thus exposed to tration was 0.28 mg/mL. Respective buffer spectra were subtracted
in each case and at least five scans were done.
the solvent, as well as NBS reagent, and are oxidized. The
other three tryptophans having low ASA values are
Unfolding of Erythrina Cristagalli Lectin
Table 1. Accessible Surface Area (ASA) of Tryptophan Residues and Number of Tryptophans Modified by NBS in Different
States of ECL
System
ASA (Å 2) for tryptophan residues


Trp 45
Trp 60

Native Dimer
78.27
36.31

ECL Monomer (Intermediate in 1.6 M GdnHCl)
78.27
36.31

Unfolded ECL in 6M GdnHCl



a
Calculated per subunit of the protein.
presumably buried, and are thus inaccessible to NBS. In the
unfolded state, all tryptophans, however, become fully ex-
posed to the solvent, and are therefore oxidized.
Red edge excitation shift (REES) studies. REES refers
to a shift in fluorescence emission maximum towards higher
wavelength on changing the excitation wavelength towards
the red edge of the absorption band. The red edge effects for
tryptophans in proteins have been attributed mainly to the
slow rates of solvent relaxation around the excited state
tryptophan(s) due to motional restriction imposed on the
surrounding solvent molecules [33,34]. REES thus offers to
probe the tryptophan environment in different structural
states in protein unfolding reaction. Fig. 5 shows the REES
plot for ECL dimer, monomer, and denatured ECL. As the
excitation wavelength is changed from 280 to 300 nm, the
emission maximum of ECL dimer is shifted from 329 to 339
nm, which corresponds to a REES of 10 nm. Similar REES
effect is observed for ECL monomer. The unfolded ECL,
however, exhibits no excitation wavelength dependence.
ECL is a multitryptophan protein containing five tryptophan
residues per monomer. REES effect may therefore be
attributed to the average environment experienced by the
tryptophans. The observation of similar REES effect for ECL
dimer and monomer implies similar average tryptophan
Figure 5. Effect of changing excitation wavelength on the emission
maximum at pH 7.2 for native ECL dimer (filled circle), intermedi-
ate monomer in 1.6 M GdnHCl (filled triangle), and unfolded ECL
in 6 M GdnHCl (solid square). Protein concentrations of samples
were 1.67
M. Excitation and emission band pass, 5 nm each; scan
rate, 60 nm/min.
Protein & Peptide Letters, 2014, Vol. 21, No. 1 5
No. of Trp oxidizeda

Trp 135
Trp 207
Trp 231


43.99
109.05
1.89
2

43.99
109.05
1.89
2




5

environment for these species in which the immediate
vicinity of at least some of the tryptophans is highly rigid.
This is in excellent agreement with chemical modification
studies described before. No REES is observed for the
unfolded ECL due to the high flexibility of the tryptophan
residues, when all of them become solvent-exposed.
Acrylamide quenching studies. Acrylamide quenching
of tryptophan fluorescence is a convenient method to exam-
ine tryptophan environments in proteins [35]. Table 2 shows
the quenching parameters obtained by analyzing the Stern-
Volmer plots. The Stern-Volmer constant (KSV) is related to
the degree of exposure (accessibility) of the tryptophans. The
KSV for native ECL is found to be 2.1 M-1, which is similar
to that for the intermediate monomer in 1.6 M GdnHCl.
These results indicate the very similar tryptophan environ-
ment in them. However, the value of KSV for the unfolded
form in 6 M GdnHCl is 14.4 M-1 indicating a large increase
in solvent exposure of tryptophan residues. These results
agree well with the findings from NBS oxidation.
Table 2. Acrylamide Quenching of Tryptophan Fluorescence of
ECL
System
KSV (M-1)

Native ECL
2.1±0.2

Intermediate Monomer in 1.6 M GdnHCl
2.3±0.2

Unfolded ECL in 6 M GdnHCl
14.4±0.3

Phosphorescence studies at 77 K. Phosphorescence of
proteins at low temperature provide structured spectra show-
ing a characteristic (0,0) band, which signify the immediate
environment of tryptophan(s) in proteins [36]. Fig. 6 shows
the phosphorescence spectra at 77 K for native ECL, inter-
mediate monomer and fully unfolded protein. Native ECL
gives a distinct (0,0) band located at 412.4 nm without any
splitting. The intermediate in GdnHCl exhibits a single (0,0)
band at about the same position as for native protein. This
probably reflects the average tryptophan environment when
two tryptophans are exposed and three remain buried in both
dimer and monomer. However, the position of (0,0) band is
blue-shifted to 408.8 nm for the fully unfolded protein. The
blue shift of phosphorescence is attributed to the less
stabilization of the triplet state by rigid solvation geometry
for the exposed tryptophans whereas the red-shifted phos-
6 Protein & Peptide Letters, 2014, Vol. 21, No. 1 Sen and Mandal

Figure 6. Phosphorescence spectra for ECL in native state (a), in-

termediate in 1.6 M GdnHCl (b), and unfolded form in 6 M
GdnHCl (c). Protein concentration was 20
M. Excitation wave-
length, 280 nm; excitation and emission band passes were 10 and
1.5 nm, respectively.

phorescence emission occurs due to the tryptophan residues

located in a buried polarizable environment that stabilizes
the triplet state more than the ground state [37].

Unfolding of ECLin Fluoroalcohols: Role of TFE and

HFIP Figure 7. Relative change of fluorescence intensity of ECL at 330
nm as a function of concentration of A) TFE B) HFIP. Protein con-
The unfolding of ECL in presence of TFE and HFIP has centrations were 5
M. The spectra were corrected by subtracting
been monitored using tryptophan fluorescence and ANS the blank in each case. Excitation wavelength, 280 nm; excitation
binding studies. Native ECL dimer shows emission and emission band pass, 5 nm each; scan rate 60 nm/min.
maximum at 328±1 nm. In presence of TFE, there occurs
gradual red shift of emission maximum with increasing TFE
concentration, which finally levels off at 341±1 nm in ~30 %
TFE, indicating ECL unfolding with Trp exposure to the
aqueous environment. In contrast, in presence of HFIP, the
emission maximum undergoes red shift to level off at 335±1
nm in ~ 10 % HFIP, signifying less exposure of Trp residues.
The relative change of fluorescence intensity at 330 nm as a
function of TFE concentration is shown in (Fig. 7A), which
appears to depict two transitions with a possible intermediate
around 30 % TFE. However, HFIP-induced unfolding of
ECL indicates a single transition (Fig. 7B).
To confirm the involvement of an intermediate in TFE-
induced unfolding, ANS binding studies were performed.
Fig. 8 shows the ANS fluorescent spectra of ECL in native
state, 25 % TFE and 8 % HFIP. As shown, the ANS fluores- Figure 8. ANS fluorescence spectra at pH 7.2 of ECL in native
cence for native ECL or ECL in 8 % HFIP at 470 nm is state (a), 25 % TFE (b), and 8 % HFIP (c). Protein concentration
small, but a dramatic enhancement of ANS fluorescence was 4
M and ANS was 100
M. Excitation wavelength, 370 nm;
intensity occurs for the species in 25 % TFE. These results excitation and emission band pass, 5 nm each; scan rate, 120
clearly suggest that an intermediate with exposure of hydro- nm/min.
phobic patches is involved in TFE-induced unfolding of
ECL, and thereby show differing roles and characteristics of To determine whether the TFE-induced intermediate is
ECL unfolding by TFE and HFIP. It may be mentioned that formed by subunit dissociation, the first transition of TFE-
a similar intermediate was obtained in the unfolding of ECL induced unfolding has been investigated under low and high
in GdnHCl as described above. protein concentrations. Fig. 9 shows two transition profiles at
Unfolding of Erythrina Cristagalli Lectin
Figure 9. Plot of relative fluorescence intensity of ECL as a func-
tion of TFE concentration with 30 μg / mL (solid triangle) and 400
μg / mL (solid circle) of protein.
ECL concentrations of 30 and 400 μg/mL obtained by meas-
uring relative change of fluorescence intensity as a function
of TFE concentration. At 30 μg/mL ECL, the transition mid-
point is obtained at 18 % TFE while the midpoint occurs at
25 % TFE for 400 μg/mL protein. The transition midpoint is
shifted to the right at higher protein concentration signifying
protein concentration dependence for the first transition. This
result indicates that the transition may involve dissociation
of two subunits of ECL.
TFE- and HFIP-induced Secondary Structure Change of
ECL as Monitored by far-UV CD
Crystal structure of native ECL dimer shows a predomi-
nantly -sheet secondary structure (Fig. 1). Fig. 10A shows
the far-UV CD spectra of ECL at 20 oC at different TFE
concentrations. In absence of TFE, ECL exhibits a negative
peak at 226 nm along with a positive peak at 197 nm
characteristic of an atypical -sheet structure. This atypical
-sheet band shape as secondary structure element is well
known for other legume lectins, for examples, concanavalin
A [8] and soybean agglutinin [23]. With addition of TFE,
this band shape is transformed to a typical band shape with a
negative peak at 215 nm in ~ 20% TFE. The signal intensity
of typical -sheet is increased with rise in TFE concentration
up to 30 %, and thereafter, some broadening of the negative
extremum occurs. When TFE concentration is around 50%,
the far-UV CD spectra begin to change to an
-helical
conformation with negative peaks at 220 and 208 nm. The
-
helical band shape persists with further rise in TFE concen-
tration with gradual enhancement of intensity, which
indicates more helical conversion. The far-UV CD spectra of
ECL at various HFIP concentration is shown in (Fig. 10B).
In contrast to TFE, limited addition of 5 % HFIP induces
change in spectral band shape which broadens with increase
in negative intensity. The CD spectra acquire a prominent
-
helical band shape at HFIP concentration of 30 % - a much
lower amount compared to TFE perturbation. This implies
Protein & Peptide Letters, 2014, Vol. 21, No. 1 7
that HFIP exerts more perturbing influence than TFE for
helical conversion. Although no intermediate is detected in
HFIP-induced unfolding monitored by fluoresecence, the far-
UV CD pattern might suggest the possibility of intermediate
state in the unfolding process.
Deconvolution of far-UV CD spectra using CDNN soft-
ware [38] gives an estimate of the secondary structure com-
ponents, which shows that native ECL has ~ 40% -sheet
structure. In 60% TFE, native -sheet structure is completely
lost and ~26%
-helix is produced. The
-helical content
continues to increase with further rise in TFE concentration
to 53 % in 90 % TFE. The high helix content in large TFE
concentration occurs presumably, at least in part, at the
expense of random coil conformation. As TFE-water
systems are less able to solvate the amide group of the
peptide backbone, the free energy of the random coil state
increases, which tends to favor states in which the backbone
amide groups form intramolecular hydrogen bonds, as in
-
helix [39]. In presence of HFIP, the maximum
-helical
conversion is 65 % in 80 % HFIP which is more compared to
TFE-induced transformation.
The -sheet to
-helix transformation was examined at
different temperatures within 5-70 oC. Fig. 10C represents
far-UV CD spectra of ECL in 90% TFE at different tempera-
tures. As shown, the spectra exhibit
-helical band shape at
all temperatures (5-70 oC). However, the signal intensity of

-helical conformation diminishes with rise in temperature.
These results suggest that induced
-helix has less thermal
stability.
TFE-induced Secondary Structure Change of ECL as
Monitored by FTIR.
The secondary structure content of proteins could be
monitored using FTIR spectroscopy primarily by the
characteristic amide I absorption band which signifies
mainly stretching vibrations of the peptide C=O linkage.
This is possible as secondary structure elements, -sheet and

-helix, have definite pattern of hydrogen bonding with C=O
groups [40,41]. The FTIR spectra of ECL without and with
TFE-OD are shown (Fig. 11). ECL shows the characteristic
amide I band (N-deuterated) at 1636 cm-1 for -sheet struc-
ture. Analysis by estimation software (JASCO, version
1.01.03) gives 54 % -sheet with no
-helix (Table 3). In 90
% TFE-OD, a new amide I
peak at 1652 cm-1 is obtained,
which is indicative of
-helix conformation. Even under this
high concentration of TFE-OD, the -sheet to
-helix
transformation is not complete (Table 3). It is seen that 35%
helix is formed solely from the -sheet as the amount of
-
helix obtained is almost same as the loss of -sheet content
(Table 3). It may be mentioned that the induced helix content
obtained from FTIR is smaller compared to that from far-UV
CD. This may arise due to the difference in sample
conditions, such as protein concentration - 3 mg/mL for
FTIR compared to 0.28 mg/mL necessary for far-UV CD.
Tryptophan Environment of TFE- and HFIP-induced
States of ECL
As mentioned before, out of five tryptophan residues per
monomer of ECL, two are solvent exposed and oxidized by
8 Protein & Peptide Letters, 2014, Vol. 21, No. 1 Sen and Mandal

Figure 10. Far-UV CD spectra of ECL in presence of A) 0 % (1), 20 % (2), 30 % (3), 40 % (4), 50 % (5), 60 % (6),70 %(7), 80 % (8), 90 %
(9) TFE at 20 oC; B) 0 %, 5 %, 20 %, 30 %, 40 %, 50 % and 80 % HFIP at 20 oC; C) 90 % TFE at different temperatures (5-70 oC). ECL and
TFE were mixed after equilibration of each at the desired temperature. Protein concentration was 0.28 mg / mL in all cases. Respective
buffer spectra were subtracted in each case and at least five scans were carried out.

Figure 11. FTIR amide I' spectra of ECL in presence of 0 % (a) and
90 % (b) TFE-OD. Protein concentration was 3 mg / mL. Spectra
were recorded at 4 cm-1 resolution as an average of 512 scans.
Respective buffer spectra were subtracted in each case.
NBS in the native ECL while all five are susceptible to oxi-
dation in the completely unfolded state in 6 M GdnHCl. In
the HFIP-induced helix in 70 % HFIP, three tryptophans are
oxidized while for TFE-induced
-helical state in 90 % TFE,
four tryptophans undergo oxidation. These results are sup-
ported by phosphorescence measurements, which show pro-
gressively blue-shifted (0,0) band at 411 nm and 409.8 nm in
70 % HFIP and 90 % TFE, respectively, indicating more
exposure of tryptophan residues. In the TFE-induced mono-
meric intermediate in ~25 % TFE, NBS oxidation could not
be performed because of precipitation; however, phospho-
rescence result under this condition gives a (0,0) band at
409.8 nm as obtained in 90 % TFE. Further, the fluorescence
emission maximum in the range of 25-90 % TFE, remains
practically same at 341 nm. These results seem to indicate
that the tryptophan environment in the TFE-induced inter-
mediate is similar to that in
-helical form. This implies that
in TFE, transformation of the -sheet intermediate state into

-helical conformation does not induce change in tryptophan
environment.
Unfolding of Erythrina Cristagalli Lectin
CONCLUSION
The unfolding characteristics of ECL, with concomitant
structural changes and perturbations, have been investigated
and compared under different denaturing conditions in pres-
ence of a chaotrope (GdnHCl) and fluoroalcohols (TFE and
HFIP). The GdnHCl-induced unfolding of ECL depicts a
three-state mechanism:
N2  2N  2U
where N2 is the native ECL dimer, N is the intermediate
monomer with native-like secondary and tertiary structure
and U is the completely unfolded monomer. The fluoroalco-
hols, TFE and HFIP show different denaturation characteris-
tics. In presence of TFE, but not in HFIP, the denaturation
process of ECL appears to involve a molten globule-like
intermediate, and the perturbation process for dimeric ECL,
D(), could be represented as in the following scheme:
D()  I()  M(
)
where I() is a monomeric molten globule-like intermediate
with nonnative typical -secondary conformation, and M(
)
is the final TFE-induced state comprising a major nonnative

-helix with partial loss of tertiary structure. In HFIP, the
denaturation process may be represented as
D()  M*(
)
where M*(
) denotes the final HFIP-induced state similar to
M(
) but differing in more
-helical content and less loss of
tertiary structure. The present results thus provide important
insight into the differential structural characteristics of un-
folding sequence in presence of chaotrope and fluoroalcohols
which may assume major significance in the context of in-
vestigation of structure and folding of lectins, and oligomeric
proteins in general.
CONFLICT OF INTEREST
The authors confirm that this article content has no
conflicts of interest.
ACKNOWLEDGEMENT
We gratefully acknowledge the support in the form of
research grant (No. 02/(0028)/11/EMR-II) from the Council
of Scientific and Industrial Research (CSIR), Government of
India, and a CSIR research fellowship (No. 08/155(0044)/
2012–EMR-I) to DS.
ABBREVIATIONS
ECL = Erythrina cristagalli lectin
GdnHCl = Guanidine hydrochloride
TFE = 2,2,2-Trifluoroethanol;
HFIP = 1,1,1,3,3,3-Hexafluoroisopropanol
ANS = 8-Anilino-1-naphthalenesulfonate
NBS = N-Bromosuccinimide
Protein & Peptide Letters, 2014, Vol. 21, No. 1 9
PBS = 10 mM sodium phosphate buffered with 0.15
M NaCl, pH 7.2
REES = Red edge excitation shift
CD = Circular dichroism
FTIR = Fourier transform infrared.
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