Báo cáo khóa học: Conformational changes of b-lactoglobulin in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles A fluorescence and CD study docx

Conformational changes of b-lactoglobulin in sodium bis(2-ethylhexyl)
sulfosuccinate reverse micelles
A fluorescence and CD study
Suzana M. Andrade, Teresa I. Carvalho, M. Isabel Viseu and Sı
´
lvia M. B. Costa
Centro de Quı
´
mica Estrutural, Complexo 1, Instituto Superior Te
´
cnico, Lisboa, Portugal
The effect of b-lactoglobulin encapsulation in sodium bis
(2-ethylhexyl) sulfosuccinate reverse micelles on the envi-
ronment of protein and on Trp was analysed at different
water contents (x
0
). CD data underlined the distortion of the
b-sheet and a less constrained tertiary structure as the x
0
increased, in agreement with a concomitant red shift and
a decrease in the signal intensity obtained in steady-state
fluorescence measurements. Fluorescence lifetimes, evalu-
ated by biexponential analysis, were s
1
¼ 1.28 ns and
s
2
¼ 3.36 ns in neutral water. In reverse micelles, decay-
associated spectra indicated the occurrence of important
environmental changes associated with x
0

. Bimolecular
fluorescence quenching by CCl
4
and acrylamide was
employed to analyse alterations in the accessibility of the two
Trp residues in b-lactoglobulin, induced by changes in x
0
.
The average bimolecular quenching constant <k
CCl4
q
>was
found not to depend on x
0
, confirming the insolubility of this
quencher in the aqueous interface, while <k
acrylamide
q
>
increases with x
0
. The drastic decrease with x
0
of k
q
, asso-
ciated with the longest lifetime, k
CCl4
q2
, comparatively to the

increase of k
acrylamide
q2
, emphasizes the location of b-lacto-
globulin in the aqueous interfacial region especially at
x
0
‡ 10. The fact that k
acrylamide
q2
(x
0
¼ 30) ) k
acrylamide
q2
(water) also confirms the important conformational changes
of encapsulated b-lactoglobulin.
Keywords: b-lactoglobulin; conformation; quenching;
reverse micelles.
Many biological phenomena occur at interfaces rather than
in homogeneous solution, and protein–surfactant inter-
actions play a key role in the reactions involving membrane
proteins [1,2]. The role of reverse micelles (RM) has been
pointed out as a convenient membrane-mimetic medium
for the study of interactions with bioactive peptides [3]. In
particular, RM formed using the anionic surfactant, sodium
bis(2-ethylhexyl) sulfosuccinate (AOT), have been widely
reported for extractive separation and purification of
proteins [4,5].
Briefly, RM can be described as water nanodroplets

dispersed in water-immiscible apolar solvents, stabilized by
a monolayer of surfactant with its nonpolar tails protruding
into the oil and the polar headgroups in direct contact with
the central water core [6]. The droplet size can be altered
with a concomitant change on the properties of the water
inside the RM. As water is added, the radius of the water
pool (range 1.5–10 nm) increases as a function of the
water : surfactant ratio (x
0
). RM are protein-sized and,
consequently, proteins and other biopolymers can be
accommodated in different microenvironments according
to their physico-chemical nature and the properties of the
interfacial layer. The presence of proteins results in struc-
tural changes in both the biomolecules and the micellar
aggregates.
Milk proteins are widely valued within the food industry
for their emulsifying and emulsion-stabilizing properties.
These proteins become rapidly adsorbed at the oil/water
interface generated during emulsification [7]. b-Lactoglo-
bulin (bLG), is a globular, acid-stable protein of 162
residues, which constitutes approximately two-thirds of the
whey fraction of ruminant milk. The structural similarity
of bLG to retinol-binding protein has been noted, and
crystallography confirmed the typical lipocalin topology,
containing a b-barrel or calyx composed of eight antiparallel
b-strands, b
A
to b
H

[8]. bLG exists as a dimer in solutions of
physiological pH, but exhibits complex association equili-
bria, shifting between monomer, dimer, tetramer, octamer,
and monomer again, upon lowering the solution pH from
8.5 to 2.0 [9].
It is of special interest that bLG has a marked high
a-helical propensity [10,11] and an afib transition was
detected, by time-resolved CD spectroscopy, during its
folding process [12]. Thus, bLGmayserveasamodelforthis
conformational change associated with the prion diseases or
with Alzheimer’s disease [13]. In spite of the vast number of
studies, involving bLG, which have been carried out over the
past 60 years, the biological function of this protein is still
unclear. Its inclusion in the lipocalin family led to the
suggestion of a transport role. In fact, bLG exhibits affinity
for a variety of hydrophobic ligands, such as retinol, fatty
acids, etc. [14,15]. The fact that bLG increases lipase activity
Correspondence to S. M. Andrade, Centro de Quı
´
mica Estrutural,
Complexo 1, Instituto Superior Te
´
cnico, 1049–001 Lisboa Codex,
Portugal. Fax: + 351 21 8464455, Tel.: + 351 21 8419389,
E-mail:
Abbreviations: AOT, sodium bis(2-ethylhexyl) sulfosuccinate; bLG,
b-lactoglobulin; DAS, decay associated spectra; GdnHCl, guanidine
hydrochloride; NAT, N-acetyltryptophan; NATA, N-acetyltrypto-
phanamide; RM, reverse micelles; x
0

, water : surfactant ratio.
(Received 8 October 2003, revised 4 December 2003,
accepted 22 December 2003)
Eur. J. Biochem. 271, 734–744 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03977.x
and contributes to the removal of free fatty acid suggested
that bLG could facilitate the digestion of milk fat [16].
The main goal of the present investigation was to obtain
information on the conformation of bLG when it is
encapsulated in AOT RM at a wide range of water-pool
sizes. The study of the interaction between bLG and AOT
RM was carried out using CD, and steady-state and time-
resolved fluorescence techniques. In spite of the widespread
use of the intrinsic fluorescence of Trp as a probe of
microenvironmental changes, only a few publications have
reported on the photophysics of proteins in RM [17–19].
bLG has two Trp residues that are differently exposed to the
water solvent (Scheme 1): Trp19, facing into the base of
the hydrophobic pocket, is essentially inaccessible to the
solvent, whereas Trp61, at the end of strand b
C
,isrelatively
exposed [8]. The guanidino group of Arg124 lies only 3–4 A
˚
from the indole ring of Trp19, and Trp61 is close to a
disulfide bridge. As both groups can be efficient quenchers
of Trp fluorescence, some discrepancy has been found in the
literature as to which residue the bLG fluorescence can be
attributed [8,20]. Quenching studies involving acrylamide
and CCl
4

provided evidence of a different accessibility of
these quenchers to Trp residues, which depended on the
quencher location in the RM and on x
0
.
Materials and methods
Sample preparation
Bovine bLG (AB mixture), chromatographically purified
and lyophilized to ‡ 90% purity (Sigma; catalogue no.
L-3908), N-acetyltryptophanamide (NATA) (Sigma; cata-
logue no. A-6501) and AOT of 99% purity (Sigma; catalogue
no. D-4422), were used without further purification. Acryl-
amide (99% purity, electrophoresis grade) (Aldrich; cata-
logue no. 14,866–0) and guanidine hydrochloride (GdnHCl;
99% purity) (Aldrich; catalogue no. 177253–100G) were
both used as received. All solvents were of spectroscopic
grade.
A stock solution of 0.1
M
AOT/iso-octane was prepared
and checked for fluorescence emission, which was negligible
at the experimental conditions used. RM solutions were
then prepared by the direct addition of bidistilled water to
the surfactant/hydrocarbon mixture. The protein was added
by the injection method and freshly prepared prior to use.
All the volume injected was considered as water and used
to calculate x
0
(x
0

¼ [H
2
O]/[AOT]). A transparent solution
was always obtained after shaking for a few seconds. The
amount of water in dry micelle solution (x
o
¼ 0.15) was
determined by the Karl-Fischer method. bLG concentra-
tions were determined spectrophotometrically, using the
molar extinction coefficient e
280nm
¼ 17 600
M
)1
Æcm
)1
[21]
for the bLG protein monomer. The final concentration of
bLG was calculated relative to the total volume of the RM
solution and was kept small to ensure that (a) the
absorbance (A) was never > 0.1 and (b) multiple occupancy
would be statistically unlikely (assuming a Poisson distri-
bution). All measurements were made at 24 ± 1 °C.
Absorption and CD spectroscopy
A Jasco V-560 spectrophotometer, together with a 10 mm
quartz cuvette, was used in UV-Vis absorption measure-
ments. CD spectra were obtained using a Jasco J-720
spectropolarimeter (Hachioji City, Tokyo). The protein
spectra were measured using 10 mm (for near-UV) and
2 mm (for far-UV) quartz cells. The solutions containing

5 l
M
(far-UV) or 14 l
M
(near-UV) bLG were scanned at
20 nmÆmin
)1
, with a 0.2 nm step resolution, a 1 nm band-
width and a sensitivity of 10 millidegrees (mdeg). An average
of 5–10 scans was recorded and corrected by subtracting the
baseline spectrum of unfilled RM of the same composition.
The CD signal (in mdeg) was converted to molar ellipticity [h]
(deg cm
2
Ædmol
)1
), defined as [h] ¼ h
obs
(10cl)
)1
,whereh
obs
(mdeg) is the experimental ellipticity, c (molÆdm
)3
)isthe
monomeric protein concentration, and l (cm) is the cell path
length. The secondary structure content was evaluated by
using the
SELCON
3 program [22] from the

DICROPROT
2000
package (release 1.0.4), available free from the Internet
().
Steady-state and time-resolved fluorescence
spectroscopy
Fluorescence measurements were recorded using a Perkin-
Elmer LS 50B spectrofluorimeter, with excitation at
295 nm. The instrumental response at each wavelength
was corrected by means of a curve obtained using appro-
priate fluorescence standards together with the standard
provided with the instrument. The quantum yields of
NATA and bLG were determined relative to that of Trp
alone, at pH 7.0 and in aerated aqueous solution (/ ¼ 0.13)
[19], with appropriate corrections for the refractive index of
the solvent in AOT solutions. Steady-state fluorescence data
of bLG obtained at different water concentrations were
fitted to the following equation:
F ¼
F
o
þ F
w
K
À1
d
½H
2
O
n

1 þ K
À1
d
½H
2
O
n
Eqn ð1Þ
where F is the fluorescence intensity (corrected for absorp-
tion at the excitation wavelength) and F
o
and F
w
are,
respectively, the fluorescence intensities in the absence and
Scheme 1. Ribbon diagram of a single unit of bovine b-lactoglobulin
(bLG) drawn using
SWISS PDBVIEWER
, version 3.7, with PDB file 1BEB.
The locations of Trp19 and Trp61 are indicated.
Ó FEBS 2004 Conformational changes of bLG in reverse micelles (Eur. J. Biochem. 271) 735
presence of water; K
d
is the dissociation constant for the
interaction of water with the protein; and n is the Hill
coefficient which accounts for the system heterogeneity, as
described previously [23].
Fluorescence decay profiles were obtained using the time-
correlated single-photon counting method [24] with a
Photon Technology International (PTI) instrument. Exci-

tation of Trp at 295 nm was made with the use of a lamp
filled with H
2
, and sample emission measurements were
performed until a maximum of 10
4
counts was obtained.
NATA lifetime (s
F
¼ 2.85 ± 0.05 ns) was used as a
standard to check the apparatus response on a daily basis.
Data analysis was performed by a deconvolution method
using a nonlinear least-squares fit programme, based on the
Marquardt algorithm. The goodness of fit was evaluated by
statistical parameters (reduced v
2
and Durbin–Watson) and
graphical methods (autocorrelation function and weighted
residuals).
The decay associated spectra (DAS) of Trp fluorescence
in bLG were obtained using the following equation [25]:
F
i
ðkÞ¼F
SS
ðkÞ
a
i
s
i

R
i
a
i
s
i
¼ F
SS
ðkÞf
i
ðkÞ Eqn ð2Þ
where s
i
are the fluorescence lifetimes and a
i
(k)arethe
normalized pre-exponential factors of the exponential
functions used for the global fit analysis. For each k
em
,
the steady-state intensity F
SS
(k) is the weighted sum of the
intensities F
i
(k) associated with each decay component.
The decay profiles were obtained at 10 nm intervals in the
wavelength range of the steady-state spectra (310–400 nm).
For fluorescence quenching experiments, a 3
M

stock
solution of acrylamide was used and the protein fluores-
cence (F) was monitored at 340 nm. The following correc-
tion factor:
f
c
¼
OD
total
OD
bLG
1 À 10
ÀOD
bLG
1 À 10
ÀOD
total
was applied to F to account for the fact that acrylamide
absorbs at the excitation wavelength (e
295 nm
¼ 0.27 ±
0.03
M
)1
Æcm
)1
)[19].TheCCl
4
extinction coefficient at
295 nm was not measurable and so inner filter effects were

negligible. Quenching data were analysed using the Stern–
Volmer equation [26]:
F
0
=F ¼ð1 þ K
sv
½QÞe
V½Q
¼ðs
0
=sÞe
V½Q
Eqn ð3Þ
where F
0
and F are the fluorescence intensities in the absence
and in the presence of the quencher Q, respectively; K
sv
and
V are related, respectively, to the fluorescence extinction rate
constant for the dynamic (K
sv
¼ k
q
s
0
,wheres
0
is the
fluorescence lifetime in the absence of the quencher) and

static processes. In the case of different ground state sites,
individual components of static quenching would contribute
to the quenching given by the following equation:
F
0
F
¼
X
n
i¼1
f
i
ð1 þ K
SVi
½QÞe
V
i
½Q
"#
À1
Eqn ð4Þ
where K
SVi
and V
i
are, respectively, the dynamic and static
quenching constants for each fluorescent component i,and
f
i
is its corresponding fractional contribution to the total

fluorescence [26].
The errors of the calculated parameters were accessed
using the propagation theory, and the distribution F of
Snedcor was used to confirm, with 99% confidence, the
relationship among the variables [27].
Results
CD spectra of bLG in AOT RM
The effect of the amount of water (x
0
) inside AOT RM on
bLG far-UV CD spectra was followed at a pH
ext
of % 6.5
(pH of the aqueous solution containing the protein)
(Fig. 1A). The band with a minimum at 216 nm, charac-
teristic of bLG in water [28], gradually broadened and
deepened as x
0
increased, so that the minimum shifted to
lower wavelengths. This suggests some change in the bLG
native structure. The spectra showed increased noise below
Fig. 1. CD spectra. (A) CD spectra of b-lactoglobulin (bLG) in
sodium bis(2-ethylhexyl) sulfosuccinate (AOT) RM and in pure water.
Far-UV CD spectra in: 1, water; 2, AOT, x
0
¼ 3; 3, AOT, x
0
¼ 5;
4, AOT, x
0

¼ 15; 5, AOT, x
0
¼ 30; and 6, GdnHCl (6
M
). (B) CD
spectra of bLG in AOT (1–4) and SDS (5 and 6) aqueous solutions at
different surfactant concentrations. Inset: % of a-helix obtained with
SELCON
3 for SDS (d)andAOT(h) aqueous solutions.
736 S. M. Andrade et al. (Eur. J. Biochem. 271) Ó FEBS 2004
210 nm, which made it difficult to detect a reliable CD
signal below 200 nm. AOT itself is chiral and although a
background subtraction was performed the spectra had
significant noise, which could not be well discounted. On the
other hand, the presence of the protein may cause changes
on the AOT chirality and also on the RM size, which might
contribute to incorrect background compensation as the
average scattering intensity of RM is highly dependent on
the micelle size. Therefore, a quantitative appreciation of the
spectral changes was not possible. Curiously, the data
obtained at a lower x
0
are more similar to those of the
native aqueous structure than the ones obtained at a higher
x
0
. A study carried out in hexane, at different hydration
levels, showed that proteins retain their native conformation
for lower concentrations of water (% 10%) than at higher
water concentrations [29]. Structural changes occur owing

to the collapse of water clusters, at the surface of the protein,
into larger clusters; this provides a medium for ion diffusion
and ion pair formation, which leads to the movement of the
charged groups of the protein in order to keep themselves
neutral.
As a means of testing the role of the surfactant on the
protein conformations, bLG was dissolved in aqueous
AOT. The far-UV CD spectra obtained at different
concentrations of AOT (Fig. 1B), show (a) the appearance
of a minimum at around 208 nm and a shoulder at around
222 nm increasing with AOT concentration and (b) less
noisy spectra, which allows for quantitative analysis down
to 190 nm. Both the values of ellipticity obtained at 222 nm,
which can be converted into a-helix content [30], as well as
the results obtained by applying the
SELCON
3program,
indicate the same trend of increasing a-helix content as
AOT concentration increases. Above 6 m
M
,aplateauseems
to be reached (Fig. 1B, inset). Curiously, this value is in the
range of the critical vesicle concentration (5–8 m
M
) deter-
mined for aqueous AOT in the presence of different
concentrations of poly(ethylene glycol) [31].
Aqueous solutions of an analogous anionic surfactant,
SDS, were also tested and the analysis of far-UV CD data
using

SELCON
3 (Fig. 1B, inset) confirms that the observed
spectral changes (Fig. 1B) are the result of an increase in
bLG a-helical content, when the SDS concentration
increases, similar to that obtained in AOT/water.
Changes in the secondary structure are accompanied by
tremendous alterations in near-UV CD signals (Fig. 2). The
CD spectrum of bLG in its native conformation presents
two peaks, at 286 and 293 nm, arising from the vibrational
fine structure of Trp residues [28]. These peaks are absent in
RM, even at high x
0
, thus suggesting that Trp residues in
such altered conformation are in a much less specific (more
symmetrical) environment and have a higher mobility than
in the native bLG. However, even at a x
0
of 3.0, the CD
signal between 260 and 300 nm is greater than that of bLG
in 6
M
GdnHCl, implying the existence of some ordered
tertiary structure, even at this low hydration level. A plot of
the ellipticities at 293 nm vs. x
0
(inset of Fig. 2 or Fig. 4)
shows a nearly sigmoid behaviour, which could be indicative
of a two-state transition. The mid-transition (x
0,mid
)of

around 6–7 corresponds to the level where AOT headgroups
are fully hydrated and water molecules start to be free for
the protein hydration. Increasing x
0
also leads to a decrease
in [h]
270
(Fig. 2, inset), with the appearance of a broad band
in the 265–280 nm region, which is not detectable in
aqueous solution or in ethanol/water mixtures.
Fluorescence of bLG in AOT RM
Steady-state fluorescence. The fluorescence spectra
obtained depend strongly on the amount of solubilized
water, Fig. 3. There is a concomitant red shift, and a
significant decrease in the fluorescence quantum yield, as
x
0
increases. Comparatively to free aqueous solution
(k
max
¼ 338 nm), the spectra at x
0
<10 are blue-shifted
(up to 5 nm), suggesting that Trp residues are less exposed
at lower x
0
. This may be associated with a decrease in the
local dielectric constant and consequent lowering of the
average polarity of the Trp environment and/or with con-
formational changes of the protein that are accompanied by

Fig. 2. CD spectra. Near-UV CD spectra in: 1, water; 2, sodium bis
(2-ethylhexyl) sulfosuccinate (AOT), x
0
¼ 5; 3, AOT, x
0
¼ 30; and
4, GdnHCl (6
M
). Insets: molar ellipticities at 270 and 293 nm as a
function of x
0
andinwater.
Fig. 3. Fluorescence spectra. Fluorescence spectra of b-lactoglobulin
(bLG) (k
exc
¼ 295 nm) in sodium bis(2-ethylhexyl) sulfosuccinate
(AOT) reverse micelles (RM) at x
0
¼ 5 (1) and 30 (2); in pure water
(3); in 6
M
GdnHCl(4)andatatemperature(T)of75°C(5).Inset:
Wavelengths of maximum emission (j) and fluorescence quantum
yields (s) as a function of x
0
andinwater.
Ó FEBS 2004 Conformational changes of bLG in reverse micelles (Eur. J. Biochem. 271) 737
dislocation of local quenchers. The inset of Fig. 3 shows a
decrease in the intrinsic Trp fluorescence as x
0

increases.
The line through the data points was fitted to Eqn (1).
The value of K
d
¼ 0.16 ± 0.05
M
,withaQ
max
¼ [(F
0
–
Fw)/F
0
] · 100 ¼ 41 ± 5%, implies that both Trp residues
in bLG are probably not effectively quenched. The free
energy of this interaction (DG° ¼ –RT · lnK
d
,molar
standard state) at 25 °Cis)4.6±0.1kJÆmol
)1
(equivalent
to the energy of one conventional hydrogen bond).
Data from both CD and steady-state fluorescence
spectroscopies in RM were converted to a normalized scale
between 0 and 1, to compare their variation and the mid-
point transition (Fig. 4). The ensemble of data provides
evidence of a common x
0,mid
between 6 and 7.
Time-resolved fluorescence. Fluorescence lifetime analysis

fitted well to a biexponential model throughout all studied
x
0
, similarly to water. Both lifetimes decreased upon
increasing the water content, although never reaching the
values obtained in free aqueous solution (Fig. 5), followed by
changes in the population associated with each lifetime
component. The weight of the shorter lifetime, which is the
major component in water (f
1
¼ 0.86), is reduced in RM,
becoming the major component only at x
0
‡ 10 and reaching
f
1
¼ 0.61 at x
0
¼ 30. As for the long component, taking into
account the CD results we may invoke the existence of
conformational changes affecting the Trp environment, in
such a way that quenching groups (e.g. disulfide bridges) may
no longer be effective and thus contribute to a longer lifetime
ofTrpinAOTRMthaninwater.
Decay associated spectra. More detailed information
about the individual environments of Trp residues in the
protein was obtained from DAS (Fig. 6, Table 1), which
were constructed across the emission spectrum (see Mate-
rials and methods). In free water (Fig. 6C), almost the entire
fluorescence intensity (% 80%) was caused by the DAS

of the short-lifetime component emitting at 338 nm
(s
1
¼ 1.28 ns), linked to the more hydrophobic region (less
polar and/or less accessible to water). In AOT RM, DAS
were obtained at x
0
¼ 5(Fig.6A)andx
0
¼ 30 (Fig. 6B),
providing evidence of quite different features. At x
0
¼ 5,
there was a larger contribution of the long-lifetime compo-
nent (s
2
¼ 4.07 ns) which emits more in the red
(k ¼ 340 nm) and with the highest fractional intensity
(f
2
¼ 0.53). The short component (s
1
¼ 1.61 ns) was
similar to that in free water but contributed less to the
overall fluorescence and was blue shifted (k ¼ 330 nm).
This implies that upon encapsulation, some changes
occurred in the vicinity of the Trp residues. At x
0
¼ 30,
the short-lifetime component (s

1
¼ 1.43 ns) became the
Fig. 5. Fluorescence lifetimes and fraction of the short-lived component.
Fluorescence lifetimes, s
1
(j)ands
2
(m),andfractionoftheshort-
lived component, f
1
(s), of b-lactoglobulin (bLG) in sodium bis(2-
ethylhexyl) sulfosuccinate (AOT) reverse micelles (RM) as a function
of x
0
andinpurewater.
Fig. 4. Comparison between ellipticities at 270 and 293 nm, and fluor-
escence quantum yields (k
exc
¼ 295 nm) for b-lactoglobulin (bLG) in
sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles (RM) as
afunctionofx
0
; and ellipticity at 222 nm for bLG in AOT/water. These
parameters were normalized to a scale between 0 and 1.
Fig. 6. Decay associated spectra (DAS). DAS for b-lactoglobulin (bLG) fluorescence in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse
micelles (RM) and in water, calculated using Eqn (2). The two spectra correspond to two lifetime components, s
1
(s)ands
2
(j). The dotted lines

were obtained by fitting to a Gaussian function (see Table 1 for details). (A) AOT, x
0
¼ 5; (B) AOT, x
0
¼ 30; (C) water.
738 S. M. Andrade et al. (Eur. J. Biochem. 271) Ó FEBS 2004
major contributor (f
1
¼ 0.60, k
1
¼ 335 nm), as observed
in water, although still far from the latter. The long-lived
component (s
2
¼ 3.87 ns) was more quenched at this x
0
but
was still longer than in free water and was red-shifted by
5nm(k ¼ 345 nm), perhaps as a result of greater exposure
to the surrounding water.
Fluorescence quenching of Trp residues in bLG. In order
to further investigate the physical association of lifetimes with
individual fluorophores in different sites, time-resolved and
steady-state quenching studies were performed with the
neutral quenchers acrylamide and CCl
4
, which are preferen-
tially located in water and oil, respectively. In these experi-
ments, the concentrations of acrylamide refer to the water
pool, whereas those for CCl

4
refer to the bulk organic phase.
Fluorescence quenching by acrylamide. The fluorescence
quenching of bLG by acrylamide has previously been
studied in free aqueous solution [20,32]. An upward
curvature in the Stern–Volmer plot, using fluorescence
intensity data, has been identified with the existence of static
contributions, similar to those found for free Trp [32] and
derivatives, N-acetyltryptophan (NAT) [19,32] or NATA
[32,33].
Stern–Volmer plots of steady-state fluorescence quenching
(F
0
/F)ofbLG by acrylamide in AOT RM at different x
0
are
presented in Fig. 7. All representations show upward
curvature. The decay profiles were analysed by a two-
exponential model, and the individual Stern–Volmer con-
stants K
SV
(i), i ¼ 1, 2 are presented in Table 2. At low x
0
values, dynamic quenching, associated with lifetime decrease,
was only detectable for the long component (s
2
¼ 4.1 ns).
Nevertheless, the dynamic rate constant was very low,
k
q2

¼ 2.8 · 10
7
M
)1
Æs
)1
, probably reflecting the high local
viscosity (g P 30 cP) and/or low polarity (e % 5–10) of the
medium [34,35]. Dynamic quenching increased with x
0
,
although the contribution of the short-lifetime component
was only measurable at x
0
P 10, when f
1
P 60%. The
k
q2
was constant at x
0
¼ 20–30 with a value of
0.3 · 10
9
M
)1
Æs
)1
,whereask
q1

at x
0
¼ 30 (% 0.54 ·
10
9
M
)1
Æs
)1
) is close to the value in free water
(% 0.58 · 10
9
M
)1
Æs
)1
). The slight blue shift detected in
fluorescence spectra at x
0
¼ 20–30 for the higher acrylamide
concentrations indicated that the fluorescence from the more
exposed residue is quenched first, thus confirming the ground
state heterogeneity with two components and different
quenching trends. Thus, Eqn (4) was used to fit the data.
At the lowest x
0
studied (x
0
¼ 5) the total quenching was
caused mainly by static contributions (as a result either of a

complex formation or of quenchers within the quenching
sphere of action). The first hypothesis was ruled out in the
absence of spectral changes. The values of V
i
are similar at
x
0
6 10, whereas above this x
0
there is a huge difference
between V
1
and V
2
and they become highly dependent on the
water content. The radius of the volume element [R
i
¼ (3V
i
/
4pN
a
)
1/3
] gives a measure of the proximity of the quencher
molecule to the fluorophore. The calculated values are
almost within the van der Waals contact (6–7 A
˚
)atx
0

6 10.
Increasing x
0
leadstoanincreaseofR
1
up to values
resembling that of indole in free aqueous solution, where a
quenching radius of % 9A
˚
has been obtained as a result of
the fast diffusion of acrylamide in this medium.
Fluorescence quenching by CCl
4
. CCl
4
remains in the
organic nonpolar phase, including the outer micelle inter-
face [36]. Thus, its uptake by AOT RM is negligible.
Quenching of bLG fluorescence by CCl
4
in AOT RM
produced downward deviations in the Stern–Volmer plot
(Fig. 8A) at all water contents studied. A similar behaviour
Table 1. Spectral resolution of the two lifetime components (s
1
and s
2
)of
Trp in b-lactoglobulin (bLG). Fitting parameters were obtained using a
Gaussian function where a represents the normalized fractional con-

tribution of each component and l
)1
is a distribution parameter rep-
resenting the emission wavelength of maximum intensity.
x
0
a l
)1
(nm)
s
1
(ns) s
2
(ns)1212
5 0.470 0.530 330 340 1.61 4.07
30 0.600 0.400 335 345 1.43 3.87
Water 0.795 0.205 338 340 1.28 3.36
Fig. 7. Stern–
–
Volmer plots. Stern–Volmer plots for the quenching of
b-lactoglobulin (bLG) by acrylamide (k
exc
¼ 295 nm) in water (e)
and in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles
(RM) at x
0
¼ 5(d), 10 (n), 20 (j) and 30 (*). The solid lines represent
the best fits of the data to Eqn (4), assuming a different k
q
for each Trp

residue.
Table 2. Stern–
–
Volmer constants and bimolecular rate constants for the
dynamic and static quenching of b-lactoglobulin (bLG) by acrylamide in
water and in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse
micelles (RM) at different x
0
, using Eqn (4) with two differently
accessible fluorophores (i ¼ 2). (k
exc
¼ 295 nm; k
em
¼ 340 nm,
T ¼ 24 °C)
x
0
K
sv1
(
M
)1
)
K
sv2
(
M
)1
)
k

q1
· 10
)9
(
M
)1
Æs
)1
)
k
q2
· 10
)9
(
M
)1
Æs
)1
)
V
1
(
M
)1
)
V
2
(
M
)1

)
R
1
(A
˚
)
R
2
(A
˚
)
5 % 0 0.094 – 0.025 0.83 0.74 6.9 6.6
10 % 0 0.55 – 0.16 0.72 0.88 6.6 7.0
20 0.55 0.95 0.53 0.30 1.28 0.06 8.0 3.0
30 0.53 0.87 0.54 0.30 2.02 0.09 8.9 3.3
Water 1.09 – 0.58 < 0.005 1.44 % 0 8.3 –
Ó FEBS 2004 Conformational changes of bLG in reverse micelles (Eur. J. Biochem. 271) 739
was reported for indole and Trp, included in the AOT/
heptane/water/RM at x
0
> 5 [36]. Time-resolved quench-
ing studies, at a range of 0–0.4
M
CCl
4
, showed no apparent
change in the measured lifetime of the short component,
while the lifetime of the long component decreased such that
K
SV2

% 2.8
M
)1
(k
q
% 1.1 · 10
9
M
)1
Æs
)1
). Steady-state data
also pointed to an effective static quenching of this
component (V
2
% 0.55
M
)1
, i.e. R % 6.0 A
˚
). A similar
pattern was observed at x
0
¼ 10, while at higher x
0
values
significant changes were detected. Fluorescence spectra
show a notorious red shift (% 10 nm) at high concentrations
of CCl
4

(Fig. 8B).
Discussion
CD data
The secondary structure of bLG in its native conformation
is that of a predominantly b-sheet protein (% 50% b-sheet,
% 10% a-helix and % 40% random coil) [8,37]. However,
some segments of the amino acid sequence show a
significant propensity to form a-helices, including the
three N-terminal b-strands, as mentioned previously. The
structure of the a-helix intermediate resembles that of a
molten globule which has been described as having a
compact size, globular shape and pronounced secondary
structure, but little rigid tertiary structure and a hydropho-
bic core exposed to the solvent [38]. The increase in a-helix
content, detected in AOT and SDS aqueous solutions at
increasing surfactant concentration, is similar to that
reported in other aqueous surfactant solutions [39] and in
alcoholic mixtures [40–44]. This conformation is quite
different from that obtained in the presence of chaotropic
solutes, such as GdnHCl, where the broad band at 216 nm
disappears as a consequence of the loss of most of the helical
and sheet secondary structure.
This apparent bfia transition cannot be interpreted only
in terms of the decrease of the dielectric constant of the
surrounding medium [45] because, in several alcoholic
mixtures, the cluster (micelle-like assembly) formation of
each solvent in the excess of the other [42,45] may take place
and interact with the polypeptide chains. This would
stabilize local hydrogen bonds and consequently induce
helical conformation.

In the case of AOT RM, in spite of the rather noisy CD
signals obtained, the pattern followed upon increasing x
0
was different from that in AOT/water or SDS/water
systems. Although the spectra seem to indicate a loss in
b-sheet structure, the concomitant blue shift and almost
invariable signal at 220 nm do not lead to a typical a-helix
CD spectrum. Similar spectra have been reported for bLG
at high temperatures (70–86 °C) [46,47] and induced by high
pressure (600 MPa) [48]. Analysis of the temperature
induced spectra, based on
SELCON
, indicated a decrease in
both helical and sheet contents with an increase in random
structure, suggesting a direct conversion from regular to
irregular structures [46]. However, the total structure
content is smaller in the pressure-induced than in the
temperature-induced partial unfolded state [48]. The hypo-
thesis that this may be a molten globule state has been raised
and seems consistent with near-UV CD data obtained,
which indicated an increase in the protein flexibility. This
conformation might be related to intermolecular aggrega-
tion changes that may be induced by high temperature as
well as by encapsulation in AOT RM. A decreased ratio of
native dimers to monomers, and the loss of H-bonding
involving the strands b
I
of bLG [47], could be promoted in
both situations. A CD spectrum assigned to distortions of
b-strands showed a band with a minimum at around

195 nm [22], which could account for the blue shift obtained
in bLG far-UV CD spectra at high x
0
. The fact that a helical
structure is not stabilized in AOT RM, in contrast to what
occurs in the aqueous AOT or SDS solutions, suggests that
the spatial confinement in RM might play an important role
in bLG conformational changes.
Near-UV CD arises from the chirality of the environ-
ments of the side-chains of aromatic residues (Trp, Tyr and
Phe) and disulfide bonds [49]. The two deep peaks found at
286 and 293 nm arise from Trp residues. In the case of bLG,
X-ray crystallography showed that while the indolic side-
chain of Trp19 is within the hydrophobic-binding cavity [8],
Fig. 8. Stern–Volmer plots and fluorescence emission spectra.
(A) Stern–Volmer plots for the quenching of b-lactoglobulin (bLG)
by CCl
4
(k
exc
¼ 295 nm) in sodium bis(2-ethylhexyl) sulfosuccinate
(AOT)reversemicelles(RM)atx
0
¼ 5(h), 10 (m), 20 (s)and30(r).
The solid lines represent the best fits of the data to Eqn (4) assuming a
different k
q
for each Trp residue. (B) Fluorescence emission spectra of
bLGinthepresenceofCCl
4

(0–0.8
M
) in AOT RM at x
0
¼ 30. Inset:
dependence of the wavelength of maximum emission on the concen-
tration of CCl
4
.
740 S. M. Andrade et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Trp61 lies at the surface (Scheme 1) and has considerable
rotational freedom [47], thus implying that near-UV CD
signals arise mainly from the former Trp residue. Moreover,
data concerning both the porcine [47] and the equine [50]
sources of bLG, which do not contain Trp61, show CD
spectra near 290 nm, which are similar to that of bovine
bLG.
As mentioned previously, disulfide bonds also contribute
to the near-UV CD spectrum, giving a broad band near
260 nm that seems to be related to the dihedral angle of the
bond [49]. Changes in this angle result in the splitting of this
band into two broad bands, one of which appears at 270–
280 nm [38] and the other, shifted to the blue, lying below
the intense peptide bond absorption band causing no
changes in the far-UV CD spectra. So, the broad band
centred at 270 nm obtained in bLG CD spectra upon
encapsulation on AOT RM may arise from the changes in
nature of the intra- and intermolecular disulfide bonds. A
relationship between the CD signal at 270 nm, and bLG
aggregation, has been previously established [47,51]. Surface

denaturation of bLG, induced by oil contact in an oil-
in-water emulsion, led to an increase in droplet flocculation
[52]. Surface denaturation exposes the protein sulphydryl
groups to the aqueous phase, leading to disulfide inter-
change reactions.
The pH of encapsulated water inside RM changes with
x
0
and must have a radial distribution [53]. Previous studies
suggest that a probe located near the interface senses a more
acidic environment than that of the aqueous solution of
departure owing to favourable electrostatic interactions of
H
+
with the anionic AOT headgroups [54]. However,
comparatively to bLG in aqueous solution (pH 6.0), CD
spectra of bLG at pH 2.0 showed great similarity in the far-
UV region and a minor decrease in intensity in the Trp
absorption region, which does not account for the differ-
ences obtained in AOT RM.
Fluorescence data
Emission from Trp is highly sensitive to the polarity of its
microenvironment. The transfer of Trp from an aqueous to
a lipid medium is characterized by a blue shift and an
increase in intensity of the emission maximum [55].
Changes induced on Trp fluorescence, upon the x
0
increase within AOT RM, provide evidence of considerable
alterations in the bLG tertiary structure, but point to
different unfolded forms of bLG compared with those

observed in the presence of GdnHCl or induced by
temperature (Fig. 3). Upon denaturation with 6
M
GdnHCl,
ashiftto353nm(% 15 nm red-shift relative to water) is
observed, showing the greater exposure of Trp residues to
water, together with an increase in fluorescence intensity
owing to less effective quenching of Trp61 by the nearby
disulfide bridge. The temperature effect leads only to 5 nm
red-shift and a considerable decrease in intensity, corrobor-
ating further the different unfolded states for each agent.
The environment in RM at low x
0
is not very fluid and
has characteristics of a hydrophobic medium. Thus, an
increase in fluorescence intensity is observed coupled with a
blue shift relative to Trp fluorescence in water. However, as
the water content increases, its properties approach those
of free water and therefore a fluorescence red shift and a
concomitant intensity decrease are observed. As fluores-
cence data at a large x
0
are still different from that in free
water, it is probable that the tertiary structure of bLG is less
constrainedthaninwater.Infact,Trpderivatives,suchas
NATA, are commonly observed to be quenched by water
molecules, probably by proton transfer [56]. It should be
noted that these water-induced conformational changes in
bLG may cause fluorescence quenching by mechanisms
other than those involving close contact between Trp and

water. A variety of chemical groups present in proteins
(including histidine, cysteine, proline or the peptide bond)
are capable of quenching the Trp fluorescence if induced
changes alter their proximity to the indole ring.
Ionic strength within these RM also changes with x
0
.Salt
solutions at moderate concentrations (0.01–1
M
) and neu-
tral pH are known to affect the structure and properties of
proteins (solubility, denaturation, dissociation, etc.), the
effect being dominated by anions. However, the fluores-
cence of bLG was found to be independent of the Na
2
SO
4
concentrationupto0.4
M
(data not shown).
bLG location on AOT RM
As mentioned above, at x
0
< 10 there is not sufficient water
to solvate both the surfactant polar headgroups and the
protein, which remain poorly hydrated. Close to neutral pH,
bLG is negatively charged and will not establish attractive
electrostatic interactions with the anionic AOT headgroups.
Although at low x
0

bLG will probably locate close to the
interface, it is expected that at higher water contents (and
thus larger water-pools) the protein will interact more closely
with water. A similar pattern was found in both fluorescence
and near-UV CD data, although the conditions of free
aqueous solution were not attained. Major changes, caused
by the presence of bLG, are not expected in AOT RM mainly
because the micellar concentration is always several orders of
magnitude higher than that of the protein and also the
prevalence of hydrophobic interactions contributes only to
weak perturbations on the AOT micelle interface [57].
Although some AOT may bind to bLG, this does not lead to
an unfolded state similar to that obtained in the presence of
GdnHCl. bLG could even bind iso-octane; however, fluor-
escence quenching dependence on x
0
obtained with both
agents (acrylamide and CCl
4
) point to an aqueous interfacial
location for bLG in AOT RM.
Encapsulation effect on the Trp environment
The amino acid residue in proteins is NATA (not Trp),
which has a single exponential decay (s
f
% 2.95 ns at 20 °C
and pH 5.0) [58]. Therefore, for a single Trp residue in a
protein, in a unique conformation and with no time-
dependent spectral relaxation, one would expect a single
exponential decay. Any deviations from this behaviour

would have to be attributed to multiple conformations,
protein dynamics, spectral relaxation, or the presence of
intrinsic nearby quenchers. The fact that bLG has two Trp
residues and shows two distinct lifetimes makes it tempting
to inter-relate it, as in the case of lac repressor from
Escherichia coli [59]. However, in water, DAS showed
that the emission maxima of both lifetimes are very close
to each other, supporting the existence of ground state
Ó FEBS 2004 Conformational changes of bLG in reverse micelles (Eur. J. Biochem. 271) 741
heterogeneity or the location of both Trp residues in protein
regions of similar polarity. The latter hypothesis does not
seem to apply, based on recent data of X-ray crystallogra-
phy [8] and NMR [60]. The increase in the average lifetime is
a manifestation of the increasing amplitude of the longer
decay component at longer wavelengths; nevertheless, some
contribution from solvent relaxation to the fluorescence
dynamics may also occur, alerting for the danger of an
overinterpretation of DAS [61]. At x
0
¼ 5, encapsulation in
AOT RM alters drastically the environment of Trp residues,
leading to a blue shift of 8 nm of the species associated with
the shorter component whose lifetime increases with a
smaller contribution (Table 1). This may be indicative of
conformational changes or alterations in the degree of
protein self-association. As no apparent dependence was
found on bLG concentration (5–14 l
M
), we believe that an
increase in the hydrophobicity of the shorter component

takes place. On the other hand, the longer component seems
to be protected from polarity changes of the medium and
thus the approach of effective quenching groups is not
sensed. In agreement with steady-state data, fluorescence
quenching by solubilized water occurs at x
0
¼ 30, and both
residues sense a more polar medium than at x
0
¼ 5, which
is still far apart from that of free water. The advantages and
disadvantages of distributed vs. discrete analysis in under-
standing protein fluorescence have been addressed in detail
previously [62]. Here, there seems to exist an apparent
physical significance in the analysis of bLG decays as a sum
of two exponentials, which was therefore pursued to further
investigate the protein structure. To test the physical
association of lifetimes with individual fluorophores in
different sites, the accessibility of bLG by distinct quencher
molecules (acrylamide or CCl
4
) was evaluated. Although
CCl
4
and acrylamide locate preferentially in the bulk oil and
water, respectively, a partition between these sites and the
interface may occur. In water, the quenching of bLG by
acrylamide showed heterogeneity in the fluorophore’s
accessibility, giving downward deviations to the Stern–
Volmer plot. However, in AOT RM, at all studied x

0
,only
upward deviations were obtained, which could be translated
by bimolecular quenching rate constants of similar magni-
tude (that is, equivalent access) at higher x
0
and still an
important static quenching at lower x
0
(Table 2). These
differences could be coupled to changes in the bLG tertiary
structure in such a way that accessibility to Trp residues
becomes different as the water content changes.
More pronounced differences were found in the case of
the residue associated with the long-lived component, when
comparing the values of k
acrylamide
q
in water and at x
0
¼ 30.
Data obtained in water showed that this residue was almost
inaccessible to acrylamide (k
acrylamide
q2
£ 10
7
M
)1
Æs

)1
, thus
justifying the downward curvature obtained). Viscosity
decrease would account for the increase of k
acrylamide
q2
with
x
0
and thus confirm the location of the quenching process
in the aqueous side. The carbonyl groups of AOT were
proposed [63] and confirmed [19,36] to be quenchers of Trp
and its derivatives. Moreover, this anionic interface attracts
H
+
to its vicinity, which would contribute to further
quenching of Trp. However, both fluorescence lifetime
components are longer for the lower x
0
.
On average, the rate constant for the dynamic quenching
by CCl
4
does not depend on x
0
(k
CCl4
q
% 1.06 ·
10

9
M
)1
Æs
)1
). This value is similar to that reported for
NATA at x
0
¼ 15–22 (% 1.12 · 10
9
M
)1
Æs
)1
) [18], but
lower than that for indole at x
0
¼ 5(% 15.6 · 10
9
M
)1
Æs
)1
)andatx
0
¼ 22 (% 6.6 · 10
9
M
)1
Æs

)1
) [36]. On the
other hand, the fact that k
CCl4
q
> k
acrylamide
q
at all the x
0
studied seems to confirm the idea of an aqueous interface
with higher viscosity than the oil interface [36]. In the case of
acrylamide, the dynamic quenching in AOT is more
effective for derivatives such as NAT (k
acrylamide
q
%
1.1 · 10
9
M
)1
Æs
)1
at x
0
¼ 20) [19] or indole (k
acrylamide
q
%
1.2 · 10

9
M
)1
Æs
)1
) [36] than for the Trp residue in the
protein matrix. This may be associated with a decrease in
the translational diffusion coefficient of Trp, when included
in the protein, and with a lower rotational mobility of the
macromolecule, causing steric limitations. It is conceivable
that, in this case, the diffusion of the quencher in the protein
matrix may be more difficult, requiring a penetration
mechanism. In the case of CCl
4
, the indole group may be
more accessible to collisions with the quencher owing to a
certain unfolding of the protein. Although a red shift of the
fluorescence maxima is observed at x
0
¼ 20–30, this does
not seem to cause major alterations because neither s
1
nor s
2
are very different in the presence of acrylamide or CCl
4
.
bLG fluorescence quenching by the latter always produces
downward curvatures. The individual bimolecular rate
constants obtained show that k

CCl4
q2
decreases drastically
with x
0
, being almost null at x
0
¼ 30. This seems to point
to an inaccessibility to CCl
4
of the residue emitting more to
the red and with longer lifetime, probably because it faces a
more aqueous environment (inaccessible to the quencher)
imposed by conformational changes in the protein tertiary
structure. This picture agrees with the decrease in s
f
and the
red shift obtained in DAS, as well as with the increase in
k
acrylamide
q2
with x
0
. In turn, the residue associated with the
shorter lifetime might be buried in the protein matrix in a
more hydrophobic region and therefore not accessible to
collisional quenching by both quenchers at lower x
0
,in
agreement with the blue shift obtained in DAS. Neverthe-

less, both acrylamide and CCl
4
can locate in the vicinity and
promote static quenching, less prone in the case of the latter,
probably owing to geometric restrictions.
Conclusions
Encapsulation of bLG in AOT RM leads to important
conformational changes of the protein. However, the bfia
transition that occurs in the aqueous AOT vesicle system
is not observed in the RM system. In the latter, bLG
secondary structure seems to evolve to a distorted b-sheet.
Such distortion probably involves strand b
I
and may be
related to changes in the intermolecular aggregation, i.e.
the dimer«monomer equilibrium might be affected upon
encapsulation on AOT RM more clearly at higher x
0
.
Near-UV CD spectra point to the loss of chirality on the
environment of Trp residues, whereas surface denaturation
by contact with the oil phase may occur, leading to
disulfide interchange reaction. Fluorescence data also
support these findings, reflecting a less constrained tertiary
structure of bLG within these aggregates.
Time-resolved fluorescence decays follows biexponential
kinetics with lifetimes of 1.28 ns and 3.36 ns in water.
Encapsulation at a x
0
of 5 suggests an increase in the

742 S. M. Andrade et al. (Eur. J. Biochem. 271) Ó FEBS 2004
hydrophobicity of the Trp residue associated with the
shorter component, and a less efficient approach of
quenching groups to the Trp residue associated with the
longer component. The fact that, even at x
0
¼ 30, the
lifetimes are still different from those found in water
suggests that this protein may be located in the water-pool
interfacial region, especially at x
0
values (x
0
‡ 7) for which
the RM have hydrodynamic radii higher than that of the
protein. Such a location is in agreement with the differ-
ences found in quenching measurements with CCl
4
and
acrylamide.
Acknowledgements
This work was supported by Project POCTI/35398/QUI/2000 and FSE
(III Quadro Comunita
´
rio de Apoio). The authors thank Professor
J. Costa Pessoa for the use of CD spectrometer. S.M. Andrade thanks
FCT for the BPD grant, no. 18855.
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