CDdata underlined the distortion of the b-sheet and a less constrained tertiary structure as the x0 increased, in agreement with a concomitant red shift and a decrease in the signal inte
Trang 1Conformational 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 (x0) CDdata underlined the distortion of the
b-sheet and a less constrained tertiary structure as the x0
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 s1¼ 1.28 ns and
s2¼ 3.36 ns in neutral water In reverse micelles,
decay-associated spectra indicated the occurrence of important
environmental changes associated with x0 Bimolecular
fluorescence quenching by CCl4 and acrylamide was
employed to analyse alterations in the accessibility of the two
Trp residues in b-lactoglobulin, induced by changes in x0 The average bimolecular quenching constant <kqCCl4> was found not to depend on x0, confirming the insolubility of this quencher in the aqueous interface, while <kqacrylamide> increases with x0 The drastic decrease with x0of kq, asso-ciated with the longest lifetime, kq2CCl4, comparatively to the increase of kq2acrylamide, emphasizes the location of b-lacto-globulin in the aqueous interfacial region especially at
x0‡ 10 The fact that kq2acrylamide (x0¼ 30) kq2acrylamide (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 reinter-actions 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 (x0) 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, bAto bH[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 CDspectroscopy, during its folding process [12] Thus, bLG may serve as a model for this 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: sandrade@popsrv.ist.utl.pt
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)
Trang 2and 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 bC, is relatively
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 CCl4 provided evidence of a different accessibility of
these quenchers to Trp residues, which depended on the
quencher location in the RM and on x0
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; catacata-logue
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 0.1MAOT/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 x0(x0¼ [H2O]/[AOT]) A transparent solution was always obtained after shaking for a few seconds The amount of water in dry micelle solution (xo¼ 0.15) was determined by the Karl-Fischer method bLG concentra-tions were determined spectrophotometrically, using the molar extinction coefficient e280nm¼ 17 600M )1Æcm)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 ± 1C Absorption and CD spectroscopy
A Jasco V-560 spectrophotometer, together with a 10 mm quartz cuvette, was used in UV-Vis absorption measure-ments CDspectra 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 lM(far-UV) or 14 lM(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 CDsignal (in mdeg) was converted to molar ellipticity [h] (deg cm2Ædmol)1), defined as [h]¼ hobs(10cl))1, where hobs
(mdeg) is the experimental ellipticity, c (molÆdm)3) is the monomeric protein concentration, and l (cm) is the cell path length The secondary structure content was evaluated by using theSELCON3 program [22] from theDICROPROT2000 package (release 1.0.4), available free from the Internet (http://dicroprot-pbil.ibcp.fr)
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:
F¼Foþ FwK
1
d ½H2On
1þ K 1
d ½H2On Eqnð1Þ where F is the fluorescence intensity (corrected for absorp-tion at the excitaabsorp-tion wavelength) and Fo and Fw 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.
Trang 3presence of water; Kdis 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 H2, and sample emission measurements were
performed until a maximum of 104 counts was obtained
NATA lifetime (sF¼ 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 v2and 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]:
FiðkÞ ¼ FSSðkÞ aisi
R
iaisi
¼ FSSðkÞfiðkÞ Eqnð2Þ
where si are the fluorescence lifetimes and ai(k) are the
normalized pre-exponential factors of the exponential
functions used for the global fit analysis For each kem,
the steady-state intensity FSS(k) is the weighted sum of the
intensities Fi(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 3M stock
solution of acrylamide was used and the protein
fluores-cence (F) was monitored at 340 nm The following
correc-tion factor:
fc¼ODtotal
ODbLG
1 10OD bLG
1 10OD total was applied to F to account for the fact that acrylamide
absorbs at the excitation wavelength (e295 nm¼ 0.27 ±
0.03M )1Æcm)1) [19] The CCl4 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]:
F0=F¼ ð1 þ Ksv½QÞeV½Q¼ ðs0=sÞeV½Q Eqnð3Þ
where F0and F are the fluorescence intensities in the absence
and in the presence of the quencher Q, respectively; Ksvand
Vare related, respectively, to the fluorescence extinction rate
constant for the dynamic (Ksv¼ kqs0, where s0 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:
F0
F ¼ Xn
i¼1
fi ð1 þ KSVi½QÞeVi½Q
Eqnð4Þ
where KSViand Viare, respectively, the dynamic and static
quenching constants for each fluorescent component i, and
fi 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 (x0) inside AOT RM on bLG far-UV CDspectra was followed at a pHextof 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 x0increased, 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) CDspectra of b-lactoglobulin (bLG) in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) RM and in pure water Far-UV CDspectra 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
3 for SDS (d) and AOT (h) aqueous solutions.
Trang 4210 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 x0 are more similar to those of the
native aqueous structure than the ones obtained at a higher
x0 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 CDspectra 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 SELCON3 program,
indicate the same trend of increasing a-helix content as
AOT concentration increases Above 6 mM, a plateau seems
to be reached (Fig 1B, inset) Curiously, this value is in the
range of the critical vesicle concentration (5–8 mM)
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
usingSELCON3 (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 CDsignals (Fig 2) The
CDspectrum 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 x0, 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 x0of 3.0, the CD
signal between 260 and 300 nm is greater than that of bLG
in 6M GdnHCl, implying the existence of some ordered
tertiary structure, even at this low hydration level A plot of
the ellipticities at 293 nm vs x0(inset of Fig 2 or Fig 4)
shows a nearly sigmoid behaviour, which could be indicative
of a two-state transition The mid-transition (x0,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 x0also leads to a decrease
in [h] (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
x0 increases Comparatively to free aqueous solution (kmax¼ 338 nm), the spectra at x0<10 are blue-shifted (up to 5 nm), suggesting that Trp residues are less exposed
at lower x0 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 CDspectra 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 and in water.
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) and at a temperature (T) of 75 C (5) Inset: Wavelengths of maximum emission (j) and fluorescence quantum yields (s) as a function of x and in water.
Trang 5dislocation of local quenchers The inset of Fig 3 shows a
decrease in the intrinsic Trp fluorescence as x0 increases
The line through the data points was fitted to Eqn (1)
The value of Kd¼ 0.16 ± 0.05M, with a Qmax¼ [(F0–
Fw)/F0]· 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 · lnKd, molar
standard state) at 25C is)4.6 ± 0.1 kJÆmol)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 x0,midbetween 6 and 7
Time-resolved fluorescence Fluorescence lifetime analysis
fitted well to a biexponential model throughout all studied
x0, 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 (f1¼ 0.86), is reduced in RM,
becoming the major component only at x0‡ 10 and reaching
f1¼ 0.61 at x0¼ 30 As for the long component, taking into account the CDresults 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
of Trp in AOT RM than in water
Decayassociated 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 (s1¼ 1.28 ns), linked to the more hydrophobic region (less polar and/or less accessible to water) In AOT RM, DAS were obtained at x0¼ 5 (Fig 6A) and x0¼ 30 (Fig 6B), providing evidence of quite different features At x0¼ 5, there was a larger contribution of the long-lifetime compo-nent (s2¼ 4.07 ns) which emits more in the red (k¼ 340 nm) and with the highest fractional intensity (f2¼ 0.53) The short component (s1¼ 1.61 ns) was similar to that in free water but contributed less to the overall fluorescence and was blue shifted (k¼ 330 nm) This implies that upon encapsulation, some changes occurred in the vicinity of the Trp residues At x0¼ 30, the short-lifetime component (s1¼ 1.43 ns) became the
Fig 5 Fluorescence lifetimes and fraction of the short-lived component Fluorescence lifetimes, s 1 (j) and s 2 (m), and fraction of the short-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 and in pure water.
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
a function of x 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) and s 2 (j) The dotted lines were obtained by fitting to a Gaussian function (see Table 1 for details) (A) AOT, x ¼ 5; (B) AOT, x ¼ 30; (C) water.
Trang 6major contributor (f1¼ 0.60, k1¼ 335 nm), as observed
in water, although still far from the latter The long-lived
component (s2¼ 3.87 ns) was more quenched at this x0but
was still longer than in free water and was red-shifted by
5 nm (k¼ 345 nm), perhaps as a result of greater exposure
to the surrounding water
Fluorescence quenching of Trp residues inbLG 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 CCl4, 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 CCl4refer to the bulk organic phase
Fluorescence quenching byacrylamide 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 (F0/F) of bLG by acrylamide in AOT RM at different x0are 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 KSV(i), i¼ 1, 2 are presented in Table 2 At low x0
values, dynamic quenching, associated with lifetime decrease, was only detectable for the long component (s2¼ 4.1 ns) Nevertheless, the dynamic rate constant was very low,
kq2¼ 2.8 · 107M )1Æs)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 x0, although the contribution of the short-lifetime component was only measurable at x0P 10, when f1P 60% The
kq2 was constant at x0¼ 20–30 with a value of 0.3· 109M )1Æs)1, whereas kq1 at x0¼ 30 ( 0.54 ·
109M)1Æs)1) is close to the value in free water ( 0.58 · 109
M )1Æs)1) The slight blue shift detected in fluorescence spectra at x0¼ 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 x0studied (x0¼ 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 Viare similar at
x0610, whereas above this x0 there is a huge difference between V1and V2and they become highly dependent on the water content The radius of the volume element [Ri¼ (3Vi/ 4pNa)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˚) at x0610 Increasing x0 leads to an increase of R1 up to values resembling that of indole in free aqueous solution, where a quenching radius of 9 A˚ has been obtained as a result of the fast diffusion of acrylamide in this medium
Fluorescence quenching byCCl4 CCl4 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 CCl4 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)1is a distribution parameter
rep-resenting the emission wavelength of maximum intensity.
x 0
s 1 (ns) s 2 (ns)
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˚)
Trang 7was reported for indole and Trp, included in the AOT/
heptane/water/RM at x0> 5 [36] Time-resolved
quench-ing studies, at a range of 0–0.4MCCl4, showed no apparent
change in the measured lifetime of the short component,
while the lifetime of the long component decreased such that
KSV2 2.8M )1(kq 1.1 · 109M )1Æs)1) Steady-state data
also pointed to an effective static quenching of this
component (V2 0.55M )1, i.e R 6.0 A˚) A similar
pattern was observed at x0¼ 10, while at higher x0values
significant changes were detected Fluorescence spectra
show a notorious red shift ( 10 nm) at high concentrations
of CCl4(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 x0 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 CDspectrum Similar spectra have been reported for bLG
at high temperatures (70–86C) [46,47] and induced by high pressure (600 MPa) [48] Analysis of the temperature induced spectra, based onSELCON, 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 CDdata 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 bIof bLG [47], could be promoted in both situations A CDspectrum 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 CDspectra at high x0 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 CDarises 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) reverse micelles (RM) at x 0 ¼ 5 (h), 10 (m), 20 (s) and 30 (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
bLG in the presence of CCl 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
Trang 8Trp61 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 CDspectrum, 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 CDspectra So, the broad band
centred at 270 nm obtained in bLG CDspectra upon
encapsulation on AOT RM may arise from the changes in
nature of the intra- and intermolecular disulfide bonds A
relationship between the CDsignal 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
x0and 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 x0
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 6MGdnHCl,
a shift to 353 nm ( 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 x0is 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 x0are still different from that in free water, it is probable that the tertiary structure of bLG is less constrained than in water In fact, Trp derivatives, such as 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 x0 Salt solutions at moderate concentrations (0.01–1M) 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 Na2SO4
concentration up to 0.4M(data not shown)
bLG location on AOT RM
As mentioned above, at x0< 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 x0bLG 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 CDdata, 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 x0obtained with both agents (acrylamide and CCl4) 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 (sf 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
Trang 9heterogeneity 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 x0¼ 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 lM), 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 x0¼ 30, and both
residues sense a more polar medium than at x0¼ 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 CCl4) was evaluated Although
CCl4and 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 x0, only
upward deviations were obtained, which could be translated
by bimolecular quenching rate constants of similar
magni-tude (that is, equivalent access) at higher x0 and still an
important static quenching at lower x0 (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 kqacrylamidein water and at x0¼ 30
Data obtained in water showed that this residue was almost
inaccessible to acrylamide (kq2acrylamide £ 107
M )1Æs)1, thus justifying the downward curvature obtained) Viscosity
decrease would account for the increase of kq2acrylamidewith
x0and 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 x0
On average, the rate constant for the dynamic quenching
by CCl4 does not depend on x0 (kCCl4 1.06 ·
109M )1Æs)1) This value is similar to that reported for NATA at x0¼ 15–22 ( 1.12 · 109
M )1Æs)1) [18], but lower than that for indole at x0¼ 5 ( 15.6 · 109
M )1Æs)1) and at x0¼ 22 ( 6.6 · 109
M )1Æs)1) [36] On the other hand, the fact that kqCCl4> kqacrylamide at all the x0 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 (kqacrylamide 1.1· 109M )1Æs)1at x0¼ 20) [19] or indole (kqacrylamide 1.2· 109M )1Æs)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 CCl4, 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 x0¼ 20–30, this does not seem to cause major alterations because neither s1nor s2 are very different in the presence of acrylamide or CCl4 bLG fluorescence quenching by the latter always produces downward curvatures The individual bimolecular rate constants obtained show that kq2CCl4 decreases drastically with x0, being almost null at x0¼ 30 This seems to point
to an inaccessibility to CCl4of 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 sfand the red shift obtained in DAS, as well as with the increase in
kq2acrylamidewith x0 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 x0, in agreement with the blue shift obtained in DAS Neverthe-less, both acrylamide and CCl4can 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 bI 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 x0 Near-UV CDspectra 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 x0 of 5 suggests an increase in the
Trang 10hydrophobicity 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 x0¼ 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 x0values (x0‡ 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 CCl4 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 CDspectrometer S.M Andrade thanks
FCT for the BPDgrant, no 18855.
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