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: 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) 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 (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: 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. 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Andrade,. 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