Binding of Molten Globule-like Conformations to Lipid Bilayers STRUCTURE OF NATIVE AND PARTIALLY FOLDED (cid:97) -LACTALBUMIN BOUND TO MODEL MEMBRANES*

.O. The effect of membrane binding on the structure and stability of several conformers of (cid:97) -lactalbumin was studied by infrared spectroscopy, circular dichroism, and fluorescence spectroscopy. In solution, under experimental conditions where all conformers interact with negatively charged membranes, they show signifi-cant conformational differences. However, binding to negatively charged membranes, which causes consider-able changes in the structure of these conformers, leads to a remarkably similar protein conformation. The mem-brane-associated conformations are characterized by 1) a high helical content, greater than any of those found in solution, 2) a lack of stable tertiary structure, and 3) the disappearance of their thermotropic transition. These observations indicate that association with negatively charged membranes induces a conformational change within (cid:97) -lactalbumin to a flexible, molten globule-like state.

The effect of membrane binding on the structure and stability of several conformers of ␣-lactalbumin was studied by infrared spectroscopy, circular dichroism, and fluorescence spectroscopy. In solution, under experimental conditions where all conformers interact with negatively charged membranes, they show significant conformational differences. However, binding to negatively charged membranes, which causes considerable changes in the structure of these conformers, leads to a remarkably similar protein conformation. The membrane-associated conformations are characterized by 1) a high helical content, greater than any of those found in solution, 2) a lack of stable tertiary structure, and 3) the disappearance of their thermotropic transition. These observations indicate that association with negatively charged membranes induces a conformational change within ␣-lactalbumin to a flexible, molten globule-like state.
There is a large class of proteins (and peptides) which, although soluble in water, during the course of their action interact with plasma or intracellular membranes (1). The transition from the water-soluble to the membrane-associated state is believed to involve large structural changes (1)(2)(3)(4). The nature of this conformational change is a central issue to understand the problem of protein folding in membrane environments. In this context, the important role of lipid-protein interactions in protein translocation across biological membranes and protein insertion into lipid vesicles has been recognized both in vivo and in vitro (5)(6)(7). The use of phospholipid biosynthetic mutants in Escherichia coli has revealed that anionic lipids are essential for efficient membrane insertion and translocation of newly synthesized proteins through both the "Sec"-dependent and -independent pathways (8). Investigations on simple defined model membranes have shown that anionic lipids are required for membrane insertion and translocation of proteins (9 -11). Changes in protein structure, facilitated by interaction with negatively charged phospholipids, allows membrane insertion and subsequent translocation of proteins (8 -12). The experimental difficulties encountered in conformational studies of membrane-associated proteins have led to the present lack of knowledge on the structural transition involved in such association processes.
To further examine the effect of membrane binding on protein structure we focus in this study on the interaction of native and several folding intermediates of ␣-lactalbumin (␣LA) 1 with model membranes. ␣LA is able to interact with lipid bilayers (13,14) and provides a suitable model to characterize the structural transition involved in membrane association of water-soluble proteins, since its conformational properties have been extensively studied (15,16). Moreover, the possibility of trapping stable reduction intermediates of this protein offers the opportunity to follow the conformational changes associated with binding of different folding intermediates to membranes (16,17). As experimental techniques we use infrared (IR) spectroscopy, circular dichroism (CD), and fluorescence spectroscopy. The complementary information provided by these biophysical methods reveals that binding to negatively charged membranes induces a different structural change in the ␣LA conformers which leads to a very similar, membranebound flexible conformation.

Methods
Selective reduction and carboxyamidomethylation of the disulfide bond between cysteine 6 and 120 (3SS cam ) or all disulfides (R cam ) of ␣LA was achieved according to Shechter et al. (17). Protein concentration was determined spectrophotometrically as described previously (16). The freeze-dried proteins were dissolved in 20 mM Na 2 HPO 4 , 100 mM NaCl, pH 7.0, and the pH of the solution was adjusted to 4.5 with citric acid. In addition to the above composition, the holo-and apobuffers contained 1 mM Ca 2ϩ and 1 mM EDTA, respectively. Large unilamellar vesicles (LUV) were prepared following the extrusion method of Hope et al. (18), using polycarbonate membranes of a pore size of 0.1 m (Nucleopore, Inc., Pleasanton, CA). For preparation of samples used to study the conformation of ␣LA and its derivatives bound to lipid vesicles, LUV suspensions and protein solutions were mixed at a starting lipid to protein molar ratio of 300 in the above buffer. After adjusting the pH, as described above, the samples were incubated at room temperature for 30 min.
IR Spectroscopy-Prior to infrared measurements, the lipid-protein complexes were collected by centrifugation (120,000 ϫ g, 2 h). Exchange of water by D 2 O was carried out by submitting the samples to three centrifugation-resuspension cycles in D 2 O buffers of identical composition to that of the original H 2 O media. Samples, at a protein concentration of Ϸ0.7 mM, were assembled between two calcium fluoride windows separated by a 50-m-thick Teflon spacer. Infrared spectra were recorded at 25°C in a Nicolet 520 spectrometer. A total of 200 scans (sample) and 200 scans (background) were taken for each spectrum, using a shuttle device. Thermal studies were carried out by a step-heating method with Ϸ4°C steps, leaving the sample to stabilize for 5 min before recording the spectra. During data acquisition, temperature was monitored with a thermocouple in contact with the win-* This work was supported in part by funds from the University of the Basque Country (042.310-EC220/94). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
dows and was stable within 0.3°C. Spectra were analyzed in a personal computer where solvent subtraction, Fourier-self deconvolution, and band position determination were performed as described previously (19).
CD-Circular dichroism experiments were performed at 25°C in a Jasco-720 spectropolarimeter. Spectra in the near-UV region were measured in a 0.5-cm quartz cylindrical cuvette at a protein concentration of 17 M. Those in the far-UV region were acquired using protein concentration of 24 M and a 0.02-cm path length cell. Blanks (buffer with or without lipid vesicles) were routinely recorded and subtracted from the original spectra. Mean residue ellipticity, (degree cm 2 dmol Ϫ1 ), values were calculated from the formula ϭ ⑀/(10Cnl), where ⑀ is the ellipticity (millidegrees), l is the path length of the cuvette (cm), C is the protein concentration (mol/liter), and n is the number of amino acid residues in the protein (123 for ␣LA).
Fluorescence Spectroscopy-Fluorescence measurements were performed on a Perkin-Elmer MPF-66 spectrofluorometer using 1-cm path length quartz cuvettes. The emission spectra were recorded at 25°C between 300 and 400 nm using 3-and 5-nm bandwidths in the excitation and emission pathways, respectively, and excitation at 295 nm. Protein concentration was 1.1 M. Backgrounds of light scattering were determined with vesicles of identical composition and concentration, but in the absence of protein. When desired, the pH of the sample was adjusted back to neutrality by the addition of NaOH and incubated for 1 h at 25°C. Fig. 1A shows the infrared spectra, in the 1800 -1525-cm Ϫ1 region, of different conformers of ␣LA in solution and bound to liposomes made of equimolar amounts of 1,2-dioleoylphosphatidylglycerol and egg yolk lecithin. Although differences between the free and membrane-bound states are clearly observed in the original spectra, a more detailed analysis can be performed on their corresponding deconvoluted spectra (Fig. 1B). Under experimental conditions where all the conformers interact with negatively charged membranes (i.e. pH 4.5), their conformational properties in solution can be summarized as follows (Fig. 1B, broken traces). The infrared spectrum of the holoprotein in deuterated buffer displays well resolved bands at 1650 and 1640 cm Ϫ1 which have been assigned to ␣-helical segments and fully hydrated, extended chains connecting different types of secondary structure elements, respectively (20,21). Minor components appearing at 1630 and 1675 cm Ϫ1 indicate the presence of ␤-structures and turns, respectively. Reduction of the Cys 6 -Cys 120 disulfide bond, in the presence of calcium, does not induce major rearrangements in the secondary structure of ␣LA. On the contrary, cation removal from ␣LA, which locally unfolds the protein, promotes a conformational change characterized by the emergence of a component band at around 1624 cm Ϫ1 , which after partial reduction of the apoconformer, becomes dominant. This component has been related to intermolecular ␤-structures originated by the aggregation of unfolded proteins segments (22,23). Interestingly, the above mentioned differences between these conformers disappear when bound to negatively charged bilayers (Fig. 1B, solid traces). Instead they show similar infrared spectra in the amide I region, with the following band components: a major one located at 1653 cm Ϫ1 , characteristic of ␣-helical structures; minor and broad components at 1640 (not always well resolved) and 1631 cm Ϫ1 , which represent "connecting loops" and ␤-structures, respectively; and a weak absorption band at around 1680 cm Ϫ1 which contains contributions from turns and the high frequency component of the antiparallel ␤-structure. A comparison between the spectra of the free and membrane-bound conformations reveals that membrane binding significantly reduces the intensity of the 1640 cm Ϫ1 component and prevents the appearance of the band at 1624 cm Ϫ1 in the spectra of the holo-and apoconformers, respectively.

Infrared Spectroscopy-
Another important difference, clearly evidenced in Fig. 1B, concerns the bands located at 1576 and 1590 cm Ϫ1 which come from the antisymmetric COO Ϫ stretching mode of the aspartic and glutamic residues (24). The intensity of these bands is drastically reduced on membrane association, indicating that neutralization of most of the protein acidic residues is required for the interaction to occur. Unfortunately, the bands corresponding to the -COOH protein groups, which normally appear between 1700 and 1750 cm Ϫ1 , cannot be detected since they overlap with the strong absorption band of the phospholipid ester groups (1730 cm Ϫ1 ; Fig. 1A).
To gain insight into the structure of ␣LA bound to lipid vesicles, we have compared the thermal stability of the different conformers in solution and associated with lipid vesicles, under identical experimental conditions (i.e. pH 4.5). The temperature-induced changes observed in the amide I band of these conformers in solution are similar to those reported previously for soluble and membrane proteins (22,23); namely, a loss of regular secondary substructures and the emergence of band components at 1618 cm Ϫ1 and 1681 cm Ϫ1 , which have been assigned to hydrogen-bonded extended structures formed upon aggregation of thermally denatured proteins (data not shown). As a consequence of these conformational changes, the amide I band undergoes an irreversible cooperative broadening with increasing temperatures (Fig. 1). Therefore, protein thermal unfolding can be easily followed by plotting the amide I bandwidth at half-height versus temperature. The "melting curves" obtained for the apo-and holonative proteins indicate that both undergo thermotropic transitions in solution (Fig. 2). In contrast, there is no evidence of a cooperative transition for the same membrane-associated conformers. Instead, the width of their amide I band follows a small, progressive temperatureinduced increase similar to that obtained for their acid compact intermediate (Fig. 2). Identical results were obtained for the 3SS cam conformers (data not shown).
To further explore the molten globule-like thermal stability of the membrane-bound conformers, we have compared their IR spectra with those of their corresponding acid compact intermediates (Fig. 3). Note that the terms molten globule and compact intermediate are used interchangeably. The deconvoluted spectrum of the acid molten globule of native, holo-␣LA exhibits two main components at 1650 and 1639 cm Ϫ1 and a broad feature at around 1680 cm Ϫ1 which are assigned as above (Fig. 3, trace 1). The modest downward shift observed for the main components, as compared with their positions in solution and in the membrane-bound state, reflects an increased accessibility of the protein backbone to solvent exchange. The presence of a less intense residual amide II band at 1545 cm Ϫ1 , partially due to unexchanged N-H groups, in the spectrum of its acid molten globule state corroborates this interpretation (Figs. 1 and 3). The width of the major components is also broader in the acid compact intermediate, suggesting an increase in the conformational motion of the different secondary structure elements. Apart from these differences and in agreement with previous structural studies (25,26), the main features of native holo-␣LA secondary structure are essentially preserved in its acid molten globule state. A comparison of the IR spectra corresponding to the acid molten globule states of the different conformers shows that while those of the native holo-and apoproteins are very similar (Fig. 3, traces 1  and 2), the intensity at around 1631 cm Ϫ1 is slightly and clearly stronger in the spectra of holo-and apo-3SS cam , respectively (Fig. 3, traces 3 and 4). This difference could reflect a higher propensity to self-associate, at the relatively high protein concentration used for IR spectroscopy, of their acid compact intermediates. The fact that at 60°C only the spectra of the 3SS cam conformers display band components at 1618 and 1684 cm Ϫ1 , indicating protein aggregation, supports an increased exposure of hydrophobic regions in their acid states (data not shown). This finding fits in with the existence of molten globule states with different conformational flexibility, as recently proposed by Redfield et al. (27). Remarkably, none of the above mentioned differences are seen between the membrane-bound conformers.
CD Spectroscopy-The far-UV CD spectrum of holo-␣LA in solution is characteristic of a largely ␣-helical structure, in accordance with previous results (16,28), displaying strong minima at 208 and 222 nm (Fig. 4, trace 1). The ellipticity value at 222 nm of holo-3SS cam indicates that partial reduction of the Cys 6 -Cys 120 disulfide bond slightly reduces the helical content of the protein (Fig. 4, trace 3). Removal of calcium from the native and selectively reduced protein results in a further and substantial reduction of the CD signal at 222 nm, respectively (Fig. 4, traces 2 and 4). In contrast, the far-UV spectra of the membrane-bound conformers look alike, showing ellipticity values higher than any of those measured in solution (Fig. 4,  traces 5-8).
The CD spectrum in the near-UV region of native holo-␣LA in solution shows a pronounced negative ellipticity at around 270 nm and a positive peak at 293 nm which have been assigned to Tyr and Trp residues, respectively (Fig. 5, trace 1) (29). This spectrum is characteristic of a native-like conformation with a fixed orientation of the ␣LA's 12 aromatic residues, which are spread throughout the protein molecule (26). Inspection of the spectra corresponding to the other conformers indicates that the environment of the aromatic side chains becomes less rigid in the following order: holo-␣LA Ͼ holo-3SS cam Ͼ apo-␣LA Ͼ Ͼ apo-3SS cam (Fig. 5, traces 1-4). Interestingly, the spectra of the four conformers associated with negatively charged LUV are similar, lacking the fine structure characteristic of the native structure in solution (Fig. 5, traces 5-8). The general shape of these spectra resembles that of the "collapse" solution spectrum at acidic pH (15).
Fluorescence Spectroscopy-The intrinsic fluorescence of ␣LA reflects mainly the environments of its four tryptophan residues, which are spaced evenly throughout its sequence (30). We have determined the parameters max and relative fluorescence emission for ␣LA and its derivatives in solution and associated with negatively charged LUV. In solution, the spec-trum of the native, calcium-bound conformation is the most blue-shifted (Fig. 6A, trace 1), while the max of apo-3SS cam appears at the longest wavelength (Fig. 6B, trace 2). Calcium removal from native ␣LA (Fig. 6B, trace 1) and selective reduction of the holoprotein (Fig. 6A, trace 2) induces a red shift of Ϸ2 nm. The fluorescence intensity of these conformers is comparable, except for apo-3SS cam , which is higher (Table I). A red shift of the fluorescence maximum reflects a change of the Trp residues to a more polar environment, while the increase in intensity indicates the disappearance of tertiary interactions that quench the fluorescence in the native state. Binding of these conformers to negatively charged liposomes results in protein conformations with similar fluorescence properties: emission maxima at around 335 nm and fluorescence intensities ranging from 45 to 55 (Fig. 6, traces 3 and 4; Table I). Therefore, binding of holo-␣LA, apo-␣LA, and holo-3SS cam to lipid vesicles results in a red shift of the Trp fluorescence and a loosening of the protein tertiary interactions. On the contrary, association of apo-3SS cam and R cam induces a blue shift of the emission maxima, indicating transfer of at least part of the Trp residues into a more hydrophobic environment. The fluorescence intensity of the membrane-bound conformers increases to a value closer to that measured for R cam , which is not conformationally restricted by disulfide bonds, in solution (Table I). It should be mentioned that fluorescence intensity determinations in the presence of lipid vesicles are not as accurate as in solution, due to light scattering. After pH neutralization, the previously membrane-associated protein almost completely regains the fluorescence properties of the free conformer, indicating that the interaction is largely reversible (data not shown). A comparison between the fluorescence properties of the membrane-bound conformers and their acid compact intermediate states reveals that the max of the later are red-shifted, while their relative fluorescence values are significantly lower (Fig. 6, traces 5 and 6; Table I). Protonation of Aspartate and Glutamate Residues of the Protein-After an initial binding step, driven by electrostatic and/or hydrophobic interactions, the more acidic interfacial pH would mediate neutralization of acidic residues of ␣LA and thus facilitate subsequent membrane partial insertion of the protein. In particular, protonation of the side-chain carboxylates of three aspartates which participate in calcium binding, could cause cation removal from the protein and the conformational transition to a state competent in membrane binding. The ability of the different ␣LA conformers to partially insert into negatively charged membranes is confirmed first by leakage experiments that show a pH-dependent release of encapsulated contents from LUV in the presence of the protein 2 and second by an increased protection of the membrane-bound proteins against solvent exchange (see "Results"). Our data provide a direct experimental observation of this process that has been postulated for several proteins, specially bacterial toxins, which in vitro penetrate into membranes upon exposure to acidic pH (14,31).
Increase of the ␣-Helical Content of the Protein-The increased helicity of the membrane-associated conformers indicates that upon membrane insertion, relatively unstructured and/or flexible segments of the protein adopt a helical conformation (see Figs. 1 and 4). This is specially pronounced for the apoconformers, which have a less ordered structure and are prone to aggregation in solution. A similar folding behavior has been described for the interaction of apocytochrome c with negatively charged membranes (10,32). Apocytochrome c lacks ordered secondary structure in solution and becomes partly ␣-helical upon interacting with anionic lipids.
Loosening of the Protein Tertiary Structure-The lack of a stable tertiary structure in the membrane-bound states is evidenced by the collapse of the near-UV CD spectra (Fig. 5), the loss of the interactions responsible for the cooperative thermal unfolding (Fig. 2) and the increase of the relative fluorescence intensity (Fig. 6). This membrane-associated "unfoldase" activity becomes more efficient as the protein conformation in solution is more stable. Formation of less stable folding intermediates at the interface of anionic phospholipid vesicles has also been described for cytochrome c (22,33), human complement protein C9 (34), and the thermolytic fragment of colicin A (12).
Taken together, these results indicate that negatively charged membranes bind a reduced number of similar highly helical and flexible protein conformations, which share struc-tural properties with the acid molten globule state and the GroEL-bound protein (35). The fact that the conformations of the membrane-bound and acid compact intermediate states of ␣LA and its derivatives are not identical supports the view of the molten globule state not as a single conformation but rather as a family of more or less "ordered" conformers (27).
The membrane-bound, highly dynamic competent state, distinct from the native or aggregated state, could facilitate protein insertion into and/or protein translocation across membranes, assembly of membrane protein complexes, or proper interaction of the protein with the transport machinery for its subsequent translocation. This would be consistent with the observation that protein translocation requires partial unfolding of its mature part (36). Negatively charged membranes could play a complementary role to that exerted by the variety of proteins which are directly or indirectly involved in protein translocation (37), as it has been proposed recently for the mitochondrial protein import (36). a The pH of the above buffer was adjusted to 2.5. b Identical results were obtained at pH 5.0 for the membrane-bound form.