Annexin A1 Interaction with a Zwitterionic Phospholipid Monolayer: A

We present the results of a fluorescence microscopy study of the interaction of .... This was placed on a vibration isolation table (Newport RS 3000, ...
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Annexin A1 Interaction with a Zwitterionic Phospholipid Monolayer: A Fluorescence Microscopy Study J. Alfredo Freites,† Shahla Ali,† Anja Rosengarth,‡ Hartmut Luecke,‡,§,| and Michael B. Dennin*,† Department of Physics and Astronomy and Institute for Surface and Interface Science, University of California, Irvine, California 92697-4575, and Department of Molecular Biology and Biochemistry, Department of Physiology and Biophysics, and Department of Information and Computer Science, University of California, Irvine, California 92697-3900 Received February 2, 2004. In Final Form: July 30, 2004 We present the results of a fluorescence microscopy study of the interaction of annexin A1 with dipalmitoylphosphatidylcholine (DPPC) monolayers as a function of the lipid monolayer phase and the pH of the aqueous subphase. We show that annexin A1-DPPC interaction depends strongly on the domain structure of the DPPC monolayer and only weakly on the subphase pH. Annexin A1 is found to be line active, with preferential adsorption at phase boundaries. Also, annexin A1 is found to form networks in the presence of a domain structure in the monolayer. Our results point toward an important contribution of the unique N-terminal domain to the organization of the protein at the interface.

Introduction The annexins are a multigene family of proteins characterized by their capacity of reversibly binding to anionic phospholipids in a Ca2+-dependent manner.1,2 Their common folding motif is a disk-shaped C-terminal core domain that contains four (eight in annexin A6) homologous repeats of five R-helices each, with the calcium-binding sites located on the convex side of the disk. In contrast, the N-terminal domain is variable and is thought to confer specific properties to each annexin. Although no unambiguous physiological role has been determined for this family, annexins have been associated with various membrane-related phenomena, including membrane organization, membrane trafficking, fusion, and ion-channel formation.1-3 Annexin A1 is characterized by an N-terminal domain that comprises its first 41 residues, with residues 2-17 forming an amphipathic R-helix. As is the case with other annexins with large N-terminal domains, annexin A1 exhibits membrane aggregation properties.2 Results from vesicle aggregation studies with annexin A1,4,5 annexin A1-A5 chimeras,6,7 and truncated annexin A1 mutants,8-10 * Author to whom correspondence should be addressed. † Department of Physics and Astronomy and Institute for Surface and Interface Science. ‡ Department of Molecular Biology and Biochemistry. § Department of Physiology and Biophysics. | Department of Information and Computer Science. (1) Gerke, V.; Moss, S. E. Physiol. Rev. 2002, 82, 331-371. (2) Gerke, V.; Moss, S. E. Biochim. Biophys. Acta 1997, 1357, 129154. (3) Creutz, C. E. Science 1992, 258, 924-931. (4) de la Fuente, M.; Parra, A. V. Biochemistry 1995, 34, 1039310399. (5) Bitto, E.; Li, M.; Tikhonov, A. M.; Schlossman, M. L.; Cho, W. Biochemistry 2000, 39, 13469-13477. (6) Hoekstra, D.; Buist-Arkema, R.; Klappe, K.; Reutelingsperger, C. P. M. Biochemistry 1993, 32, 14194-14202. (7) Andree, H. A. M.; Willems, G. M.; Hauptmann, R.; Maurer-Fogy, I.; Stuart, M. c. A.; Hermens, W. T.; Frederick, P. M.; Reutelingsperger, C. P. M. Biochemistry 1993, 32, 4634-4640. (8) Bitto, E.; Cho, W. Biochemistry 1998, 37, 10231-10237. (9) Bitto, E.; Cho, W. Biochemistry 1999, 38, 14094-14100. (10) Wang, W.; Creutz, C. E. Biochemistry 1994, 33, 275-282.

as well as structural studies,5,11,12 suggest that annexin A1 possesses two distinct membrane binding sites. One is the canonical calcium-dependent binding site to anionic phospholipids that is part of the core domain. The other one is calcium independent and nonspecific for anionic lipids. On the basis of high-resolution structural studies of annexin A1 in the presence12 and the absence11 of calcium, Rosengarth and Luecke have proposed a twostep model for the annexin A1-membrane interaction leading to membrane aggregation. Starting with the protein in its inactive form, the first step would be the calcium-mediated binding to anionic phospholipid headgroups of one membrane. This process involves a change in conformation of the C-terminal core that results in the previously buried N-terminal domain becoming solvent accessible. The second step would be the binding of a second membrane via hydrophobic interactions with the now exposed amphipathic N-terminal domain. To evaluate the hypothesis of a direct interaction between lipid membranes and annexin A1, Rosengarth et al.13 conducted a study of the interaction of annexin A1 with DPPC, DPPS, and DPPC-20 mol% DPPS monolayers. Tensiometry measurements were carried out both in the presence and in the absence of calcium ions. A monotonic increase in surface pressure as a function of time was considered as an indication of protein penetration into the phospholipid monolayer. It was shown in that study that annexin A1 is capable of penetrating phospholipid monolayers in the absence of calcium and in the absence of calcium and anionic phospholipids. The penetration process kinetics were found to be best described as first-order in the presence of calcium and DPPS and second-order in the absence of calcium. Similar experiments conducted with annexin A5, an annexin with a short N-terminal domain of only 16 amino acids, did not show any indication of penetration of this protein into any of the monolayers. Also, no penetration into the DPPC monolayer was found when a proteolytic fragment of (11) Rosengarth, A.; Gerke, V.; Luecke, H. J. Mol. Biol. 2001, 306, 489-498. (12) Rosengarth, A.; Luecke, H. J. Mol. Biol. 2003, 326, 1317-1325. (13) Rosengarth, A.; Wintergalen, A.; Galla, H.-J.; Hinz, H.-J.; Gerke, V. FEBS Lett. 1998, 438, 279-284.

10.1021/la049713b CCC: $27.50 © 2004 American Chemical Society Published on Web 11/19/2004

Annexin A1 Interaction with a Phospholipid Monolayer

annexin A1 lacking the N-terminal domain was tested. These results demonstrated the possible occurrence of a calcium-independent hydrophobic interaction between the annexin A1 N-terminal domain and phospholipid membranes. The emerging model of the cell membrane14 pictures an inhomogeneous medium that is organized into well-defined domains. The domain structure is dependent upon local composition and ordering. In this context, an alternative point of view to conventional binding experiments is to consider the influence of spatial organization on membrane-protein interaction. In the case of calcium-mediated annexin interaction with model membranes, microscopy studies of lipid monolayer systems at the micrometer15,16 and sub-micrometer17 scales have revealed the formation of microstructural domains that are specific, in distribution and morphology, to the calcium-mediated binding event. Such behavior is manifested as formation of condensed domains in single component fluid monolayers16 or formation of anionic lipid-rich domains in mixed monolayers.17 A similar characterization has not been reported for the calcium-independent annexin A1 membrane interaction. In this work, fluorescence microscopy results are reported on the evolution of the interaction process of annexin A1 with dipalmitoylphosphatidylcholine (DPPC) monolayers as a function of the monolayer phase state and the composition of the aqueous subphase. DPPC is zwitterionic, and it has already been established that a calcium-independent annexin A1 interaction with DPPC exists.13 Central to this work is the possibility of testing the protein behavior in a heterogeneous medium by exploiting the well-understood phase behavior of DPPC monolayers. Both the zwitterionic nature of DPPC and its domain structure are potentially relevant to the identified secondary protein membrane binding site. The phase behavior of the annexin A1-DPPC monolayer system is found to be consistent with a mean-field theory proposed by Netz et al.18 Annexin A1 is line active, and its phase behavior suggests that, upon adsorption, it undergoes a form of self-assembly. Although DPPC is not the best representative of a cell membrane phospholipid, it serves as an excellent model system for probing the calcium-independent membrane binding. The results presented here highlight the importance of the nature of the annexin A1 secondary binding site and the heterogeneous nature of the monolayer organization. This provides a motivation for more-direct studies that better resemble biological conditions. Experimental Dipalmitoylphosphatidylcholine (DPPC) was purchased from Avanti Polar Lipids (Alabaster, AL). The monolayer fluorescent probe employed was chain-labeled nitrobenzoxadiazole phosphatidylcholine (NBD-PC) from Molecular Probes (Eugene, OR). Both lipids were specified to be more than 99% pure and were used as received. Spreading solutions contained less than 0.7 mol% (with respect to total lipid) of fluorescent probe and were prepared with chloroform (HPLC grade, EM Science, Gibbstown, NJ) in concentrations of 1.4-1.5 g/L. Monolayer subphase buffers contained 50 mM MES/NaOH, 100 mM NaCl, pH 6.0 adjusted (14) Edidin, M. Nat. Rev. Mol. Cell Biol. 2003, 4, 414-418. (15) Koppenol, S.; Tsao, F. H. C.; Yu, H.; Zografi, G. Biochim. Biophys. Acta 1998, 1369, 221-232. (16) Wu, F.; Gericke, A.; Flach, C. R.; Mealy, T. R.; Seaton, B. A.; Mendelsohn, R. Biophys. J. 1998, 74, 3273-3281. (17) Janshoff, A.; Ross, M.; Gerke, V.; Steinem, C. ChemBioChem 2001, 2, 587-590. (18) Netz, R. R.; Andelman, D.; Orland, H. J. Phys. II 1996, 6, 10231047.

Langmuir, Vol. 20, No. 26, 2004 11675 by NaOH, or 50 mM Tris/HCl, 100 mM NaCl, pH 7.4 adjusted by HCl. Additionally, buffers contained either 1 mM ethyleneglycol bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) or 1 mM CaCl2. The water used throughout all the experiments was filtered using a Milli-Q device (Millipore) and had a resistivity greater than 18 MΩ. Expression and purification of full-length porcine annexin A1 was performed according to Rosengarth et al.19 Protein fluorescent labeling was performed by incubating a mixture of 1-5 mg of protein dialyzed against 0.1 M sodium carbonate/bicarbonate (pH 9.0) and 1 mg of Texas Red Sulfonyl Chloride (Pierce, Rockford, IL) on ice for 1 h. The mixture was then dialyzed against 20 mM sodium phosphate (pH 7.5), 150 mM NaCl (three times), and finally, against the buffer used in the monolayer experiment. All the experiments were performed in a NIMA Technologies (Coventry, UK) 601M Langmuir trough equipped with a PS-4 pressure sensor, and controlled by a desktop computer. Trough temperature was controlled at 20.0 ( 0.5 C. The trough and tensiometer were mounted on a Olympus BX-60 (Olympus America, NY) epifluorescence microscope. This was placed on a vibration isolation table (Newport RS 3000, Irvine, CA). Microscopy images were captured with a monochrome CCD camera (Cohu 5515, San Diego, CA) connected to a video monitor and a videocassette recorder. Selected video frames were digitized to 640 pixels × 480 pixels 8-bit gray scale images with a desktop computer using a frame grabber, cropped to the desired size and, except for Figure 2a and b, presented without further processing. Quantitative image analysis was performed using ImageJ (National Institute of Health, Washington, DC). The phospholipid solution was spread with a Hamilton microsyringe to form a monolayer at the air/buffer interface. After the spreading, 30 min were allowed for solvent evaporation and overall system relaxation. Isotherm experiments were conducted with a barrier speed of 4.0 Å2/(molecule min). Protein/lipid monolayer imaging experiments were conducted at constant surface pressure. Initially, the monolayer was compressed to a specific trough area, and then the motion control of the trough barriers was set to maintain a constant surface pressure. Once the surface pressure and the trough area had reached stationary values, the annexin A1 solution was injected underneath the monolayer at the bottom of the trough, using a bent long-needle Hamilton syringe. This procedure was conducted without perturbing the monolayer. All the microscopy results on protein/lipid monolayer systems presented were obtained during dual-label experiments, in which both the protein and the phospholipid monolayer contained a fluorescent probe. Imaging was carried out with two different sets of excitation-observation fluorescent cubes that were manually switched during the experiment. For NBD-PC imaging, an Olympus U-MWB fluorescent cube (exictation filter, wideband blue 450-480 nm; long-pass barrier filter, 515 nm) was employed, herein identified as wide-band filter. Texas Red imaging was performed with a band-pass filter set (Omega O-5732; excitation filter, 560 ( 20 nm; emission filter, 635 ( 27.5 nm), herein identified as Texas Red filter. DPPC monolayer imaging was conducted during isotherm experiments (i.e., under compression). Experiments on protein adsorption to the bare air/buffer interface were conducted at constant trough area by injecting the annexin A1 solution at the bottom of the trough.

Results This section is organized as follows. We will first present the phase behavior and kinetics of the DPPC monolayer and annexin A1 separately. We will then focus on the interaction between the annexin A1 and the liquid expanded-liquid condensed (LE-LC) coexistence phase of DPPC. This is the central result of the paper, and we will report on the results for two different pH values. To support our interpretation of the LE-LC results, we will discuss separately the interaction between annexin A1 and the pure LC phase of the monolayer and the (19) Rosengarth, A.; Rosgen, J.; Hinz, H.-J.; Gerke, V. J. Mol. Biol. 1999, 288, 1013-1025.

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interaction between annexin A1 and the pure LE phase of the DPPC monolayer. Surface Activity of Individual Components. The results of the phase behavior of DPPC-0.7 mol% NBDPC at 20 °C are shown in Figure 1. Both the Π vs A isotherm and the corresponding fluorescence micrographs are in good agreement with those reported in the literature for DPPC monolayers.20-23 As a function of surface pressure, DPPC monolayers exhibit three phases at room temperature: a low-density gaslike phase (G), a liquid isotropic phase, known as liquid expanded (LE), and a hexatic phase with a tilted director, known as liquid condensed (LC). NBD-PC localizes preferentially in the LE phase, as it is excluded from the LC phase due to the acyl chain ordering. Therefore, it only produces a significant signal in the LE phase. The onset of the monophasic LE region occurs at values of specific area in the range of 90-100 Å2/molecule, as indicated by the appearance of a uniformly bright, featureless image under the fluorescent filter. After further compression, a first-order transition occurs from LE to LC, between 4.5 and 5.0 mN/m, as indicated by the isotherm plateau and the observation of dark domains on the fluorescence microscopy image (see Figure 1a). These curved, multilobular LC domains are characteristic of monolayers of enantiomeric phospho-

lipids.24 In phospholipid monolayers, for a given cycle of compression and expansion, the nucleation and growth of the LC phase depend on a series of factors: the composition of the subphase dominates the nucleation and early stages of growth, whereas the subsequent domain shape evolution and growth are primarily determined by the compression rate history.22,23,25,26 The LC monophasic region appears in fluorescence microscopy images with the LC domain boundaries flattened and in contact with each other (see Figure 1b). The fluorescent probe is segregated to the interboundary regions. As the pressure increases, the fluorescent probe is increasingly excluded from the air/ water interface. The overall microstructure formation of phospholipid monolayers in a condensed biphasic state and the morphology of LC domains has been successfully described by a simple phenomenological model,27 where mesoscopic phenomena emerge from the interplay between electrostatic, interfacial, and chiral effects. Domain arrangement and the lack of secondary nucleation events are considered to be due to a long-range repulsion between collinear effective electric dipoles of neighboring LC domains. These electric dipoles represent the net electrostatic effect arising from the ordering of the lipid acyl chains. Domain morphology and growth are understood in terms of a balance between the line tension associated with the LELC boundaries, which favors compact shapes of low perimeter-to-area ratio, and the long-range dipolar repulsion within LC domains, which favors more-extended morphologies. It has been shown24 that in the case of enantiomeric lipids both aspects are governed by molecular chirality. The surface-active character of annexin A1 was first reported by Rosengarth et al.13 Here, the tensiometry characterization is complemented with fluorescence microscopy. Figure 2 shows the evolution of the surface pressure after injection of annexin A1 into pH 6.0 buffer solution. A nonzero surface pressure is observed after a time lag of 1500 s. After a transient period, the surface pressure begins to plateau around 10 mN/m 6000 s after injection. The presence of protein at the surface was first observed 200 s after injection. During the induction period and during the first half of the transient period, fluorescence microscopy reveals extended condensed phase protein domains arranged in an inhomogeneous frothlike pattern, as shown in Figure 2a and b. Eventually, these domains coalesce, yielding a featureless, bright image (see Figure 2c). The adsorption of proteins to fluid interfaces reflects the amphipathic nature of the polypeptide chain. However, in contrast to simple amphiphilic molecules, the mechanism of adsorption is determined not only by the intrinsic gradient of chemical potential but also by a complex interrelation between entropic (conformational), hydrophobic, electrostatic, and van der Waals interactions. The surface of the annexin C-terminal core is mostly hydrophilic. As a consequence, adsorption to the fluid interface implies conformational changes that expose hydrophobic segments to the nonpolar medium. Several studies by neutron reflectivity on the adsorption to the air/water interface of rigid28,29 and nonrigid30 globular proteins have

(20) Albrecht, O.; Gruler, H.; Sackmann, E. J. Phys. 1978, 39, 301313. (21) Kane, S. A.; Compton, M.; Wilder, N. Langmuir 2000, 16, 84478455. (22) McConlogue, C. W.; Vanderlick, T. K. Langmuir 1997, 13, 71587164. (23) Klopfer, K. J.; Vanderlick, T. K. J. Colloid Interface Sci. 1996, 182, 220-229.

(24) Kruger, P.; Losche, M. Phys. Rev. E 2000, 62, 7031-7043. (25) Mohwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441-476. (26) Mohwald, H. Phospholipid Monolayers. In Handbook of Biological Physics; Lipowsky, R., Sackmann, E., Eds.; Elsevier Science: Amsterdam, 1995; Vol. 1. (27) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171-195. (28) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J.; Webster, J. J. Chem. Soc., Faraday Trans. 1998, 94, 3279-3287.

Figure 1. Surface pressure vs specific area isotherm for DPPC on MES/NaOH buffer at 20 °C. Corresponding fluorescence micrographs: (a)Π ) 4.7 mN/m, (b)Π ) 9.1 mN/m. The scale bar is 20 µm. Imaging performed with a wide-band blue excitation filter and a long-pass green emission filter, sensitive to both NBD-PC and Texas Red (herein identified as wideband filter). In the micrographs, the bright regions correspond to the monolayer LE phase and the dark domains to the LC phase.

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Figure 2. Surface pressure evolution after the injection of annexin A1 in MES/NaOH buffer to a final concentration of 24 nM. Corresponding fluorescence micrographs: times after injection (a) 1120, (b) 1330, and (c) 2800 s. The scale bar is 20 µm. Imaging performed with a narrow band-filter selective for Texas Red (herein identified as Texas red filter). The bright regions in the micrographs correspond to annexin A1 adsorbed at the air/water interface.

revealed that most of the conformational changes associated with adsorption tend to conserve secondary structure. This is achieved by the promotion of specific forms of aggregation or assembly that are consistent with the tertiary structure in solution.28,29 A recent fluorescence microscopy study of lysozyme,31 consistent with this model, presents a similar phase behavior to the one reported here for annexin A1. Our fluorescence microscopy results confirm the formation of a protein condensed phase accompanying the surface tension relaxation, suggesting that a similar behavior can be expected for annexin A1. Annexin A1 interaction with DPPC Biphasic Monolayers. To study the interaction between annexin A1 and the DPPC monolayer, fluorescence microscopy was conducted while the monolayer was held at stationary values of surface pressure, as shown in Figure 3. Constant surface pressure experiments, as opposed to constant area, present the protein with a phospholipid monolayer that has a stationary phase distribution. Because the LC phase of DPPC is metastable,26 a monolayer held at fixed specific area experiences a surface pressure relaxation and accompanied partial dissolution of LC domains. The extent and specific evolution of this relaxation process will depend on the specific compression/expansion history.23 In consequence, to achieve the desired nearly constant phase distribution, the magnitude of the surface pressure has to be maintained stationary by continuously adjusting the trough area. (29) Lu, J. R.; Su, T. J.; Howlin, B. J. J. Phys. Chem. B 1999, 103, 5903-5909. (30) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. J. Chem. Soc., Faraday Trans. 1995, 91, 2847. (31) Erickson, J. S.; Sundaram, S.; Stebe, K. J. Langmuir 2000, 16, 5072-5078.

Figure 3 shows the evolution of the surface pressure and trough area throughout a complete experiment. Region I corresponds to the initial compression to a specific area in the LE-LC biphasic region after which the trough barriers are controlled so as to keep a constant surface pressure. The experiments were performed at an LC area fraction of 33 ( 1%. Region II corresponds to a transient stabilization period. The asterisk marks the time of injection of annexin A1. The first indication of annexin A1 at the air/buffer interface occurs about 800 s after injection. During the period identified as Region III, the presence of annexin A1 at the air/buffer interface is observed in isolated locations. These small domains have no measurable impact on the trough area. Only after a uniform distribution of small protein domains at the LELC boundaries exists is a monotonic trough area increase observed (region IV in Figure 3). This monotonic increase in the trough area, with a corresponding constant surface pressure, can only be explained as a displacement of the phospholipid by the adsorbed protein due to the penetration of annexin A1 into the monolayer. Figure 4 shows a sequence of micrographs obtained with the Texas Red filter corresponding to the interaction process of the protein with the phospholipid monolayer at pH 6.0 containing EGTA in the subphase. The initial nucleation and uniform distribution of protein domains at the LE-LC boundaries is shown in Figure 4a (see also Figure 1 in Supporting Information). The subsequent growth by coalescence of the initial domains consists of a wetting of the LE-LC boundary. Only after complete coverage of the LE-LC boundaries are protein domains observed in the LE phase (see Figure 4b). Occasionally, what appear to be small protein domains can be observed associated to the location of the monolayer LC domains

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Figure 3. Surface pressure and trough area evolution for the system DPPC monolayer-annexin A1 at pH 6.0. The asterisk indicates the time of injection of annexin A1 to a final concentration of 24 nM. Points a-d correspond to the fluorescence microscopy images shown in Figure 4. See text for explanation of the regions labeled I-IV.

Figure 4. Fluorescence micrographs for the system DPPC monolayer-annexin A1 at pH 6.0 and with the phospholipid monolayer in a biphasic state (LC area fraction is 33%). Imaging performed with the Texas red filter (see Figure 2 and text for more details on the filter set). The scale bar is 20 µm. Times after protein injection are: (a) 1604, (b) 1731, (c) 1917, and (d) 2203 s. The white regions correspond to adsorbed annexin A1.

(see Figure 2 in Supporting Information). The protein domains at the boundaries grow toward the LE phase keeping a circular interface with it. Micrographs (Figure 5) taken with the wide-band filter during this stage, revealing the protein domains in light gray (red in the visual observation), confirm the growth of the protein domains and the wetting of the LE-LC boundary. During the initial stages of adsorption, there are no apparent changes in shape or size of the LC domains,

suggesting that annexin A1 has displaced the LE phospholipid phase without compressing it to form a new LC phase, hence, the observed increase in trough area. The next step of the interaction process is the coalescence of protein domains located on different LE-LC boundaries. This results in the formation of a continuous protein network with the LC domains as nodes (Figures 4c and 5b and c). During this process, an increase of LC domain size of 35% on average was also observed. As a consequence

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Figure 5. Fluorescence micrographs for the system DPPC monolayer-annexin A1 at pH 6.0 and with the phospholipid monolayer in a biphasic state (LC area fraction is 33%). Imaging was performed with the wide-band filter (see Figure 1 and text for more details on filter set). White and gray regions appeared green and red, respectively, in the visual observation. The scale bar is 20 µm. Times after protein injection are: (a) 1626, (b) 1802, (c) 1915, and (d) 2441 s. White or light gray regions correspond to the monolayer LE phase, gray regions correspond to adsorbed annexin A1, and dark domains correspond to the monolayer LC phase.

of this process, the LE regions are fully confined and the protein domains appear to occupy most of the LE area. The formation of this network and the nucleation and growth of protein domains in the LE phase region occur independently. The completion of the interdomain protein network is followed by a loss of curvature and an overall change in shape of the LC domains. It is worth noting that this shape transition coincides with the formation of the protein network and not with the complete wetting of the LC domains. Notice also that, during the LC domain shape change process, the fastest average rate of trough area increase is about 56% of the slowest compression rate (that of region I in Figure 3) usually employed for the generation of surface pressure vs area isotherms. As no morphology changes are observed due to any motion caused by compression, it is reasonable to expect that the LC domain shape transition and other phenomena associated with the protein-monolayer interaction are not the result of the slower barrier motion necessary to maintain a constant surface pressure. In the final stage of adsorption, the fluorescence signal originating from the annexin A1 essentially fills the viewing field (Figure 4d). However, comparison with Figure 5d confirms that the LE phase is still present in the monolayer. Additionally, after the size increase observed in the previous stage, the size of the LC domains remains unchanged within the experimental uncertainty (LC average domain size relative uncertainty per image is between 15% and 16%) until the end of the protein adsorption process. These facts suggest that the features revealed by fluorescent microscopy during the late stages of adsorption do not reflect a process that occurs entirely

at the air/buffer interface but immediately underneath. Also, contributing to the features in the Texas Red images is the larger fluorescence intensity of Texas Red. This tends to amplify the size of the protein domains, such as the ones observed in Figure 4. This effect was verified by contrasting these images with those taken with the wideband filters on the same areas. A pH of 6.0 for the aqueous subphase was selected on the basis of previously reported results,19 indicating that annexin A1 shows it highest thermodynamic stability between pH 5.0 and 6.0. To investigate a potential dependence of the protein surface activity on pH, experiments were also performed at pH 7.4 with a similar LC area fraction. Taking the rate of change in trough area as a qualitative measure of kinetics, the comparison of the graph in Figure 6 with Figure 3 reveals similar penetration kinetics at pH 6.0 and 7.4, once full coverage of LC domains occurs. Fluorescence microscopy revealed mostly similar microstructural features and overall interaction processes between annexin A1 and the DPPC monolayer for pH 7.4, as observed in the pH 6.0 experiments (see Figure 6). Comparison of the graph in Figure 6 with Figure 3 reveals that the length of time spent in region III, the initial adsorption of the protein into the monolayer, differs by approximately 3000 s, with it being longer for the pH 7.4 system. However, consistent with the pH 6.0 systems, the nonzero rate of change of area (region IV) for pH 7.4 occurs when a uniform distribution of small protein domains covers the LE-LC boundaries (see Figure 6a and c). At this point, the rate of trough area expansion is very similar at both values of pH. It is also noticeable that the time interval between the first protein adsorption

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Figure 6. Surface pressure and trough area evolution for the system DPPC monolayer-annexin A1 at pH 7.4 with the phospholipid monolayer in a biphasic state (LC area fraction is 33%). The asterisk indicates the time of injection of annexin A1 to a final concentration of 24 nM. Corresponding fluorescence micrographs: times after injection (a) 3212, (b) 4280, (c) 3087, and (d) 3917 s. (a-b) White regions correspond to adsorbed annexin A1, imaging performed with the Texas red filter. (c-d) White or light gray regions correspond to the monolayer LE phase, gray regions correspond to adsorbed annexin A1, and dark domains correspond to the monolayer LC phase, imaging performed with the wide-band filter. The scale bar is 20 µm.

events and the full coverage of LE-LC domain boundaries in all of these experiments is within the same time scale (between 1500 and 2000 s) as the onset of surface pressure increase for annexin A1 at the bare air/water interface. For both systems at pH 6.0 and 7.4, the microstructural organization consists of a network of protein domains. Also, at the late stages, the LC domains undergo a shape transition to long, skinny domains (see Figure 6d). The only real difference is the magnitude and extent of the domains. For pH 7.4, the growth by coalescence of protein domains at the LE-LC boundaries does not progress to a full extent before the experiment is terminated. Similarly, in contrast to the observations at pH 6.0, the interdomain network formed is not uniformly extended (Figure 6b and d). Therefore, the LE regions are not confined by the protein network (Figure 6b). Additionally, as a consequence of the protein domain formation, it appears that the distribution of LC domains becomes clustered or at least less uniform. Given that the chosen values of pH are on opposite sides of the protein calculated isoelectric point, these results

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suggest that there is not a strong pH dependence for the protein-monolayer interaction. For both pH values, the initial and final trough areas are approximately equal. Therefore, the observed difference in protein coverage in the late stages may reflect the amount of protein aggregated below the interface and may not be related to the monolayer-protein interaction. Moreover, differences in LC domain size and morphology were observed for monolayers spread over the two buffer solutions in the absence of protein. Therefore, the possibility that the observed differences in overall microstructural organization between the two systems are due to constitutional differences introduced by the different buffer solutions and intrinsic to the phospholipid monolayer themselves cannot be discarded. Further experiments are needed to completely understand the late-time difference in network coverage. Experiments were conducted substituting EGTA in the subphase with CaCl2 at both pH 6.0 and 7.4 (results not shown). No substantial or systematic differences were found in either microstructural features or overall kinetics. In contrast to the canonical behavior of annexins in the presence of calcium ions and anionic phospholipids, the results presented so far suggest that the annexin A1DPPC monolayer interaction is predominantly nonelectrostatic. The adsorption of annexin A1 to the phospholipid monolayer is likely to be accompanied by a change in protein conformation in a manner consistent with the adsorption behavior of the protein at the bare air/buffer interface. In both cases, specific domains in the protein chain are more likely to be attracted to the lipid interface through hydrophobic interactions. The formation of a condensed phase in discrete domains by the adsorbed protein suggests an aggregation processes regulated by this change in conformation at the surface. The presence of the LE-LC domain boundaries appears to modulate both the nucleation and the growth of the protein domains, as indicated by the occurrence of wetting and networking. One consequence of this modulated growth could be differences in protein chain packing between the domains at the boundary and those that nucleate in the interior of the LE phase. This would explain why the first coalescence occurs only between domains nucleated at the boundaries since it is the process that leads to the network formation. The difference in protein domain morphology between the present case and the adsorption to the bare aqueous interface suggests that the protein domains are insoluble in the LE phase since the formation of circular domain boundaries minimizes the contact between the protein and the lipid LE phase. This idea is reinforced by the fact that, even though the LE regions are being compressed due to the penetration of the protein into the monolayer, no secondary nucleation of LC domains is observed in the interior of the LE phase. Any additional contribution to the state of stress arising from the fine compressibility of the LE phase is being relaxed by an increase in the trough area. Immiscibility between the protein domains and the LE phase could also explain why those protein domains nucleated in the interior of LE phase seem to participate in the coalescence process only at a late stage when their surface coverage is high and/or the network of domains nucleated at the LE-LC boundaries is sufficiently thick. Annexin A1 Interaction with Monophasic Monolayers. To further confirm the previous assessment, the specific interaction of annexin A1 with each phospholipid monolayer phase was studied through constant-pressure experiments conducted above the onset of the LE-LCto-LC transition at 9.0 mN/m and below the onset of the LE-to-LE-LC at 3.5 mN/m. These results confirm the

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preference for protein adsorption at domain boundaries (the line activity of annexin A1) and the insolubility of annexin A1 in the LE phase. At 9.0 mN/m, the monolayer consists of fully grown LC domains with flattened boundaries which are in contact with all their neighbors. In other words, the LC monophasic region is characterized by a granular texture. As indicated before, achieving a steady state starting from a monophasic LC state involves some relaxation. In this case, the relaxation process introduces small isolated domains of LE phase (see Figure 3 in Supporting Information). Notice, however, that this microstructure is not the same as the one for monolayers in the biphasic region. After injection of annexin A1, an image with the Texas red filter shows that these boundaries are fully decorated with small protein domains (see Figure 4 in Supporting Information) after 1000 s. As in the case of the biphasic monolayer, the protein domains at the LCLE boundaries grow by coalescence forming a continuous interphase among the LC domains (see Figure 7a and c). Subsequently, the LC domains become completely isolated from each other and the protein layer thickens. At the same time, the LC domains are elongated until both lipid and protein form a striped pattern (see Figure 7b and d). Penetration kinetics are substantially slower than for the biphasic experiments (see Figure 7). The striped microstructural pattern was maintained with minimum change until the experiments were stopped. Similar experiments performed with labeled protein but without fluorescent label in the monolayer (results not shown, see Figure 5 in Supporting Information for a comparison between experiments with and without fluorophore in the monolayer) produced a consistent behavior for the adsorption and domain formation of the protein. These results confirm the preferential adsorption at monolayer domain boundaries and the line-active character of annexin A1. The lack of extended regions of LE phase confirms that the line activity is a distinct characteristic of the quasi two-dimensional protein domains. The complete alteration of the DPPC monolayer microstructure can only be achieved through a change of the electric dipole field distribution over and across the amphiphilic monolayer. This suggests again specificity in conformation of the protein domains either at a mesoscopic level or at the level of chain conformation. At 3.5 mN/m, the protein penetrates the LE monolayer forming circular domains (see Figures 8a and c), and ultimately, an emulsion-like pattern forms between the annexin A1 and the LE phase. The initial protein domains nucleate uniformly in regions of about 5 µm and grow by coalescence. This should be contrasted, particularly, with the morphologies observed during the early stages of the adsorption of annexin A1 to the bare air/water interface (see Figure 2a and b). No apparent condensation of the LE phase to LC phase was observed. At the end of the experiment, individual protein domains were on the order of 70-80 µm in diameter (see Figure 8b and d). At that time, these large domains collapse onto each other to form larger extended regions. The formation of such large domains and extended regions is consistent with the behavior observed for the adsorption to the bare aqueous interface. These observations are consistent with the results obtained with the biphasic monolayers and confirm the immiscibility of the protein domains in the phospholipid LE phase. No domain networking was observed on the LE phase in the monophasic experiment, confirming that the growth of protein domains in the biphasic monolayer system is modulated by the presence of the LE-LC

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Figure 7. Surface pressure and trough area evolution for the system DPPC monolayer-annexin A1 at pH 6.0. The protein was injected, while the monolayer was held at 9.0 mN/m with the phospholipid monolayer in the LC phase. The asterisk indicates the time of injection to a final concentration of 24 nM. Corresponding fluorescence micrographs: times after injection (a) 3683, (b) 4733, (c) 3472, and (d) 4716 s. (a-b) White or light gray regions correspond to adsorbed annexin A1, imaging performed with the Texas red filter. (c-d) White or light gray regions correspond to the monolayer LE phase, gray regions correspond to adsorbed annexin A1, and dark domains correspond to the monolayer LC phase, imaging performed with the wide-band filter. The scale bar is 20 µm.

boundaries. In the same way, it can be asserted that nucleation of protein domains in the interior of the LE phase is an independent state from the preferential adsorption to the LE-LC boundaries. (This will be discussed in more detail in the next section in the context of the model by Netz et al.18) Additionally, as was observed in the biphasic monolayer systems, the protein domains that nucleate in the LE phase reach a critical size before starting coalescence. This behavior is consistent with a specific pattern of aggregation for the adsorbed protein at the scale of tertiary structure. Discussion We have presented fluorescence microscopy results on the interaction of annexin A1 with DPPC monolayers as a function of the lipid monolayer phase state. The central features are that annexin A1 preferentially adsorbs to LC-LE domain boundaries and that it ultimately induces a shape change of the LC domains. Both of these results indicate that the annexin A1 is line active, relative to

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Figure 8. Surface pressure and trough area evolution for the system DPPC monolayer-annexin A1 at pH 6.0. The protein was injected while the monolayer was held at 3.5 mN/m with the phospholipid monolayer in the LE phase. The asterisk indicates the time of injection to a final concentration of 24 nM. Corresponding fluorescence micrographs: times after injection (a) 748, (b) 5113, (c) 795, and (d) 5160 s. (a-b) White domains correspond to adsorbed annexin A1, imaging performed with the Texas red filter. (c-d) White or light gray regions correspond to the monolayer LE phase and gray domains correspond to adsorbed annexin A1, imaging performed with the wide-band filter. The scale bar is 20 µm.

LC-LE domains. The adsorption in the presence of LCLE domains results in the formation of a protein network, something that does not occur for adsorption in the absence of the monolayer or in the LE phase. This suggests that two different adsorbed states exist in the monolayer. Finally, some protein fluorescent signal was also occasionally observed at the location of the LC domains. This last feature appeared to originate from underneath the monolayer since it could be observed even below the monolayer focal plane. Also, the late-time protein images suggest the existence of protein aggregates below the air/ water interface. These findings suggest that it is necessary to consider the possibility that the complexity of the observed adsorption process and surface behavior could imply that the protein domains are only partially at the surface and that the phenomena of conformational change and aggregation have a multilayer character. Preferential adsorption to the LE-LC domain boundaries of phospholipid monolayers has been reported for other proteins that present interfacial activity, such as

Freites et al.

concanavalin A,25 bacterial surface layer proteins,32 fibronectin,33 and surfactant protein A.34 Netz, Andelman, and Orland18 have developed a Flory-Huggins type meanfield theory that is able to account for this phenomenon. According to this model, the preferential adsorption of a protein to LE-LC domain boundaries is an entropic effect due to the constitutional differences between the adsorbed protein phase and the phospholipid monolayer. The model predicts a reduction of the line tension associated with the LE-LC boundary due to the protein adsorption. This is consistent with the observed wetting of the LC phase by the annexin A1 domains located at the LE-LC. The change in shape of the LC domains can also be explained in this context. The full coverage of the LE-LC boundary by coalesced protein domains could screen the dipoledipole interaction between neighboring LC domains. At the same time, a reduction of the line tension allows the LC domain morphology to be dominated by the repulsive dipole-dipole interaction within the domains. It has been predicted35,36 that under these circumstances the LC domains would assume elongated shapes, as was observed during the last stages of the annexin A1-DPPC monolayer interaction process. The screening of the LC interdomain dipolar interaction by the adsorbed protein could also explain the clustering of LC domains observed at pH 7.4. The theory by Netz et al.18 also accounts for the observed nucleation of new protein domains in the LE phase, as an event that could occur due to a change in the protein chemical potential at the surface. This is consistent with the reported observation that the adsorption in the interior of the LE phase occurs after the protein domains have completely covered the LE-LC boundaries. Consequently, nucleation of new protein domains in the interior of the LE phase could be attributed to a critical increase of protein surface concentration. The model of Netz et al. is based only on pairwise interactions between the system components, which justifies the assumption that hydrophobic interactions are dominant in the annexin A1-DPPC monolayer system. These ideas can be connected to the three different penetration kinetics reported by Rosengarth et al.13 for annexin A1 phospholipid monolayer systems: first-order kinetics for the system containing both calcium ions and DPPS, second-order kinetics for the systems containing DPPS in the absence of calcium ions, and slower secondorder kinetics for the DPPC monolayer system. It can be speculated that the occurrence of the first-order kinetics characterizes unambiguously the canonical electrostatic interaction between annexins and anionic phospholipids. On the other hand, the mixed monolayer DPPC-DPPS tested in that study has been reported to present DPPSrich domains in the absence of chelator agents,17,37 it is then possible that the second-order kinetics correspond to the kind of complex interfacial phenomena described here, whereby protein aggregation and line activity play a dominant role. The reported differences in kinetics between the DPPC and the monolayers containing DPPS could be attributed to the different nature of the domains formed in these systems. (32) Diederich, A.; Sponer, C.; Pum, D.; Sleytr, U. B.; Losche, M. Colloids Surf., B 1996, 6, 335-346. (33) Baneyx, G.; Vogel, V. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12518-12523. (34) Ruano, Miguel, L. F.; Nag, K.; Worthman, L.-A.; Casals, C.; PerezGil, J.; Keough, K. M. Biophys. J. 1998, 74, 1101-1109. (35) Keller, D. J.; McConnell, H. M.; Moy, V. T. J. Phys. Chem. 1986, 90, 2311-2315. (36) de Koker, R.; McConnell, H. M. J. Phys. Chem. 1993, 97, 1341913424. (37) Ross, M.; Steinem, C.; Galla, H.-J.; Janshoff, A. Langmuir 2001, 17, 2437-2445.

Annexin A1 Interaction with a Phospholipid Monolayer

There is good evidence that the phase behavior reported here for annexin A1 is directly linked to interactions involving the N-terminal domain. No penetration into DPPC monolayers was observed by Rosengarth et al.13 for a proteolytic fragment of annexin A1 lacking the amphipathic N-terminal domain and for annexin A5, which lacks an N-terminal domain. Further evidence for the role of the N-terminal domain comes from considering the association to membranes of annexin A12 and annexin A5 in the absence of calcium. Under acidic conditions (pH below 5.0), these annexins appear to refold and insert into bilayers, yielding a transmembrane configuration.38-40 This phenomenon has been shown to depend on hydrophobic interactions between the protein and zwitterionic components of the model membranes.39,40 It is highly (38) Langen, R.; Isas, J. M.; Hubbell, Wayne, L.; Haigler, H. T. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 14060-14065. (39) Isas, J. M.; Cartailler, J.-P.; Sokolov, Y.; Patel, D. R.; Langen, R.; Luecke, H.; Hall, J. E.; Haigler, H. T. Biochemistry 2000, 39, 30153022. (40) Ladokhin, A. S.; Isas, J. M.; Haigler, H. T.; White, S. H. Biochemistry 2002, 41, 13617-13626.

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sensitive to the protonation state of the C-terminal core. This is in contrast to the results reported here. The fact that the interaction of annexin A1 with zwitterionic phospholipid monolayers presents the same microstructural features at neutral and acidic pH allows us to speculate that this behavior is not related to the C-terminal core conformation but rather, in accordance with the results of Rosengarth et al.,13 is related to the amphipathic nature of the N-terminal domain. Acknowledgment. J.F., S.A., and M.D. thank the Petroleum Research Fund (Grant No. 39070-AC9) for support of this research. A.R. and H.L. thank NIH (Grant No. GM56445) for support. The authors also thank Nathan Benedict for his contribution to initiating this collaboration. Supporting Information Available: Additional images are available as referred to in the text. This material is available free of charge via the Internet at http://pubs.acs.org. LA049713B