Protein-Directed Spatial Rearrangement of ... - ACS Publications

Nov 23, 2009 - Haifu Zheng and Xuezhong Du*. Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination ...
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J. Phys. Chem. B 2010, 114, 577–584

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Protein-Directed Spatial Rearrangement of Glycolipids at the Air-Water Interface for Bivalent Protein Binding: In Situ Infrared Reflection Absorption Spectroscopy Haifu Zheng and Xuezhong Du* Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination Chemistry, and School of Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093, People’s Republic of China ReceiVed: September 4, 2009; ReVised Manuscript ReceiVed: October 31, 2009

Lateral rearrangement of lipids on the surfaces of cell membranes plays an important role in multivalent interactions because a ligand for the second and subsequent binding events can be delivered through the lateral rearrangement. The binary monolayers containing glycolipids with mannose moieties at the air-water interface before and after binding of concanavalin A (Con A) have been investigated in detail using infrared reflection absorption spectroscopy (IRRAS). The spatial rearrangement of glycolipids in the binary monolayers directed by Con A in the subphase facilitated to match with protein binding pockets and minimize steric hindrance of neighboring carbohydrate ligands for bivalent protein binding. The amounts of specifically bound proteins were almost independent of surface glycolipid density at the air-water interface, different from the dependence of the amounts on surface ligand density at the solid-water interface with limited glycolipid rearrangement. Besides, hydrocarbon chains of the glycolipids in the monolayers were even reoriented favorable to the access of the ligands to the proteins for enhanced binding. Introduction Protein-carbohydrate interactions play an important role in a variety of cellular processes including cell adhesion, trafficking, metastasis, and immune response.1-4 These specific interactions take place through glycoproteins, glycolipids, and polysaccharides on cell surfaces and carbohydrate-binding lectins.5 The intricacy of the cell membrane structures along with the highly dynamic nature of lipid-lipid and lipid-protein interactions in the cell membranes make the biophysical interactions very difficult to investigate and understand in real time.6 Langmuir monolayers have one-half a structure of cell membranes and offer a simple system to mimic cell surface rearrangement due to the lateral mobility of the lipid components at the air-water interface.7-10 Lateral mobility is expected to play a key role in free rearrangement of ligand molecules in the membranes,10-13 because a ligand for the second and subsequent binding events can be delivered through the lateral rearrangement of lipid components. Langmuir monolayers at the air-water interface have been successfully used not only as a simple model for cell membranes but also as a powerful means to control molecular orientation and packing necessary for molecular devices, because the lipid monolayers are very well-defined and stable two-dimensional systems with planar geometry.6,7,14 However, lectins typically possess shallow binding pockets that are solvent-exposed, and monovalent protein-carbohydrate interactions are often of low affinity.2,15,16 It is possible for the lectins to engage in multivalent binding or several simultaneous binding events to improve interaction strength and specificity.2 Multivalent protein binding may achieve higher binding affinity, allow signaling through oligomerization, and induce changes in the distribution of molecules at the membrane interface.17,18 On the other hand, multivalent interactions are crucial for * To whom correspondence should be addressed. Fax: +86-25-83317761. E-mail: [email protected].

discovering a broad range of biosensors with a variety of functions for use in medicine, environment, and food processing.18 Concanavalin A (Con A) is usually selected as a model protein, because its multivalent binding to carbohydrate ligands such as mannose moieties in the presence of Ca2+ and Mn2+ has been extensively investigated, and a number of methods have been developed to evaluate this binding.8,9,19,20 Con A (pI 4.5-5.6) is a multivalent binding protein found in jack bean and at pH < 6.0 exists as a dimer and at pH > 7.0 as a tetramer (molecular weight, 104 kDa for tetramer).21,22 Con A tetramer has four carbohydrate binding sites and presents two binding sites on each face. Con A is capable of forming two attachment points to the surfaces to engage in bivalent interactions, and the distance between these points on the proteins is approximately 6.5 nm.23 Surface plasmon resonance (SPR) studies by Kiessling and co-workers indicate each Con A tetramer can only interact with two mannose residues simultaneously,2,24 with a high affinity (binding constants of 106-107 M-1)2,8,16,25 in contrast to the monovalent interactions of low affinity (binding constant of 103-104 M-1).24,26 Self-assembled monolayers (SAMs),25,27-29 supported lipid bilayers (SLBs),24,30 and vesicles17,19,31 have been also used as model systems to study protein-carbohydrate interactions. It is shown that surface density (mole fraction) and spatial arrangement of carbohydrate ligands play an important role in protein binding.2,25,27 However, the surface densities of the ligands in these model systems cannot be precisely controlled because they are formed from bulk solutions in comparison with Langmuir monolayers at the interface, and covalent attachment of glycolipids in SAMs obviously suppresses their spatial rearrangement for bivalent protein binding and amplification of ligand affinity. Moreover, the influence of spatial arrangement of ligands on protein binding is still largely unknown. The favorable spatial arrangement of the glycolipids in Langmuir monolayers through lateral mobility could facilitate enhancement of protein binding.

10.1021/jp908559n  2010 American Chemical Society Published on Web 11/23/2009

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CHART 1: Chemical Structures of Glycolipid DPEM and Protein-Resistant Lipid DPE

In situ infrared reflection absorption spectroscopy (IRRAS) has been one of the leading structural analyses for the monolayers at the air-water interface.32-46 The IRRAS technique not only allows for nondestructive and sensitive characterization of chain conformations and headgroup structures,34,35,41,45,46 but also provides quantitative information about molecular orientation.33,36-38,42-44 Moreover, the IRRAS is the most commonly used technique able to directly monitor protein secondary structures in situ in Langmuir monolayers.47 In this Article, binary Langmuir monolayers consisting of double-chained glycolipids with mannose epitopes linked through oligo(ethylene glycol) (OEG) spacers (DPEM) and lipids with the proteinresistant OEG chains (DPE), the chemical structures of which are shown in Chart 1. The two synthetic glycolipid and lipid have identical hydrocarbon chains and glyceryl skeletons except for the nonionic hydrophilic moieties. To facilitate lateral rearrangement of glycolipids at the interface, amide linkages for synthetic glycolipids and lipids were avoided from the formation of intermolecular hydrogen bonds between adjacent glycolipids/lipids from the molecular design point of view. Con A binding to the binary monolayers with different surface ligand densities at the air-water interface was investigated in detail using the IRRAS technique. The spatial rearrangement of the glycolipids in the binary monolayers directed by proteins facilitated bivalent protein binding and minimized steric hindrance of neighboring ligands for enhanced protein binding, even along with reorientation of alkyl chains. Experimental Section Materials. The syntheses of DPEM and DPE were reported very recently.48 Stock solutions of the two compounds were prepared in chloroform (analytical grade), which was pretreated to remove quite a bit water and acid, at a concentration of 1 mM and stored at -20 °C prior to use. The binary mixtures of DPEM and DPE were prepared volumetrically from their stock solutions. Con A from CanaValia ensiformis (Type V) was purchased from Sigma. Water used was double-distilled (pH 5.6, resistivity 18.2 MΩ cm, surface tension 73.06 mN/m at 22 °C) after a deionized exchange. Aqueous Con A solutions and

Zheng and Du subphases were prepared from phosphate buffered saline (PBS, 10 mM phosphate, 150 mM NaCl, pH 7.4) containing 0.1 mM Mn2+ and 0.1 mM Ca2+. Both a transition metal ion and Ca2+ must be bound before Con A was capable of binding carbohydrate.22 Monolayer Spreading and Isotherm Measurements. The surface pressure-area (π-A) isotherms were recorded on a Nima 611 Langmuir trough (Nima Technology, England) equipped with a computer control. The maximum available surface area was 30 cm × 10 cm and could be varied continuously by moving two Teflon barriers. A Wilhelmy plate with a small piece of rectangular filter paper was used as the surface pressure sensor with an accuracy of (0.1 mN/m. Chloroform solutions of DPEM, DPE, and their mixtures at various molar ratios were spread on the PBS solutions containing Mn2+ and Ca2+, and then 15 min was allowed for solvent evaporation. Two barriers compressed symmetrically at the same rate of 5 mm/min. The subphase temperature was kept at 22 °C. Each sample was run at least three times to ensure reproducibility. IRRAS Spectrum Measurements. In situ IRRAS spectra of the monolayers at the air-water interface were recorded on a Bruker Equinox 55 FTIR spectrometer connected to an XA511 external reflection attachment (Bruker, Germany) with a shuttle trough system and a narrow band mercury-cadmiumtelluride (MCT) detector. Sample (film-covered surface) and reference (film-free surface) troughs were fixed on a shuttle device driven by a computer-controlled stepper motor for allowing spectral collections from the two troughs in an alternating fashion. A KRS-5 polarizer was used to generate perpendicularly polarized lights. The IRRAS experiments were carried out at 22 °C. The film-forming molecules were spread from 26-28 µL of chloroform solution depending on lipid component, and 20 min was allowed for solvent evaporation. The measurement system was then enclosed for humidity equilibrium and monolayer relaxation for 4 h prior to compression. The monolayers were compressed discontinuously to the desired surface pressure of 30 mN/m from ∼0 mN/m. After 30 min of relaxation, the two moving barriers were stopped, and the monolayer areas were kept constant. Upon protein binding, concentrated Con A solutions were injected into the unstirred subphase underneath the compressed monolayers at a surface pressure of 30 mN/m behind the barriers with an L-shaped syringe to reach a final protein concentration of 40 µg/mL. During the period of protein binding, the surface pressures of the monolayers changed slightly within 1-2 mN/m. The external reflection absorption spectrum of the PBS solution containing Ca2+ and Mn2+ was used as a reference. The spectra were recorded with a resolution of 8 cm-1 by coaddition of 1024 scans. A time delay of 30 s was allowed for film equilibrium between trough movement and data collection. Spectra were acquired using a p-polarized radiation followed by data collection using s-polarized radiation. The IRRAS spectra were presented without any smoothing or baseline correction. The angles of incidence measured by the instrument were corrected to increase by 1.0° in comparison with the preset angles of incidence. Results and Discussion Protein-Directed Spatial Rearrangement of Glycolipids and Bivalent Protein Binding. The π-A isotherms of the monolayers of DPEM, DPE, and their binary mixtures at the air-water interface are shown in Figure 1. All of them displayed typical liquid expanded-liquid condensed phase transitions with

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Figure 1. Surface pressure-area isotherms of the monolayers of DPEM, DPE, and their binary mixtures on the PBS solutions (pH 7.4) containing Ca2+ and Mn2+ at 22 °C.

plateaus primarily representing the mushroom-like to brush conformation transition of the OEG spacers/chains49 and the disordered to ordered conformation transition of alkyl chains. The miscibility of the binary monolayers has been studied recently.48 DPEM and DPE were miscible, and their surface behaviors were close to the ideal mixtures to some extent due to their compatible chemical structures. Note that the behavior of the isotherm at the mole fraction of DPEM (XDPEM) of 0.2 was different from others to some extent, which regularly changed with the increase of XDPEM, but close to the DPEM one. Our recent SPR studies showed that the amount of specifically bound Con A on the preimmobilized binary monolayers was highest at XDPEM ) 0.1 followed by a drop at XDPEM ) 0.2, and then increased gradually upon further increase of XDPEM.48 These results suggest that the binary monolayer at XDPEM ) 0.2 had a different spatial arrangement of lipids from others. IRRAS data are defined as plots of reflectance-absorbance (RA) versus wavenumber. RA is defined as -log(R/R0), where R and R0 are the reflectivities of the film-covered and film-free surfaces, respectively. The selection rules of the IRRAS for the monolayers at the air-water interface are first presented. For s-polarization, the electric field vector is perpendicular to the plane of incidence, that is, parallel to the water surface. The bands are always negative, and their intensities decrease with increasing angle of incidence.33,36 For p-polarization, the electric field vector is parallel to the plane of incidence. For the vibrations with their transition moments parallel to the water surface, the bands are initially negative and their intensities increase with increasing angle of incidence to reach a maximum, and then a minimum in the reflectivity is found at the Brewster angle. Above the Brewster angle, the bands become positive, and their intensities decrease upon further increase in angle of incidence.33,36 For the vibrations with their transition moments perpendicular to the water surface, the bands are positive first and then become negative above the Brewster angle.33,38,40,42,50 If the vibrational transition moments are tilted to the air-water interface, the intensities of the bands are weak and even zero.51 Figure 2 shows the p-polarized IRRAS spectra of the monolayers of DPEM, DPE, and their binary mixture at XDPEM ) 0.1 at the air-water interface at various surface pressures, respectively, and other spectra at XDPEM ) 0.2, 0.3, and 0.4 are shown in Figure S1 in the Supporting Information. These

Figure 2. The p-polarized IRRAS spectra of the monolayers of DPEM, DPE, and their binary mixture at XDPEM ) 0.1 on the PBS solution (pH 7.4) containing Ca2+ and Mn2+ at various surface pressures at 22 °C: (a) DPE; (b) DPEM; (c) XDPEM ) 0.1.

spectral behaviors were very similar for different monolayers due to the compatible chemical structures of the glycolipids/ lipids and their similar surface behaviors. The spectral baselines (positive bands) were distorted in the region between 1750-1600 cm-1 because of the altered structure of the water adjacent to the headgroups of the film constituents, and the distortions were maximal for small angles of incidence. In the vicinity of about 0 mN/m, two weak bands around 2926-2924 and 2856-2855 cm-1 were assigned to the antisymmetric and symmetric CH2 stretching vibrations [νa(CH2) and νs(CH2)] of hydrocarbon chains, respectively. With increasing surface pressure up to 10

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Figure 3. Time-dependent p-polarized IRRAS spectra of the individual monolayers of DPE on the PBS solution (pH 7.4) containing Ca2+ and Mn2+ at the surface pressure 30 mN/m at 22 °C upon Con A injection, along with the difference spectrum after and before protein injection.

mN/m (except for DPEM up to 15 mN/m), the νa(CH2) and νs(CH2) bands gradually increased in intensity, and upon further increase of surface pressure, the two bands had a significant increase in intensity and shifted to 2918 and 2851 cm-1, respectively. It is well-known that the νa(CH2) and νs(CH2) frequencies are sensitive to the conformation order of alkyl chains, especially for the νa(CH2) modes. Lower wavenumbers are characteristic of all-trans conformations in highly ordered chains, while higher wavenumbers are indicative of gauche conformations in highly disordered chains.52 It is clear that the chain order in these monolayers increased progressively with surface pressure, which was consistent with their respective isotherms. Above 15 mN/m, a peak at 1468 cm-1, attributed to the CH2 scissoring mode [δ(CH2)], appeared and intensified with surface pressure. The appearance of the singlet peak at 1468 cm-1 indicated that the alkyl chains in the monolayers were probably packed in a hexagonal subcell structure where each chain could freely rotate around its axis.53 At the same time, a weak band around 2958 cm-1 was clearly observed due to the antisymmetric CH3 stretching vibrations [νa(CH3)]. The significant increase of these band intensities with surface pressure was related not only to molecular densities upon compression but also to molecular orientation. At high pressures, the alkyl chains adopted an orientation almost perpendicular to the water surface. It has been shown that the monolayers at 30 mN/m were enough to inhibit proteins from penetrating into the hydrophobic chain regions besides being capable of lateral mobility.54 The surface pressure of 30 mN/m is equivalent to the lateral pressure estimated for cell membranes under physiological conditions.55-57 The monolayers at 30 mN/m were chosen for protein binding as follows. Figure 3 shows time-dependent IRRAS spectra of the DPE monolayer at the air-water interface at 30 mN/m after injection of Con A. The spectrum after 24 h was virtually identical to that prior to protein injection, which indicates that Con A could not be adsorbed at the surface of the monolayer in the vicinity of 30 mN/m. It is clear that the OEG chains in the DPE monolayer with the brush conformations were protein resistant. This kind of short OEG chains has been demonstrated to provide even higher resistance against protein adsorption than long poly(ethylene glycol) (PEG) ones.58,59 On the other hand, this also verifies that Con A could not penetrate into the monolayer at 30 mN/m.

Zheng and Du Con A binding was clearly observed for the binary monolayers of DPEM and DPE with different XDPEM (Figure 4). Protein injection resulted in the appearance of amide I and amide II bands around 1640 and 1535 cm-1, respectively, together with an increase in intensity of the positive bands at approximately 3600 cm-1 (Figure S2), which were primarily assigned to the OH stretching vibration of water at the interface. The contribution from the OH groups attached to the polar headgroups of the lipids depended on the orientation of their vibrational transition moments. The corresponding OH stretching bands usually were negative-oriented at small angles of incidence such as 30°, and their intensities were relatively weak whether positive peaks or negative ones considering the discrepancy in intensity between the positive bands around 3600 cm-1 and the negative νa(CH2) and νs(CH2) bands. On the other hand, the areas of the monolayers remained constant upon Con A binding, and the contribution from the polar headgroups in the monolayers almost remained unchanged. The increasing positive bands obviously came from the water at the interface and indicated an increasing surface layer thickness due to protein binding.60 Blume and co-workers investigated the relation between intensity of the OH stretching band around 3600 cm-1 and film thickness at the air-water interface and correlated the increase in the band intensity directly with the increase of film thickness.61 It is known that amide I bands originate primarily from the peptide bond CdO stretching mode, and amide II bands result from the mixed modes of C-N stretching and N-H bending vibrations. It has been shown that there are wellestablished empirical correlations between amide I band frequencies and protein secondary structures.62-64 Second derivatives of the IRRAS spectra in the region of 1700-1600 cm-1 after protein binding saturation are shown in Figure 5. Both a strong band e1640 cm-1 (1640-1630 cm-1) and a weak band g1680 cm-1 are characteristic of β-sheet structures.65,66 The appearance of the amide I bands suggested a great amount of antiparallel β-sheet conformations, which was consistent with the secondary structures of native Con A (predominant β-sheet structures without R-helix one).22,65,66 These spectral features indicate that the secondary structures of the proteins were basically maintained when they bound to the hydrophilic headgroup regions of the binary monolayers. Because neither amide I nor amide II band was observed from the DPE monolayer, it is suggested that the amide I and amide II bands observed from the binary monolayers reflected specific protein binding. It is known that specific Con A-mannose interactions occur through the access of carbohydrate ligands to protein binding pockets. Note that the four panels of Figure 4 were drawn to the same scale. Seen from the difference spectra after and before protein binding with different XDPEM, the amide I bands had very comparable intensities for these binary monolayers. That meant that the amounts of specifically bound proteins in the binary monolayers at the air-water interface were almost independent of XDPEM. This was different from Con A binding to the corresponding immobilized binary monolayers at the solid-water interface by the horizontal Langmuir-Blodgett (LB) technique investigated using SPR.48 The amount of specifically bound proteins on the preimmobilized binary monolayers was highest at XDPEM ) 0.1 followed by a drop at XDPEM ) 0.2, and then increased gradually upon further increase of XDPEM.48 It has been shown that in SAMs and LB films the surface density and spatial arrangement of the carbohydrate ligands play a crucial role in Con A binding.2,25,27,48 Low surface ligand densities would limit bivalent protein binding because the separation distance between

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Figure 4. Time-dependent p-polarized IRRAS spectra of the binary monolayers of DPEM and DPE on the PBS solution (pH 7.4) containing Ca2+ and Mn2+ at the surface pressure 30 mN/m at 22 °C upon Con A binding, along with the difference spectra after and before protein binding, with different XDPEM: (a) 0.1; (b) 0.2; (c) 0.3; (d) 0.4.

Figure 5. Second derivatives of the IRRAS spectra at protein binding saturation of the binary monolayers of DPEM and DPE with different XDPEM at the air-water interface.

two binding pockets of Con A is about 6.5 nm,23,67 while high surface densities resulted in steric hindrance of neighboring ligands, which would inhibit access of the ligands to protein binding pockets. It is obvious that favorable spatial arrangement of the glycolipids can alleviate the steric hindrance of neighboring ligands and facilitate the bivalent protein binding even at a given surface ligand density. The glycolipids in the binary

monolayers at the air-water interface underwent a lateral reorganization to constitute a new spatial arrangement directed by Con A in the subphase. The optimal spatial arrangement of the ligands at the interface could match well with the protein binding pockets and simultaneously minimize the steric hindrance of neighboring ligands, which is schematically illustrated in Scheme 1. The spatial rearrangement of the glycolipids at the air-water interface promoted the formation of a great number of bivalent binding sites to meet the separation distance between the protein binding pockets, so that the amounts of specifically bound proteins were accordingly increased.48 Seen from the difference spectrum at XDPEM ) 0.2, two weak positive peaks at 2918 and 2951 cm-1 indicate that the alkyl chains increased in tilt angle with respect to the normal of the water surface after protein binding. Our recent SPR studies showed that the preimmobilized binary monolayer at XDPEM ) 0.2 had a low affinity to Con A, and the amount of specifically bound proteins was substantially enhanced after protein binding to the corresponding binary monolayer at the air-water interface.48 At XDPEM ) 0.2, the spatial rearrangement of the glycolipids in the monolayer not only matched with the protein binding pockets and reduced the steric hindrance of neighboring ligands but also adjusted molecular orientations to the development of bivalent protein binding. Chain Orientations before and after Protein Binding. Gericke et al. first applied the Kuzmin and Michailov’s optical model68,69 to describe IRRAS band intensities.33,36-38 Orientation

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SCHEME 1: Schematic Illustration of Con A-Directed Spatial Rearrangement of Glycolipids in the Binary Monolayer at the Air-Water Interface

angles of functional groups can be determined by the simulation of theoretical calculations in the three-phase system (air, anisotropic monolayer, and isotropic liquid substrate) to experimental data. The following parameters were required to calculate a RA value using Kuzmin and Michailov’s formulation:37,68,69 angle between dipole moment vector and chain axis, R ) 90° for the νa(CH2) vibration; refractive index and extinction coefficient of air, n1 ) 1 and k1 ) 0; refractive index and extinction coefficient of H2O, n2 ) 1.402 and k2 ) 0.0155 at 2918 cm-1, obtained from the literature;70 film thickness, d, obtained by taking into account tilt angle of alkyl chains, θ, and extended chain length, L ) 2.05 nm, for DPEM and DPE; ordinary and extraordinary refractive indices of alkyl chains in the mid-IR region, nord ) 1.46 and next ) 1.57,33 and corresponding directional refractive indices of the film, nx ) ny and nz, obtained from nord, next, and θ; directional extinction coefficients of the film, kx ) ky and kz, obtained for the given tilt angle and transition moment direction when the film extinction coefficient, kmax, is known; and polarizer efficiency, Γ, for the spectral measurements in this work was determined to be 98.5%. Here, only two unknowns remain, kmax and θ. A set of different combinations of kmax and θ were used by computer simulation to calculate RA values to fit the measured data. Figure 6 presents IRRAS spectra of a DPE monolayer at the air-water interface at 30 mN/m at different angles of incidence for p- and s-polarization. Although there were three ethylene oxide (EO) units in DPEM and DPE, respectively, it is known that the νa(CH2) and νs(CH2) frequencies in EO unit (2950-2930 and 2890-2865 cm-1, respectively)71,72 are different from those in the alkyl chain. Here, the νa(CH2) and νs(CH2) bands at 2918 and 2851 cm-1 at 30 mN/m were basically contributed from the alkyl chains. No obvious OEG νa(CH2) and νs(CH2) bands were observed, which was probably due to the more tilted orientations of their vibrational transition moments for the OEG spacers/chains in a couple of conformations (one OEG spacer/ chain per double alkyl chain). The fit of theoretical RA values of the νa(CH2) bands for the DPE monolayer to the experimental data against angle of incidence is shown in Figure S3. The tilt angle of the alkyl chains was estimated to be about 15°, and the value of kmax was 0.80 by the best fit. Smith and co-workers73 previously used synchrotron grazing-incidence X-ray diffraction

Figure 6. IRRAS spectra of the individual monolayer of DPE on the PBS solution (pH 7.4) containing Ca2+ and Mn2+ at the surface pressure 30 mN/m at 22 °C with different angles of incidence: (a) p-polarization; (b) s-polarization.

(GIXR) to investigate distearoylphosphatidylethanolamine (DSPE) lipid monolayers modified by chemically grafting PEG chains of different EO units of 0, 2, 8, and 17 at the air-water interface. The chain tilt angles in the cases of 2 and 8 EO units at 42 mN/m were at about 15° and 20°, respectively.73 The kmax value is related to the extinction coefficient for the vibrational mode and the density of the film-forming molecules at the air-water interface.39 The relative magnitude of the molecular density can be obtained from the appropriate π-A isotherms.39,45,46 The kmax values for the νa(CH2) bands were about 0.80 for the binary monolayers (XDPEM ) 0.1, 0.3, and 0.4) and 0.85 for the individual DPEM and binary monolayers at XDPEM ) 0.2, taking into account their molecular areas at 30 mN/m and the kmax value of 0.8 obtained in the case of individual DPE monolayer. The proteins were introduced after the monolayers were compressed to 30 mN/m and then maintained at the fixed trough areas, and thus the molecular areas remained constant and the band frequencies unchanged; that is, the chain conformations were not altered, before and after the introduction of proteins. The same kmax values were used for fitting the experimental data before and after protein binding.45,46 From the p- and s-polarized IRRAS spectra of the corresponding monolayers (parts a and b of Figures S4-S12), the tilt angles of alkyl chains were readily evaluated by the fit of theoretical calculations to the experimental data (part c of Figures S4-S12). The chain tilt angles of the monolayers of DPEM, DPE, and their binary mixtures with different XDPEM before and after Con

Protein-Directed Spatial Rearrangement of Glycolipids TABLE 1: Tilt Angles of Alkyl Chains in the Monolayers before and after Con A Binding monolayer

subphase

tilt angle (deg)

DPE DPEM XDPEM ) 0.1

water water water Con A water Con A water Con A water Con A

15 25 15 15 25 30 25 25 20 20

XDPEM ) 0.2 XDPEM ) 0.3 XDPEM ) 0.4

A binding are listed in Table 1. Prior to protein binding, the alkyl chains in the individual monolayers of DPEM and DPE were respectively oriented at the tilt angles of 25° and 15° with respect to the water surface normal depending on headgroup structure, and the chains in the binary monolayers with different XDPEM were tilted at the angles ranging from 15° to 25°. After protein binding, the chain orientations in the binary monolayers at XDPEM ) 0.1, 0.3, and 0.4 remained unchanged, while the chain tilt at XDPEM ) 0.2 was increased from 25° before protein binding to 30°. Con A-directed spatial rearrangement of the glycolipids in the binary monolayers not only matched with the protein binding pockets and minimized the steric hindrance of neighboring ligands but also adjusted molecular orientations to the formation of bivalent protein binding. These research results provide a deeper insight into the correlations between lateral rearrangement of lipids and multivalent protein binding. Conclusions The binary monolayers of DPEM and DPE with different XDPEM at the air-water interface before and after Con A binding were investigated in detail. Prior to protein binding, the studies of the π-A isotherms and IRRAS spectra indicated that the individual and binary monolayers underwent a typical liquid expanded-liquid condensed phase transition with surface pressure primarily representing the mushroom-like to brush conformation transition of the OEG spacers/chains and the disordered to ordered conformation transition of alkyl chains. The alkyl chains in the binary monolayers at 30 mN/m were oriented at the angles from 15° to 25° between the chain tilt angles of the individual monolayers of DPE and DPEM. After protein binding, the spatial rearrangement of glycolipids in the binary monolayers at 30 mN/m directed by Con A in the subphase matched with protein binding pockets and minimized steric hindrance of neighboring carbohydrate ligands for bivalent protein binding. The amounts of specifically bound proteins were almost independent of surface glycolipid density at the air-water interface, in contrast to the dependence of the amounts on surface ligand density at the solid-water interface with inhibited glycolipid rearrangement. The chain orientations in the binary monolayers at XDPEM ) 0.1, 0.3, and 0.4 remained unchanged after protein binding, while the alkyl chains at XDPEM ) 0.2 slightly increased in tilt angle favorable to enhanced protein binding. Acknowledgment. This work was supported by the National Natural Science Foundation of China (Grants 20673051, 20635020, and 20873062), the Natural Science Foundation of Jiangsu Province (Grant BK2007519), and the program for New Century Excellent Talents in University (NCET-07-0412). Supporting Information Available: IRRAS spectra of the binary monolayers of DPEM and DPE at XDPEM ) 0.2, 0.3, and

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