Influence of the Physical State of Phospholipid Monolayers on Protein

Article pubs.acs.org/Langmuir

Influence of the Physical State of Phospholipid Monolayers on Protein Binding Élodie Boisselier,† Philippe Calvez,† Éric Demers, Line Cantin, and Christian Salesse* LOEX/CUO-recherche, Hôpital du Saint-Sacrement, Centre Hospitalier Affilié de Québec, and Département d’Ophtalmologie, Faculté de Médecine, and PROTEO, Université Laval, Québec, Canada G1S 4L8 S Supporting Information *

ABSTRACT: Langmuir monolayers were used to characterize the influence of the physical state of phospholipid monolayers on the binding of protein Retinis Pigmentosa 2 (RP2). The binding parameters of RP2 (maximum insertion pressure (MIP), synergy and ΔΠ0) in monolayers were thus analyzed in the presence of phospholipids bearing increasing fatty acyl chain lengths at temperatures where their liquid-expanded (LE), liquid-condensed (LC), or solid-condensed (SC) states can be individually observed. The data show that a larger value of synergy is observed in the LC/SC states than in the LE state, independent of the fatty acyl chain length of phospholipids. Moreover, both the MIP and the ΔΠ0 increase with the fatty acyl chain length when phospholipids are in the LC/SC state, whereas those binding parameters remain almost unchanged when phospholipids are in the LE state. This effect of the phospholipid physical state on the binding of RP2 was further demonstrated by measurements performed in the presence of a phospholipid monolayer showing a phase transition from the LE to the LC state at room temperature. The data collected are showing that very similar values of MIP but very different values of synergy and ΔΠ0 are obtained in the LE (below the phase transition) and LC (above the phase transition) states. In addition, the binding parameters of RP2 in the LE (below the phase transition) as well as in the LC (above the phase transition) states were found to be indistinguishable from those where single LC and LE states are respectively observed. The preference of RP2 for binding phospholipids in the LC state was then confirmed by the observation of a large modification of the shape of the LC domains in the phase transition. Therefore, protein binding parameters can be strongly influenced by the physical state of phospholipid monolayers. Moreover, measurements performed with the α/β domain of RP2 strongly suggest that the β helix of RP2 plays a major role in the preferential binding of this protein to phospholipids in the LC state.

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INTRODUCTION Phospholipid monolayers are very useful for studying lipid− protein interactions because this model membrane allows several physical parameters such as the surface pressure and density of lipids, subphase content, and lipid composition to be controlled. (See refs 1−5, and for a review, see refs 6−9.) Moreover, there is a direct thermodynamic relationship between bilayers and monolayers.10,11 Phospholipid monolayers have been characterized by compression isotherms that are influenced by their polar headgroup as well as the length and the degree of unsaturation of their fatty acyl chains. Depending on the surface pressure and temperature, phospholipids can be found in a liquid-expanded (LE), liquid-condensed (LC), or solid-condensed (SC) state. (For a review, see refs 6 and 12−14.) The SC state is characterized by a high level of organization where fatty acyl chains adopt an alltrans conformation that minimizes the space between hydrocarbon chains and consequently maximizes their interactions. The LC state is quite similar to the SC state although it occurs over a slightly larger area per molecule. In contrast, unsaturation and shorter phospholipid fatty acyl chains favor the observation of the LE state where the fatty acyl chains are © 2012 American Chemical Society

conformationally disordered, resulting in a larger area per molecule.12 These different physical states have major biophysical significance because of their correlation with those observed in bilayers6,12 and their involvement in the formation of lipid rafts or microdomains.15,16 A study of protein binding on lipid monolayers in different physical states can thus provide useful information on their preference for such microdomains. DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) undergoes a phase transition at room temperature from the LE to the LC state,17,18 during which LC domains can be observed as previously documented by Brewster angle and fluorescence microscopy.19−21 The binding of proteins onto DPPC monolayers at room temperature has been widely studied. (For a review see, refs 6, 7, 22, and 23.) A useful method for characterizing protein binding in monolayers is based on the determination of the maximum insertion pressure (MIP).23 It consists of measuring the surface pressure increase induced by Received: March 16, 2012 Revised: May 27, 2012 Published: June 11, 2012 9680

dx.doi.org/10.1021/la301135z | Langmuir 2012, 28, 9680−9688

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desired initial surface pressure (Πi) was reached. The waiting time for the spreading solvent evaporation and for the film to reach equilibrium varied with the type of lipid, the spreading volume, the initial surface pressure, and the lipid concentration.24 RP2 (or its α/β domain) was then injected into the subphase underneath the lipid monolayer until a saturating final concentration of 20 μg/mL was achieved. The kinetics of protein binding onto phospholipid monolayers was monitored until the equilibrium surface pressure (Πe) was reached. The surface pressure increase (ΔΠ) after the injection of RP2 corresponds to Πe−Πi. The binding of RP2 onto the DMPC monolayer has been measured in both the LC and LE states at 4 and 23 °C, respectively. Indeed, a single LE state of DMPC is observed at 23 °C whereas an LC state can be seen above 5.5 mN/m at 4 °C.17 The binding of RP2 onto the DSPC monolayer has been measured in the SC and LE states at respectively 23 and 58 °C. Indeed, the surface pressure isotherm of DSPC shows a single SC state at 23 °C and a single LE state at 58 °C.26−28 It is noteworthy that buffer evaporation at this temperature has no effect on the extent of RP2 binding because the kinetics observed at 58 °C for DSPC in the LE state is very quickly reached and a highly stable plateau is obtained. (See Figure S1 for a typical example.) Finally, the binding of RP2 onto a DPPC monolayer has been measured in the LC and LE states at respectively 4 and 37 °C. Indeed, the surface pressure isotherm of DPPC shows a single LC state below 9 °C and a single LE state at 37 °C of up to ∼37 mN/ m.17,18 Moreover, measurements with the DPPC monolayer have also been performed at 23 °C in order to highlight the influence of its phase transition on RP2 binding. Determination of the Binding Parameters of RP2. The MIP of RP2 (and of its α/β domain) was determined as described previously.23,24 Briefly, RP2 (or its α/β domain) is injected into the subphase underneath the phospholipid monolayer at different Πi values. Then, the plot of the surface pressure increase (ΔΠ) as a function of Πi allows the determination of the MIP and of the ΔΠ0 by extrapolating the regression of the plot to the x and y axes, respectively. The synergy is obtained by adding 1 to the slope of the plot of ΔΠ as a function of Πi or more directly from the slope of the plot of Πe as a function of Πi.24 The uncertainty in MIP and ΔΠ0 were calculated with a confidence interval of 95% from the covariance of the experimental data on the linear regression as previously described.23 The uncertainty in the synergy was calculated as previously documented.24 Comparison of the Linear Regression of ΔΠ as a Function of Πi. The method used to compare two linear regressions (y = ax + b) was built on three conditions with a confidence interval of 95%. First, the residual variances (σ2) of the experimental data are compared by the test of Snedecor (F = σ12/σ22) with degrees of freedom of (n1 − 1) and (n2 − 2) to check the homogeneity of the data, where n is the number of data points of each regression. Second, the slopes, a, of the regressions are compared with the student’s t test using

the injection of a protein into the subphase of lipid monolayers at different initial surface pressures. The measurement of the MIP thus allows an estimation of the extent of protein binding onto lipid monolayers. It corresponds to the maximum surface pressure up to which protein binding onto a given lipid monolayer is energetically favorable. A large number of MIP values have been determined for proteins with phospholipids such as DPPC showing a phase transition at room temperature.23 However, this complicates the analysis of the data because large changes in the phospholipid molecular area and thus in the fatty acyl chain conformation take place during the phase transition. The influence of the phase transition on protein binding has not yet been analyzed in detail. We have previously shown that the plot allowing us to determine the MIP can provide additional useful information for evaluating the binding parameters of proteins such as the synergy.24 We have indeed demonstrated a positive synergy and thus a favorable binding of protein Retinitis pigmentosa 2 (RP2) in the presence of the saturated DSPC (1,2-distearoyl-snglycero-3-phosphocholine) monolayer in the SC state at room temperature. In contrast, a negative synergy of RP2 was observed in the presence of the polyunsaturated 1,2didocosahexaenoyl-sn-glycero-3-phosphocholine (DDPC) monolayer demonstrating an unfavorable binding of this protein to this particular phospholipid in the LE state.24 These data thus suggest that the binding parameters of RP2 are governed by the physical state of phospholipid monolayers. This protein is therefore suitable for determining the influence of the phospholipid monolayer phase transition on its binding parameters. In this article, we thus report the binding of RP2 onto monolayers of phospholipids bearing different saturated fatty acyl chain lengths at various temperatures to determine the influence of the physical state of lipid monolayers on protein binding. Moreover, measurements were performed with the α/β domain of RP2 to determine which structural component of this protein could play a major role in its mechanism of binding.

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EXPERIMENTAL SECTION

Materials. The deionized water used for the buffer solutions was prepared from a Barnstead Nanopure system (Barnstead, Dubuque, IA). Its resistivity and surface tension at 20 °C were respectively 18.2 MΩ·cm and 72 mN/m. Tris, NaCl, MgCl2, and β mercaptoethanol were purchased from Sigma (St. Louis, MO). The resin used for protein purification was from GE Healthcare (Uppsala, Sweden). 1,2Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), DPPC, and DSPC were purchased from Avanti Polar Lipids (Alabaster, AL). All chemicals were used as received. The phospholipid solutions were prepared in chloroform at a concentration of 0.1 mg/mL. The constructs of the complete RP2 (amino acids 1−350) and its α/β domain (amino acids 230−350) were a kind gift from Dr. Alfred Wittinghofer (Max-Planck-Institut für Molekulare Physiologie, Germany). They were expressed and purified as described previously.24,25 Surface Pressure Measurements. The surface pressure (Π) was measured by the Wilhelmy method using a tensiometer from Nima Technology (Coventry, U.K.). The experimental setup was placed in a plexiglass box with humidity control. Measurements were performed either at room temperature (23 °C), at 4 °C in a cold room, or at higher temperatures in a laboratory oven. Temperature was controlled to an accuracy of ±0.5 °C at 4, 23, and 37 °C whereas a slightly larger variation was measured at 58 °C (±1 °C). The volume of the homebuilt, round Teflon trough used for the monolayer measurements was 1200 μL (diameter of 20 mm). The subphase buffer contained 5 mM phosphate at pH 7.4 and 100 mM NaCl. The monolayer was prepared by spreading a few microliters of a solution of phospholipids until the

a1 − a 2

t= σ*

x1̅ 2

2

∑in−1 1(x1i − x1̅ )2

+

x2̅ ∑in−2 1(x 2i − x 2̅ )2

(1)

and degrees of freedom of (n1 + n2 − 4), where σ* is the common residual variance. Finally, the abscissas at the origin, b (ΔΠ0), are also compared with the student’s t test using b1 − b2

t= σ*

1 n1

+

1 n2

2

+

x1̅ ∑in−1 1(x1i − x1̅ )2

2

+

x2̅ ∑in−2 1(x2i − x 2̅ )2

(2)

and degrees of freedom of (n1 + n2 − 4). When those three conditions are satisfied, the two distributions of experimental data can be considered to follow the same linear regression. Epifluorescence Microscopy. Epifluorescence microscopy experiments were performed at room temperature in monolayers at the air−buffer interface using the DeltaPi trough setup (Kibron, Helsinki, Finland) and a Nikon epifluorescence microscope. This equipment is very similar to that reported by Meller29 and was thoroughly described 9681


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elsewhere.30 RP2 was labeled with Alexa Fluor 488 sulfodichlorophenol ester as described previously31 and injected into the subphase of a DPPC monolayer containing 2 mol % Texas Red DHPE (1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt). Images were captured with a SIT camera (Hamamatsu Photonics, Shizuoka, Japan) and analyzed with Simple PCI software (Compix Inc., Cranberry Township, PA).

Table 1. Summary of the Values of MIP, ΔΠ0, and Synergy for DMPC, DPPC, and DSPC in the Liquid-Condensed (LC) or Solid-Condensed (SC) State as Well as in the Liquid-Expanded (LE) State liquid-condensed (LC) or solid-condensed (SC) state

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phospholipid

RESULTS AND DISCUSSION Monolayer Binding Parameters of RP2: Effect of the Phospholipid Physical State. A typical example of the binding parameters of RP2 on a DPPC monolayer in the LC state at 4 °C is illustrated in Figure 1. It can be seen in the inset

temperature MIP ΔΠ0 synergy

DMPC

DPPC

DSPC

4 °C 4 °C 23 °C 20.9 ± 1.3 mN/m 32.0 ± 1.2 mN/m 36.4 ± 2.4 mN/m 10.3 ± 0.3 mN/m 14.5 ± 0.4 mN/m 20.1 ± 0.8 mN/m 0.52 ± 0.02 0.55 ± 0.02 0.45 ± 0.03 liquid-expanded (LE) state

phospholipid

DMPC

DPPC

DSPC

temperature MIP ΔΠ0 synergy

23 °C 25.5 ± 1.7 mN/m 20.2 ± 0.7 mN/m 0.21 ± 0.05

37 °C 30.8 ± 2.9 mN/m 21.0 ± 0.6 mN/m 0.32 ± 0.04

58 °C 26.7 ± 2.5 mN/m 21.1 ± 1.1 mN/m 0.21 ± 0.08

larger than zero whatever the phospholipid fatty acyl chain length and physical state, thus suggesting that the corresponding values of MIP are consistent with an insertion surface pressure. As can be seen in Figure 2A, the longer the fatty acyl

Figure 1. Determination of the binding parameters of RP2 onto a DPPC monolayer in the LC state at 4 °C from the plot of ΔΠ as a function of Πi: the MIP (intercept of the linear regression with the x axis), the synergy (slope of the linear regression +1), and the ΔΠ0 (intercept of the linear regression with the y axis). The inset shows typical isotherms of the adsorption of RP2 onto the DPPC monolayer at Πi values of 11, 17, and 25 mN/m as a function of time. These isotherms are used to build the plot of ΔΠ as a function of Πi. Only a few isotherms are shown for clarity.

of Figure 1 that the larger the initial surface pressure (Πi), the smaller the increase in surface pressure (ΔΠ). Indeed, ΔΠ values of 8.6, 6.2, and 2.5 mN/m have been measured for Πi values of 11, 17, and 25 mN/m, respectively. A negative slope is thus obtained when plotting these values of ΔΠ as a function of Πi (Figure 1), which allowed the calculation of a MIP of 32 ± 1.2 mN/m for RP2. As shown in Figure 1, the synergy can also be calculated from the linear regression of ΔΠ as a function of Πi by adding 1 to the value of the slope. A positive synergy of 0.55 ± 0.02 has thus been obtained that suggests favorable binding between RP2 and the DPPC monolayer in the LC state. Moreover, a ΔΠ0 value of 14.5 ± 0.4 mN/m has been obtained by extrapolating the regression of the plot to the y axis (Figure 1). The parameters of the binding of RP2 onto DMPC, DPPC, and DSPC monolayers in different physical states are shown in Table 1. We have previously shown that a negative synergy correlates with an unfavorable binding of a protein to a phospholipid monolayer.24 The corresponding MIP in this case is an exclusion surface pressure. In contrast, a positive synergy corresponds to a favorable binding of the protein, and the MIP in this case corresponds to an insertion surface pressure. Then, when the synergy is close to zero, the binding of the protein is neither favored nor disfavored by the lipid monolayer because Πe remains almost unchanged with increasing values of Πi.24 The MIP in this case corresponds to a stationary surface pressure. It can be seen in Table 1 that all synergy values are

Figure 2. Comparison of the values of (A) MIP and (B) ΔΠ0 obtained with RP2 in the presence of DMPC, DPPC, and DSPC monolayers. The values obtained in the liquid-expanded (LE) state or in the liquidcondensed (LC)/solid-condensed (SC) states are represented by gray squares and by black circles, respectively.

chain, the larger the MIP when phospholipids are in the LC/SC states. Indeed, values of 20.9 ± 1.3, 32.0 ± 1.2, and 36.4 ± 2.4 mN/m have been obtained for RP2 in the presence of DMPC, DPPC, and DSPC monolayers, respectively (Table 1, Figure 2A). In contrast, the fatty acyl chain length has no influence on the MIP of RP2 when binding to phospholipid monolayers in the LE state (Figure 2A). Indeed, the value of MIP obtained with DMPC (25.5 ± 1.7 mN/m) is not significantly different from that measured with DSPC (26.7 ± 2.5 mN/m), which is not significantly different from that obtained with the DPPC monolayer (30.8 ± 2.9 mN/m, Table 1). Therefore, the values 9682


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of MIP depend on the physical state of phospholipid monolayers, except for DPPC as discussed below. A strong influence of the physical state of phospholipid monolayers can thus be observed on the binding of RP2. On the basis of these data, one can postulate that RP2 membrane binding will physiologically take place in the presence of membranes composed of phospholipids in the LC/SC states at 37 °C, such as DSPC. This is consistent with the observed location of RP2 in detergent-resistant membranes,32 which are known to consist of phospholipids with long saturated fatty acyl chains such as DSPC,33 which is in the SC state at 37 °C. The values of ΔΠ0 also vary with the physical state of phospholipid monolayers and follow the same trend as the MIP. Indeed, as shown in Figure 2B, ΔΠ0 remains unchanged when increasing the fatty acyl chain length of phospholipids in the LE state. Indeed, values of ΔΠ0 of 20.2 ± 0.7, 21.0 ± 0.6, and 21.1 ± 1.1 mN/m have respectively been obtained with DMPC, DPPC, and DSPC monolayers in the LE state (Table 1, Figure 2B). In contrast, ΔΠ0 increases with the fatty acyl chain length in the presence of phospholipid monolayers in the LC/SC states. Indeed, as can be seen in Figure 2B and Table 1, the longer the fatty acyl chains, the larger the value of ΔΠ0. Indeed, values of ΔΠ0 of 10.3 ± 0.3, 14.5 ± 0.4, and 20.1 ± 0.8 mN/m have respectively been obtained for DMPC, DPPC, and DSPC in the LC/SC states. Moreover, the largest value obtained in the LC/SC states (ΔΠ0 of 20.1 ± 0.8 mN/m for DSPC) is quite similar to that obtained with the phospholipids in the LE state (varying from 20.2 to 21.1 mN/m, Table 1). In addition, the smallest value of ΔΠ0 observed in the presence of the DMPC monolayer in the LC state (10.3 ± 0.3 mN/m) is very similar to the surface activity of pure RP2 in the absence of a phospholipid monolayer (∼10 mN/m) whereas the ΔΠ0 of RP2 observed with DMPC in the LE state is much larger than this value (20.2 ± 0.7 mM/m, Table 1). As discussed previously,24 it can be postulated that when ΔΠ0 is equal to the protein surface activity this protein would not protude more deeply than the polar headgroup of the phospholipid monolayer. In contrast, when ΔΠ0 is larger than the protein surface activity, this protein would insert more deeply, presumably within the phospholipid fatty acyl chains. Figure 3 shows linear regressions of the plot of the normalized equilibrium adsorption pressure (Πe) of RP2 as a function of the initial surface pressure (Πi) of DMPC in the LE (23 °C) and LC (4 °C) states as well as DPPC in the LE (37 °C) and LC (4 °C) states and DSPC in the LE (58 °C) and SC (23 °C) states. The slope of these linear regressions corresponds to the synergy between the protein and the phospholipid monolayers. It can be seen that a larger synergy is observed between RP2 and phospholipid monolayers in the LC/SC states than when these phospholipids are in the LE state. Indeed, the synergy of RP2 in the presence of phospholipid monolayers in the LC/SC states is larger than 0.45 ± 0.03 but smaller than 0.32 ± 0.04 in the LE state (Figure 3, Table 1). The values of synergy observed in the LC/SC states are quite similar and very large. They are between 0.45 and 0.55 (Table 1), which suggests that the binding of RP2 is largely favored in the presence of phospholipid monolayers in the LC/SC states, with fatty acyl chains in the all-trans conformation. However, the binding of RP2 to phospholipid monolayers in the LE state is less favorable. Indeed, synergy values of 0.21 ± 0.05, 0.21 ± 0.08, and 0.32 ± 0.04 have respectively been obtained for RP2 in the presence of DMPC, DSPC, and DPPC monolayers in the LE state (Table 1).

Figure 3. Plot of the normalized equilibrium adsorption pressure (Πe) as a function of the initial surface pressure (Πi), which allows us to compare the synergy values of RP2 upon binding to DMPC, DPPC, and DSPC monolayers in the liquid-expanded (LE) or liquidcondensed (LC)/solid-condensed (SC) states (gray dotted lines for the LE state and black solid lines for the LC/SC states). The Πe values have been normalized by subtracting the value of ΔΠ0 from each linear regression in order to facilitate a comparison of all of the slopes illustrating the synergy measured at different temperatures and with different phospholipids.

Therefore, the synergy values obtained in the presence of phospholipid monolayers in the LE state are significantly smaller than those obtained with phospholipids in the LC/SC states. Moreover, a synergy value of close to 0.2 was shown to correspond to the upper limit of the “stationary surface pressure”.24 Hence, the interaction of RP2 with phospholipid monolayers in the LE state is very close to this regime. This is consistent with the observation that, in the LE state, the length of the phospholipid fatty acyl chains has no influence on the values of MIP or ΔΠ0 for RP2 (Figure 2). Consequently, one could postulate that the binding of RP2 is neither favored nor unfavored in the presence of phospholipids in the LE state. In addition, similar values of MIP have been obtained for RP2 in the presence of DPPC monolayers in the LE and LC states (Figure 2A, Table 1) whereas different slopes of Πe as a function of Πi have been observed and thus different synergy values (Figure 3, Table 1). Therefore, the analysis of the synergy should most appropriately allow us to highlight the influence of the phase transition of this phospholipid on the binding of RP2. Influence of the Phase Transition of the DPPC Monolayer on the Binding of RP2. The binding of RP2 has been studied in the presence of a DPPC monolayer at room temperature (23 °C) to determine the effect of its phase transition that occurs between 8 and 15 mN/m at this particular temperature.17,18 A single LE state and a single LC state are respectively observed below ∼8 mN/m and above ∼15 mN/m.17,18 A mixture of bent conformers is present in the LE state of DPPC whereas its fatty acyl chains adopt an alltrans conformation in the LC state.6,12−14 The statistical test to compare two linear regressions has been used to determine whether a correlation exists between the slope of the experimental points in the LE state and that in the LC state of DPPC upon the binding of RP2. The homogeneity of each set of data was successfully checked by Snedecor's test. Then, student t tests were performed to compare the slope of the regression of each set of experimental points (below and above the phase transition) and the values of ΔΠ0 (Figure 4). These statistical analyses have shown that these two sets of points are 9683


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Figure S2, RP2 is made of an α/β domain and a β helix.25 The α/β domain is made of α helices and β sheets and is highly soluble. In contrast, the β helix of RP2 is very hydrophobic and thus insoluble in aqueous solutions. Therefore, to determine the importance of the structural components of RP2 on its preferential binding onto phospholipid monolayers in the LC state, additional binding experiments were performed with a DPPC monolayer at 23 °C using the α/β domain of RP2. A single slope can be seen in the plot of ΔΠ as a function of Πi (inset of Figure 4) whereas two slopes were observed with the complete RP2 (Figure 4). Moreover, the binding parameters of the α/β domain of RP2 are compared in Table 3 using all data Table 3. Summary of the Values of MIP, ΔΠ0, and Synergy of the α/β Domain of RP2 in the Presence of a Monolayer of DPPC at 23 °C When Using All Data Points or Only Those Measured in the Liquid-Expanded (LE) or the LiquidCondensed (LC) States

Figure 4. Determination of the binding parameters of RP2 onto a DPPC monolayer at 23 °C from the plot of ΔΠ as a function of Πi. The LE state is represented by gray squares, the LC state, by black circles, and the phase transition, by open diamonds. The plot of ΔΠ as a function of Πi, allowing the determination of the binding parameters of the α/β domain of RP2 onto a DPPC monolayer at 23 °C, is shown in the inset.

all data points surface pressure range temperature MIP

different as illustrated in Figure 4. Different slopes of ΔΠ as a function of Πi and different values of ΔΠ0 are obtained when DPPC is in the LE (∼15 mN/m) state. These data thus suggest that the physical state of the DPPC monolayer dictates the binding of RP2. It is important to stress that the experimental points located in the phase transition (between 8 and 15 mN/m) fit well with those in the LE state and are thus significantly different from those in the LC state (Figure 4). Moreover, it must be stressed that, as shown in Figure 4, the values of MIP do not allow us to highlight the influence of the physical state of DPPC on RP2 binding because they are not significantly different (30.0 ± 3.7 mN/m in the LE state (∼15 mN/m), Table 2), in contrast to the synergy

ΔΠ0 synergy

liquid-expanded (LE) state 0−8 mN/m 23 °C 30.0 ± 3.7 mN/m 20.6 ± 0.2 mN/m 0.31 ± 0.05 liquid-condensed

0−35 mN/m 37 °C 30.8 ± 2.9 mN/m 21.0 ± 0.6 mN/m 0.32 ± 0.04 (LC) state

surface pressure range temperature MIP ΔΠ0 synergy

15−35 mN/m 23 °C 32.2 ± 2.3 mN/m 14.4 ± 1.3 mN/m 0.55 ± 0.05

5−35 mN/m 4 °C 32.0 ± 1.2 mN/m 14.5 ± 0.4 mN/m 0.55 ± 0.02

LE state 0−8 mN/m 23 °C 39.4 ± 2.6 mN/m 24.6 ± 0.1 mN/m 0.38 ± 0.02

LC state 15−35 mN/m 23 °C 40.7 ± 3.0 mN/m 25.2 ± 0.7 mN/m 0.38 ± 0.05

points or only those measured in the LE or the LC states. It can be seen that, within experimental error, almost identical results are obtained whatever the physical state. This contrasts with the behavior of the complete protein. Indeed, as shown in Table 2, the binding parameters of the complete RP2 are very different in the LC and LE states of DPPC except for the MIP, which is very similar, as discussed above. Altogether, these data thus strongly suggest that the binding of the α/β domain of RP2 is not influenced by the physical state of the DPPC monolayers. Therefore, one can postulate that it is the β helix of RP2 (Figure S2) that plays a major role in the preferential binding of RP2 to phospholipids in the LC state. The statistical test has also been used to compare the binding parameters of RP2 in the presence of a DPPC monolayer in the LE state at 37 °C (up to ∼37 mN/m) and at 23 °C (up to 8 mN/m). As shown in Figure 5A, these linear regressions can be combined into a single data set because the statistical analysis of the homogeneity of the data of the three binding parameters was found to be successful. This is very consistent with the binding parameters shown in Table 2, which are very similar. Indeed, the values of synergy (0.31 ± 0.05 at 23 °C and 0.32 ± 0.04 at 37 °C), of MIP (30.0 ± 3.7 mN/m at 23 °C and 30.8 ± 2.9 mN/m at 37 °C) and of ΔΠ0 (20.6 ± 0.2 mN/m at 23 °C and 21.0 ± 0.6 mN/m at 37 °C) are not significantly different when considering the uncertainty. These linear regressions are very similar as illustrated in Figure 5A. Similarly, the statistical analysis of the data measured in the LC state at 4 °C (entire isotherm) and at 23 °C (>∼15 mN/m) revealed that these binding data are homogeneous and can be combined into a single set of experimental data points to draw a unique linear regression as shown in Figure 5B. This is consistent with the very similar binding parameters of RP2 obtained in the presence of DPPC in the LC state at 4 and 23 °C as summarized in Table 2. Indeed, the values of synergy (0.55 ± 0.05 at 23 °C and 0.55 ± 0.02 at 4 °C) of ΔΠ0 (14.4 ± 1.3 mN/m at 23 °C and 14.5 ± 0.4 mN/m at 4 °C) as well as

Table 2. Summary of the Values of MIP, ΔΠ0, and Synergy of Complete RP2 in the Presence of a Monolayer of DPPC at 4, 23, and 37 °C in the Liquid-Expanded (LE) and LiquidCondensed (LC) States surface pressure range temperature MIP ΔΠ0 synergy

0−35 mN/m 23 °C 41.6 ± 3.7 mN/m 24.3 ± 0.2 mN/m 0.42 ± 0.02

values that are different for these two physical states (0.31 ± 0.05 mN/m in the LE state (∼15 mN/m), Table 2). The same experiments were performed with the α/β domain of RP2 in order to determine the influence of the structural components of this protein on its mechanism of binding. As can be seen in 9684


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increase in the size of the domains with no effect on their shape.34 In contrast, the diameter of the DPPC domains does not increase much in size upon RP2 binding; however, the total size of the domains slightly increases as observed by the presence of the outgrowths (Figure 6B). These data thus suggest that RP2 preferentially binds to LC domains of DPPC under these conditions. Second, a homogeneous fluorescence of the DPPC monolayer in the LE state with no LC domains can be observed before the injection of the protein (Figure 6D). However, although the contrast of the image is not very good, a heterogeneous fluorescence of the Texas Red DHPE fluorescent label can be seen after the injection of the protein into the subphase (Figure 6E), thus suggesting the formation of poorly defined DPPC domains. Nevertheless, most important is the observation of a homogeneous distribution of the protein fluorescence after RP2 binding (Figure 6F), which suggests that RP2 binds DPPC in both the LE and LC states when injected underneath a DPPC monolayer in the LE state. This behavior is thus very different from that observed when RP2 is injected underneath a DPPC monolayer in the phase transition (Figure 6A−C). Finally, experiments with the α/β domain of RP2 were performed in order to determine which structural component of this protein could modulate its preferential binding to phospholipids in the LC state. Indeed, on the basis of the binding parameters of the α/β domain of RP2 (Table 3 and inset of Figure 4), one could expect this α/β domain to show no preference for the LE or LC state of DPPC. As in Figure 6A, LC domains can be seen in Figure 6G in the phase transition of DPPC at a surface pressure of 13 mN/m. The injection of the α/β domain of RP2 underneath this DPPC monolayer led to the deformation of the domains and to a large loss of contrast (Figure 6H). However, homogeneous fluorescence of the fluorescently labeled α/β domain of RP2 can be observed in Figure 6I, thereby demonstrating the very different behavior of this structural component of RP2 compared to that of the complete protein (Figure 6A−C). Indeed, these data demonstrate that the α/β domain of RP2 binds to both the LE and LC domains of DPPC, which is consistent with the unique slope of the curve shown in the inset of Figure 4. Altogether, the fluorescence microscopy data further support the conclusions drawn from the binding parameters of RP2 (Tables 1−3) that complete RP2 preferentially binds the LC domains of DPPC.

Figure 5. Plot of ΔΠ as a function of Πi to compare the binding parameters of RP2 on a DPPC monolayer in the liquid-expanded (LE) and liquid-condensed (LC) states. (A) The measurement in the LE state was performed at 37 °C (gray squares) and 23 °C (black circles). (B) The measurement in the LC state was made at 4 °C (gray squares) and at 23 °C (black circles).

of MIP (32.2 ± 2.3 mN/m at 23 °C and 32.0 ± 1.2 mN/m at 4 °C) are almost identical. These observations are consistent with the preference of RP2 for phospholipids in the LC state as further demonstrated by fluorescence microscopy. Epifluorescence microscopy has been used to determine the effect of RP2 binding on the LC domains of DPPC in its phase transition. To illustrate the preferential binding of RP2 to phospholipid monolayers in the LC state properly, three sets of experiments were performed: (1) RP2 injected underneath a DPPC monolayer in the phase transition, (2) RP2 injected underneath a DPPC monolayer in the LE state, and (3) the α/ β domain of RP2 injected underneath a DPPC monolayer in the phase transition. First, the DPPC monolayer has been compressed until a surface pressure of 12 mN/m is obtained, where LC domains can be observed because the Texas Red DHPE fluorescent label is excluded from these domains (Figure 6A). Very early after the injection of RP2 into the subphase underneath this DPPC monolayer, a large modification of the shape of the DPPC domains could be observed (Figure 6B). Indeed, those domains became highly irregular with a large number of poorly defined outgrowths. In addition, there is no evidence for the partitioning of RP2 in the LE fluid regions surrounding the LC domains of DPPC. Indeed, no fluorescence from the fluorescently labeled RP2 could be observed in the areas corresponding to the LE state or the LC domains of DPPC (Figure 6C). One could thus postulate that RP2 is entirely located underneath the LC domains, which masks protein fluorescence by an unknown mechanism. Moreover, if RP2 binds to DPPC in the LE state, one should have observed an

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CONCLUSIONS The present data show that the binding parameters of RP2 are drastically influenced by the physical state of phospholipid monolayers. Two different behaviors are highlighted by the values of synergy upon the binding of RP2 to phospholipid monolayers in LE or LC/SC states. Indeed, larger values of synergy have been observed in the LC/SC states than in the LE state. In addition, no effect of the fatty acyl chain length has been observed upon the binding of RP2 onto phospholipid monolayers in the LE state. Indeed, MIP, ΔΠ0, and the synergy of RP2 remain unchanged upon binding to DMPC, DPPC, and DSPC monolayers in the LE state. However, these binding parameters increase with the fatty acyl chain length when phospholipids are in the LC/SC states. The analysis of the synergy allowed us to suggest that the binding of RP2 onto phospholipid monolayers in the LC/SC state results in protein insertion whereas a stationary phase takes place in the LE state. 9685


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Figure 6. Epifluorescence microscopy of DPPC with RP2 and its α/β domain. (A) Typical micrograph of the DPPC monolayer in the phase transition at 12.0 mN/m observed using 2 mol % Texas Red DHPE (TRDHPE). (B) Micrograph of the same DPPC monolayer observed 4 min after the injection of RP2. (C) Micrograph of the same monolayer as in part B obtained after quickly changing the filters, allowing the observation of fluorescently labeled RP2 with Alexa Fluor 488. (D) Micrograph of the DPPC monolayer in the LE state at 6 mN/m observed using TRDHPE. (E) Micrograph of the same DPPC monolayer as in part D observed 4 min after the injection of RP2. (F) Micrograph of the same monolayer as in part E obtained after quickly changing the filters, allowing the observation of fluorescently labeled RP2. (G) Micrograph of the DPPC monolayer in the phase transition at 13 mN/m observed using TRDHPE. (H) Micrograph of the same DPPC monolayer as in part G observed 4 min after the injection of the α/β domain of RP2. (I) Micrograph of the same monolayer as in part H obtained after quickly changing the filters, allowing the observation the fluorescently labeled α/β domain of RP2.

Therefore, on the basis of the values of MIP, ΔΠ0, and the synergy factor, the binding of RP2 is favored when phospholipids are in the LC/SC states. The synergy has been useful in analyzing the influence of the phase transition on the binding of RP2 onto a DPPC monolayer at room temperature. Two different slopes corresponding to different synergies have been observed, depending on the physical state of the DPPC monolayer. Moreover, the data obtained upon the binding of RP2 onto a DPPC monolayer at 23 °C in the LC state (>∼15 mN/m) are very consistent with those measured at 4 °C in the LC state. This preference of RP2 for phospholipids in the LC state has also been confirmed by fluoresence microscopy. Indeed, RP2 binding significantly modified the shape of the LC domains of the DPPC monolayer in the phase transition. Moreover, the analysis of the binding parameters and the fluorescence microscopy data obtained with the α/β domain of RP2 demonstrates that this structural component of RP2 is not responsible for its preferential binding to phospholipid monolayers in the LC state. One can thus postulate that the β helix of RP2 (Figure S2) could play the major role in this preferential binding.

RP2 abolishes the paradigm that protein insertion is highly facilitated by phospholipid monolayer at a high area per molecule. Indeed, this protein prefers to bind to highly packed phospholipid monolayers in the LC/SC states. The present data thus suggest that the binding of peripheral proteins could be strongly influenced by the physical state of phospholipids. Therefore, the determination of the binding parameters of proteins onto phospholipid monolayers showing a phase transition should be performed with caution. Moreover, the analysis of the MIP is not sufficient because the same MIP value can be obtained with different slopes of ΔΠ as a function of Πi. The calculation of the synergy is very helpful because it can highlight different binding behaviors below and above the phospholipid phase transition. DPPC monolayers are useful in studying the effect of different physical states on protein binding because its phase transition occurs at room temperature, but this must be done with caution.

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ASSOCIATED CONTENT

S Supporting Information *

Illustration of the kinetics of RP2 binding when injected underneath a DSPC monolayer at 58 °C and of the 3D 9686


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structure of RP2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 418-682-7569. Fax: 418-682-8000. E-mail: christian. [email protected]. Author Contributions †

These two authors contributed equally to this work. They should thus be considered cofirst authors. They are consequently listed in alphabetical order. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are indebted to the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support. E.B. holds a postdoctoral fellowship from the Fonds de Recherche en Santé du Québec. P.C. was awarded a scholarship from PROTEO (Regroupement Québécois de Recherche sur la Fonction, la Structure et l’Ingénierie des Protéines), and E.D. was awarded a joint scholarship from the Canadian Institutes for Health Research (CIHR) and the E.A. Baker Foundation from the Canadian National Institute for the Blind (CNIB).

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