How To Decipher Protein and Peptide Selectivity for Lipids in

Chapter 6

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How To Decipher Protein and Peptide Selectivity for Lipids in Monolayers Sarah C. Bernier, Éric Demers, Line Cantin, Sylvain Bussières, and Christian Salesse* CUO–Recherche, Centre de recherche du CHU de Québec - Université Laval and Département d’ophtalmologie, Faculté de médecine, and Regroupement stratégique PROTEO, Université Laval, Québec (Québec) Canada *E-mail: [email protected]. Phone: (418) 682-7569. Fax: (418) 682-8000.

Cell membranes include a large number of different types of lipids and proteins. The properties of a large share of these proteins are modulated by membrane lipids. Various model membranes have been developed to overcome the complexity of cell membranes and to gather information on protein-lipid interactions. Lipid monolayers are very useful model membranes to study these interactions. The binding of proteins and peptides to lipid monolayers can be described by three different parameters: the maximum insertion pressure (MIP), the synergy and ΔΠ0. Several examples are presented in this book chapter to illustrate the usefulness of these binding parameters to decipher the selectivity of proteins for lipid monolayers. The binding parameters obtained with the protein Retinitis pigmentosa 2 (RP2) allowed demonstrating a large affinity of this protein for phospholipids with saturated fatty acyl chains and a repulsion for those with polyunsaturated fatty acyl chains. Accordingly, the monolayer binding of RP2 was shown to be dependent on the physical state of phospholipids. In addition, although RP2 showed no selectivity for polyunsaturated phospholipids, its binding was nonetheless reduced when these phospholipids are oxidized. Moreover, measurements were performed with lecithin retinol acyltransferase (tLRAT) whose membrane anchoring N- and

© 2015 American Chemical Society Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


C-terminal segments have been truncated. tLRAT showed a slightly larger preference for saturated phospholipids. However, the MIP of tLRAT with all phospholipids assayed was larger than the lateral pressure of membranes, thereby suggesting its extensive membrane binding even in the absence of its membrane anchoring segments. Finally, data of MIP and synergy are also shown for the C-terminal transmembrane segment of the protein R9AP to illustrate their usefulness to gain information of the membrane binding of such hydrophobic peptide segments.

Introduction Biological membranes are made of a complex array of lipids and proteins. The basic unit is made of a lipid bilayer, which includes a large collection of different lipids in mammals, such as sterols, sphingolipids and glycerophospholipids. The composition of the head group and fatty acyl chains of these lipids vary so extensively that cell membranes can incorporate more than 1000 different lipids (1). Cell membranes also include a large range of different transmembrane and peripheral proteins which allow various phenomena to take place such as signal transduction, active transport and so on (2). In fact, more than half of all proteins interact with membranes (3). The number of proteins regulated by lipid-protein interactions is quickly expanding (4). Integral membrane proteins experience selective interactions with lipids (5, 6) which modulate their function such as, for example, the stability and activity of Na,K-ATPase and Ca2+-ATPase, which is influenced by their lipid environment (7, 8), or the formation of rhodopsin photointermediates, which is controlled by the fluidity of its surrounding lipids (9–11). Moreover, growing evidence suggest that cytosolic proteins can be recruited to cellular membranes through lipid-protein interactions during processes such as cell signaling and membrane trafficking (2). For a number of these proteins, hydrophobic residues which are buried from water in membrane or which are important for the membrane binding of these proteins, have been identified (3). Membrane binding of a number of peripheral proteins can also involve electrostatic interactions with negatively charged phospholipids such as phosphoserine and phosphoinositol (12–14). The mechanisms by which proteins are recruited to and interact with various cell membranes are only beginning to be resolved using in vitro membrane binding studies with model membranes (2). Indeed, the complexity of cell membranes has led to the development of various model membranes, which facilitate the study of particular proteins and lipids. Lipid monolayers are very useful model membranes to study lipid-protein interactions (for a review, see (15–22)). It allows to control several physical parameters such as the lipid composition, the density of lipids, the surface pressure, the subphase content, etc. Moreover, there is a direct thermodynamic relationship between bilayers and monolayers (23, 24). This methodology can also allow to independently determine the affinity of proteins for lipids (25) which are asymmetrically distributed in the inner or the outer leaflet 110 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


of the membrane bilayer (26). This model membrane is also very well suited to study peripheral proteins since they do not protrude in the membrane deeper than the outer (or inner) monolayer of lipid bilayers. In addition, a large number of methods have been developed to characterize monolayers (25). In the present review, the monolayer binding parameters (maximum insertion pressure (MIP), synergy, ΔΠ0) have been used to decipher the selectivity of proteins and peptides for lipids. When the MIP is larger than the lateral pressure of membranes, one can postulate that the protein binds cell membranes. As a first example, measurements with the peripheral protein Retinitis pigmentosa 2 (RP2) with several different phospholipids are presented as well as the effect of the physical state of saturated lipids and of the oxidation of polyunsaturated lipids on this binding. Moreover, the use of truncated lecithin retinol acyltransferase (tLRAT) has allowed to gather information on the membrane binding of a protein whose membrane anchoring segments have been removed in order to find out whether it would nonetheless show an affinity for membranes. Finally, results of monolayer binding of the transmembrane segment of RGS9-1-Anchor Protein (R9AP), which serves to anchor this protein to the membrane, are shown to find out whether the membrane binding of such hydrophobic segments can be modulated by specific lipids.

Materials and Methods Materials The deionized water used for the preparation of buffer solutions was filtered using a Milli-Q direct water purification system (Millipore, Billerica, MA). Its resistivity and surface tension were 18.2 MΩ•cm and 72 mN/m at 20 °C, respectively. The phospholipid solutions were prepared in chloroform at a concentration of 0.1 mg/mL. They were purchased from Avanti Polar Lipids (Alabaster, AL) : 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-didocosahexaenoylsn-glycero-3-phosphocholine (DDPC), 1-palmitoyl,2-docosahexaenoyl-snglycero-3-phosphocholine (PDPC), 1-stearoyl,2-docosahexaenoyl-sn-glycero3-phosphocholine (SDPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-didocosahexaenoylsn-glycero-3-phosphoethanolamine (DDPE), 1,2-dipalmitoyl-sn-glycero-3phosphoserine (DPPS), 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-didocosahexaenoylsn-glycero-3-phosphoserine (DDPS). These lipids were used as received. Unsaturated phospholipids were stored with butylated hydroxytoluene (BHT) at a molar ratio of 200:1 (phospholipid:BHT) to prevent the oxidation of their fatty acyl chains (27). The C-terminal segment of human R9AP (DPRKALAAILFGAVLLAAVALAVCVAKLS; purity >92.2%; molecular mass 2865.6 g/mol) (further referred to as Cter-R9AP) was purchased from Peptide 2.0 (Chantilly, VA). 111 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Cloning and Expression of RP2 and tLRAT The human RP2 (30.8 kDa) construct cloned in the pGEX-4T3 plasmid to express a GST fusion protein (GST-RP2) was a kind gift from Dr. Alfred Wittinghofer (Max-Planck-Institut für Molekulare Physiologie, Germany). Human tLRAT (amino acids 31-196, 20.8 kDa, without its N- and C-terminal segments postulated to serve for its membrane anchoring) has been cloned in the plasmid pET11a as previously described (28). Briefly, RNA from freshly dissected retinal pigment epithelium was isolated with Tri-reagent (Sigma) and used for reverse transcription reaction with the RevertAid H minus first strand cDNA synthesis kit (Fermentas). A thrombin cleavage site and a His-tag of 10 histidines were also added to the C-terminus of tLRAT (tLRAT-His-tag) to facilitate its purification. The pET11a vector was linearized and subsequently ligated with the purified PCR product corresponding to tLRAT. Plasmid DNA of RP2 and tLRAT were transformed into E. coli BL21(DE3) RIPL (Novagen) and grown overnight in the LB medium until saturation. Then, fresh LB containing 50 μg/ml ampicillin and chloramphenicol was inoculated with the transformed cell culture and incubated at 37 °C under agitation (250 rpm) until A600nm of 0.3 (RP2) or 0.6 (tLRAT) is reached. Their expression was then induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) followed by an incubation during 5 h at 37 °C (RP2) or for 5 h at 30 °C (tLRAT). Bacteria were then sedimented by centrifugation at 3275 x g for 25 min. RP2 was not acylated in our experiments. Purification of RP2 and tLRAT RP2 and tLRAT were purified as previously described (19, 29). Pellets of bacteria containing GST-RP2 were resuspended in buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl2, 3 mM β-mercaptoethanol, pH 7.5), sonicated and centrifuged at 20,000 x g for 1 hour. The supernatant was then loaded on a GSTrap FF column (GE Healthcare) of 1 ml that had been preequilibrated with the same buffer. After an extensive washing of the column (at least 10 column volumes of buffer), RP2 was cleaved from GST with thrombin directly on the column for 16 hours at room temperature. Pure RP2 was then eluted using the same buffer containing instead 500 mM NaCl. Thrombin was removed from the eluent by connecting a Hitrap Benzamidine FF sepharose column (GE Healthcare) to the GSTrap column. RP2 was concentrated and the buffer was changed to 5 mM phosphate buffer, 100 mM NaCl (pH 7.4) using Amicon Ultra15. Pellets of bacteria containing the tLRATHis-tag were first disrupted by 3 cycles of freeze–thawing in the lysis buffer (100 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.8). After sonication, bacteria were centrifuged at 13,000 x g during 20 min at 4 °C. Supernatant was discarded and membranes were resuspended in the loading buffer (500 mM Tris, 5 mM imidazole, 0.1% sodium dodecyl sulfate (SDS), pH 7.8). These resuspended pellets were shaken for 1 h at room temperature to homogenize the suspension which was then centrifuged at 100,000 g for 30 min at 21 °C. The supernatant was then loaded on a 5 ml His-Trap column preequilibrated with 5 column volumes of loading buffer. Column was washed with 10 to 20 column volumes of washing buffer (500 mM Tris, 40 mM imidazole, 0.1% SDS, pH 7.8). Elution was achieved 112 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

with a buffer containing 500 mM Tris, 150 mM imidazole, 0.1% SDS, pH 7.8. In order to remove the SDS to perform monolayer measurements, the elution buffer was exchanged for a phosphate buffer (50 mM, pH 7.0) using an Econo-Pac 10DG column (Bio-Rad) previously equilibrated with this buffer. The purity of RP2 and tLRAT was larger than 98% as judged from polyacrylamide gel electrophoresis which was carried out using a Bio-Rad Mini-protean II electrophoresis cell with 15% acrylamide.


Determination of the Binding Parameters of tLRAT, RP2, and Cter-R9AP The determination of the MIP and synergy is very useful to decipher protein and peptide selectivity for particular lipids (20, 25, 27, 29–57). The surface pressure (Π) was measured by the Wilhelmy method using a tensiometer from Nima Technology (Coventry, U.K.) for RP2 and a DeltaPi4 microtensiometer from Kibron Inc for tLRAT and Cter-R9AP. The experimental setup was placed in a Plexiglas box with humidity control at room temperature. A 1200 μL home-built round Teflon trough (RP2) and a 500 μL glass trough from Kibron Inc (tLRAT and Cter-R9AP) were used for the monolayer binding measurements (30, 32, 33). The subphase buffer was 50 mM phosphate buffer (pH 7) for tLRAT, 5 mM phosphate buffer, 100 mM NaCl (pH 7.4) in the case of RP2 and 50 mM Tris, 150 mM NaCl, 5 mM β-mercaptoethanol (pH 7.4) for Cter-R9AP. The monolayer was prepared by spreading a few microliters of a solution of phospholipids at the surface of the buffer until the desired initial surface pressure (Πi) was reached. The waiting period for the film to reach equilibrium varies between 20 and, at most, 60 min, depending on the Πi, the type of lipid, the speading volume and the lipid concentration. Then, RP2, tLRAT or Cter-R9AP was injected underneath the lipid monolayer until an optimal, saturating final concentration of 0.5 µM, 79 nM or 5 µM was achieved, respectively (30, 32, 33). The kinetics of protein binding onto the phospholipid monolayer was monitored until the equilibrium surface pressure (Πe) was reached. The MIP and synergy are determined by injecting the protein or peptide at different Πi values of the lipid monolayer as described previously (20, 29). No difference was observed when measurements were performed in the presence or the absence of N2 or Ar when using polyunsaturated phospholipids for at least 2h. In addition, the same protein or peptide adsorption isotherms were obtained in the presence or the absence of BHT in the phospholipid solution (29).

Results and Discussion Measurement of the Binding Parameters of Proteins or Peptides to Lipid Monolayers As can be seen in the inset of Figure 1, the surface pressure of the phospholipid monolayer increases after the injection of the peptide into the subphase until equilibrium is reached (Πe). The larger Πi is, the smaller the surface pressure increase (ΔΠ ; ΔΠ = Πe - Πi). For example, ΔΠ of 20.7, 15.2 and 5.9 mN/m have been obtained after the injection of Cter-R9AP into the subphase of a DSPE monolayer at Πi of 7.8, 15.4 and 26.4 mN/m, respectively (inset of Figure 1). 113 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


As a consequence, a negative slope is obtained when plotting ΔΠ as a function of Πi (Figure 1). This plot allows to determine the MIP by extrapolating the regression curve to the x-axis (20, 29). A MIP value of 34.3 ± 1.5 mN/m has thus been obtained for Cter-R9AP in the presence of a DSPE monolayer (Figure 1). The MIP corresponds to the maximum surface pressure up to which proteins or peptides can insert into the monolayer and beyond which no insertion takes place. The synergy between the lipid monolayer and the protein or peptide is calculated by adding 1 to the slope of the plot of ΔΠ as a function of Πi (Figure 1) (29). The synergy is >0 when a positive interaction between the protein or peptide and the lipid monolayer is observed, whereas the synergy is

How To Decipher Protein and Peptide Selectivity for Lipids in

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