Probing Ras Effector Interactions on Nanoparticle Supported Lipid

Aug 20, 2008 - Daniel Filchtinski,† Christine Bee,† Tudor Savopol,‡ Martin ... Many biological processes take place in close proximity to lipid ...
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Bioconjugate Chem. 2008, 19, 1938–1944

TECHNICAL NOTES Probing Ras Effector Interactions on Nanoparticle Supported Lipid Bilayers Daniel Filchtinski,† Christine Bee,† Tudor Savopol,‡ Martin Engelhard,‡ Christian F. W. Becker,‡,§ and Christian Herrmann*,† Physikalische Chemie 1, Ruhr-Universita¨t-Bochum, Fakulta¨t fu¨r Chemie and Biochemie, Universita¨tsstr. 150, 44780 Bochum, Germany, and Max-Planck Institut fu¨r Molekulare Physiologie, Abt. Physikalische Biochemie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. Received March 10, 2008; Revised Manuscript Received July 28, 2008

Many biological processes take place in close proximity to lipid membranes. For a detailed understanding of the underlying mechanisms, tools are needed for the quantitative characterization of such biomolecular interactions. In this work, we describe the development of methods addressing the dynamics and affinities of protein complexes attached to an artificial membrane system. A semisynthetic approach provides the Ras protein with palmitoyl anchors, which allow stable membrane insertion, as a paradigm for membrane associated proteins that interact with multiple effectors. An artificial membrane system is constituted by nanoparticles covered with a lipid bilayer. Such a stable suspension allows for the characterization of the interaction between membrane-bound Ras and effector proteins using conventional fluorescence-based methods.

INTRODUCTION Many biological processes mediated by the specific interaction between proteins take place in close proximity to the plasma membrane or intracellular compartments. In particular, small GTP binding proteins such as members of the Ras, Rho, and Rab protein families are attached to membranes by insertion of hydrophobic anchor molecules covalently attached to their C-terminal region (1-3). These proteins interact with various proteins such as regulator and effector proteins, respectively, in order to further propagate the desired biological signal. Ras is a small GTP binding protein playing a central role in cellular signal transduction. It interacts in a nucleotide dependent manner with downstream effectors such as Raf kinase (4), RalGDS (5), and Nore1A (6), while its activity is regulated by extra- and intracellular inputs through nucleotide exchange factors and GTPase activating proteins (7). In all these respects, Ras is the prototype for a large number of GTP binding proteins from various subfamilies (8). Crucial for function of Ras is its attachment to the inner leaflet of the plasma membrane achieved through hydrophobic anchors at its C-terminus. For example, one farnesyl group and two palmitoyl groups are linked to the isoform H-Ras by post-translational processing (1). The distinct roles of those modifications were investigated in great detail (1, 9-11). The farnesyl anchor alone is not able to stably attach Ras to the membrane (1). Therefore, H-Ras is further modified with the two palmitoyl groups. Importantly, the plasma membrane attachment of Ras correlates with its biological function. Only properly bound to the plasma membrane can * Corresponding author. Tel: +49 234 3224173. Fax: +49 234 3214785. E-mail: [email protected]. † Ruhr-Universita¨t-Bochum. ‡ Max-Planck Institut fu¨r Molekulare Physiologie. § Current address: Technische Universita¨t Mu¨nchen, Department Chemie, Lichtenbergstr. 4, 85747 Garching and Center for Integrated Protein Science Munich.

H-Ras trigger cell signaling via the activation of the mitogenactivated protein kinase. The nonpalmitoylated H-Ras mislocalizes to the cytosol and fails to promote cell signaling (10). In contrast, constructs of H-Ras allowing for palmitoylation but not farnesylation suggested that the membrane attachment can be provided without the farnesyl group (11). Nevertheless, most of the biochemical studies on Ras activity and activity of many other proteins lack these modifications neglecting their potential impact and the influence of the membrane surface on interactions with effector proteins. An important issue for understanding the complex network of Ras signaling is its interaction with a defined but varying set of effector proteins depending on the particular task of Ras, such as initiating cell division, contributing to morphology changes, or leading to apoptosis (12). In this work, we establish a general method for analyzing protein-protein interactions at membrane surfaces using the interaction between Ras proteins and effectors as a paradigm for such processes. In order to develop a widely usable experimental setup, readily available fluorescence based methods should be used that allow the quantification of both Ras/effector affinities as well as dynamics. The artificial membrane system devised for this purpose is based on lipid coated nanoparticles with a defined size. Using a suspension of such lipidated nanoparticles allows the application of fluorescence assays previously developed in our laboratory in order to characterize Ras/effector interactions. The necessary membrane anchor is incorporated into Ras by expressed protein ligation (EPL) (13) where a chemically synthesized peptide carrying two palmitoyl groups is ligated to the C-terminus of Ras (14).

EXPERIMENTAL METHODS Peptide Synthesis. Solid phase peptide synthesis was performed manually using Fmoc-chemistry (15). Cleavage of ivDde-protecting groups was achieved with 3% hydrazine in DMF. Subsequent palmitoylation was carried out with 20 equiv of palmitoylchloride, 20 equiv of N-hydroxybenzotriazole, and

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Technical Notes

22 equiv of triethylamine in dichlormethane/dimethylformamide (3:1) for 4 h. Cleavage was achieved by 2.5% H2O and 5% triisopropylsilane in trifluoracetic acid (TFA). The crude product was precipitated with diethyl ether, dissolved in 50% aqueous acetonitrile (0.1% TFA), and lyophilized. Purification was carried out by RP-HPLC on C4-columns from Vydac using linear gradients of acetonitrile with 0.08% TFA in water. Bacterial expression and purification of the effector RBDs was described earlier (16). Briefly, the resuspended bacterial cell pellet was sonicated, the cell lysate was cleared by centrifugation, and the GST fusion proteins were purified by glutathione sepharose affinity chromatography followed by size exclusion chromatography. Preparation of Double Palmitoylated Ras. The DNA insert coding for C-terminally truncated H-Ras (1-180) was subcloned into the multiple cloning site of a pTXB1 vector (New England Biolabs) to express H-Ras (1-180) as fusion protein with a C-terminal intein from M. xenopi and the chitin binding domain from B. circulans. The vector was transformed into the E. coli BL21(DE3) strain. For protein expression, the 50 mL starting culture in LB containing 100 µg/mL Ampicillin was transferred to 10 L of the same media, and the media were incubated at 37 °C until an OD600 of 0.6 was reached. Expression of the fusion protein was induced by addition of 100 µM IPTG, and the culture was incubated at 37 °C overnight. The bacteria were harvested by centrifugation, and the cell pellet was resuspended in buffer A (25 mM Tris (pH 7.9), 100 mM NaCl, and 5 mM MgCl2) and 1 mM PMSF. Cell lysis was achieved by 10 min of sonication (Bendelin), and the lysate was centrifuged to remove insoluble cell material. The supernatant was applied to a 40 mL chitin column (New England Biolabs) equilibrated with buffer A. The chitin material loaded with fusion protein was washed with 5 column volumes of buffer A and incubated for three days in buffer A containing 250 mM MESNA (sodium 2-mercaptoethane sulfonate) at 4 °C in order to form the H-Ras(1-180)-thioester. The cleaved protein was separated from the chitin beads, concentrated by ultrafiltration (Vivaspin) to a volume of 5 mL and further purified by size exclusion chromatography (Superdex 75 26/60, Pharmacia). The fractions containing H-Ras-thioester were pooled and concentrated using ultrafiltration (Vivaspin) to a final concentration of 70 mg/mL. The protein was shock frozen in liquid nitrogen and stored at -80 °C. Three hundred micromolars of the protein were incubated with 5.4 molar excess of the double palmitoylated 2Palm-3PPOpeptide in the presence of 200 mM MESNA for 4 days. The ligation product was selectively precipitated from the reaction mixture by the addition of 400 mM ammonium sulfate, redissolved in buffer A, shock frozen in liquid nitrogen, and stored at -80 °C. Peptide and protein masses were determined by electrospray ionization mass spectrometry on an LCQ Advantage Max (Finnigan) operating in positive ion mode. The molecular masses were deconvoluted from the ion spectra. Preparation of Lipidated Nanoparticles. The amount of 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC, Sigma) required in order to achieve a 40-fold excess of lipid surface over the nanoparticle surface (26 mg of DOPC) was dissolved in 2 mL of chloroform, dried under a stream of nitrogen, and subsequently dried for 2 h in vacuum. The lipid film was rehydrated in 2-4 mL of buffer A flushed with argon. The suspension was then sonicated and cooled on ice (Brenson Sonifier 250 Cup Horn) until it was clear and then ultracentrifuged (Beckman) at 100,000g. The clear supernatant containing small unilamellar vesicles (SUV) was used for nanoparticle preparation. Then, 100 µL of a 50 mg/mL stock solution of silica nanoparticles (Micromod, Germany) was washed with 1 mL

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methanol, 1 mL 1 M KOH, and five times with buffer A, respectively. Separation of nanoparticles from supernatant was achieved by centrifugation (table top centrifuge, 5000 rpm). After washing, the nanoparticle suspension was mixed with the supernatant of the lipid preparation, flushed with argon, and incubated under gentle rotation for 2 h at room temperature. After lipidation, 1 mL of the nanoparticle suspension was washed twice to remove excess DOPC and mixed with 144 µg of the double palmitoylated H-Ras to cover (calculated) 20% of the nanoparticle surface. For this calculation, a particle radius of 130 nm, a particle concentration of 2.5 mg/mL, a particle density of 2 g/cm3, and a footprint of Ras of 9 nm2 were assumed. The suspension was incubated at room temperature overnight under gentle rotation, washed twice to remove the nonmembrane bound protein, and used for the GDI-assay and stopped flow experiments in concentrations of 0.05 µM and 0.25 µM, respectively. The characterization of particle size distribution was achieved by the dynamic light scattering technique. Samples of DOPC small unilamellar vesicles and lipidated nanoparticles were filtered using 0.45 µm filters, whereas blank nanoparticles were sonicated prior to addition into the cuvette. For the measurement, a He-Ne-Laser (633 nm) with a power of 35 mW was used, and the signal was collected at 90° using a four-quadrant detector (ALV 5001, Langen, Germany). The autocorrelation traces were fitted using the ALV software package, calculating the weight averaged particle size (17). Measurement of Interactions. In order to detect the interaction between Ras proteins and effectors in solution, various fluorescence based methods were developed previously. The N-methyl-anthraniloyl (mant) group attached to the guanine nucleotide serves as a fluorescence label in these experiments. Ras, which is tightly bound to GDP after the purification, is loaded with mantGppNHp by addition of a 5-fold excess of this nucleotide and 10 mM EDTA, and overnight incubation at 4 °C. The nucleotide excess was removed by Zeba Desalt Spin Columns (Pierce) following the desalting instructions of the manufacturer. Loading was examined by C18 reversed phase HPLC. The so-called GDI-assay (guanine nucleotide dissociation inhibitor) takes advantage of a large fluorescence change upon dissociation of the mant-nucleotide after addition of a large excess (1000-fold) of nonfluorescent GppNHp. The time course of the decrease in fluorescence is measured by a Perkin-Elmer LS 55 fluorescence spectrometer with an excitation wavelength of 360 nm and an emission wavelength of 450 nm at 37 °C using buffer A. A single exponential function is fitted to the fluorescence traces yielding the observed rate constant kobs representing the rate of the dissociation of mantGppNHp and Ras. Dependent on the binding of Ras effectors such as Raf, RalGDS, and Nore1A, the nucleotide dissociation rate is decreased. The observed rate constants are plotted against the concentration of the effector, and a quadratic function is fitted to the data in order to yield the KD values of the Ras/effector interaction as described earlier (16, 18). In order to obtain, in addition to the KD values, the individual rate constants for the Ras/effector association and dissociation, the stopped flow method was used (19, 20). Again, this is a well established method for dynamic measurements of Ras/ effector interactions in solution. Using a stopped flow apparatus (Biologic, Grenoble, France), Ras loaded with mantGppNHp and effector solutions were rapidly mixed and the fluorescence traces recorded. A 10-fold excess of the effector ensures pseudofirst-order conditions. To improve the fluorescence signal-tonoise ratio, fluorescence resonance energy transfer (FRET) was employed by the use of Ras · mantGppNHp and the tryptophan mutant Nore1A M305W. The largest fluorescence changes were observed at low temperatures, and therefore, kinetic measurements were performed at 10 °C in buffer A using an excitation

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Figure 1. Synthesis of a double palmitoylated Ras variant. (A) Solid phase synthesis of a polymer-modified palmitoylated peptide (2Palm-3PPO peptide with n ) 3; mass, 3196.1 Da) and native chemical ligation of Ras1-180 thioester with this peptide. (B) SDS-PAGE of the ligation mixture. Lane 1, Ras-MESNA thioester; lane 2, ligation mixture after one day; lane 3, ligation mixture after four days. (C) ESI-MS of the purified ligation product Ras2Palm-3PPO. The deconvoluted spectrum is shown on the right and contains 2 peaks corresponding to Ras-2Palm-3PPO with a theoretical mass of 23519.3 Da and missing its N-terminal methionine with a theoretical mass of 23388.1 Da.

wavelength of 290 nm and a fluorescence light cut off filter of 420 nm. To determine the dissociation rate constant, the double palmitoylated Ras immobilized on lipidated nanoparticles was incubated with Nore1A M305W at a concentration of 2 µM and rapidly mixed in the stopped flow apparatus with Ras loaded with the nonfluorescent GppNHp to displace the effector.

RESULTS Palmitoylated Ras by Expressed Protein Ligation. In order to study Ras/effector interactions under conditions close to their native environment, Ras was equipped with an artificial membrane anchor. Figure 1A summarizes the strategy for the synthesis of double palmitoylated Ras. H-Ras encompassing residues 1-180 is expressed as a fusion construct with an intein and a chitin binding domain in E. coli and purified by affinity chromatography. By incubation with MESNA, H-Ras (1-180) is cleaved off harboring a reactive thioester at its C-terminus (21). In parallel, a peptide is synthesized with a cysteine at the N-terminus and two palmitoyl groups coupled to lysine side chains according to Figure 1A. In order to improve handling properties and solubility of this peptide, a polyethylene glycol polyamide oligomer (PPO) comprising three PPO subunits was introduced at the C-terminus without affecting the membrane binding properties (Figure 1A) (22, 23). Peptides containing shorter PPOs comprising two or only one subunit have been prepared, but these shorter PPOs do not efficiently solubilize the lipidated peptide in aqueous buffers during purification and

ligation. This water-soluble 2Palm-3PPO-peptide is ligated to Ras using a large excess of MESNA as ligation mediator (Figure 1A). The ligation reaction proceeds slowly but reaches a yield of 90% after four days (Figure 1B). LC-MS analysis and deconvolution of the resulting ESI spectrum shows two mass peaks that correspond to the ligation product Ras-2Palm-3PPO (calculated mass: 23519.3 Da) with and without the N-terminal methionine (∆mass: 131.2 Da, Figure 1C). The product was precipitated by the addition of 400 mM ammonium sulfate and separated from unligated Ras protein that remained soluble. The redissolved ligation product was loaded with the fluorescent GTP analogue mantGppNHp by incubation with a 5-fold excess of this nucleotide in the presence of 10 mM EDTA. Ras Attached to Nanoparticle Supported Lipid Bilayers. As a membrane mimic, we chose nanoparticle supported lipid bilayers that were prepared using a procedure similar to that described by Richter et al. (24). Small unilamellar vesicles (SUV) made of DOPC by sonication collapse onto silica beads. The size and integrity of the particles were characterized by dynamic light scattering studies and autocorrelation analysis (Figure 2A). The polydispersity index of 0.10 and 0.08, for blank and lipidated nanoparticles, respectively, indicates a narrow distribution of the particle size before and after lipidation. The hydrodynamic radius of the lipidated particles was measured to 130 nm (Figure 2B). This nanoparticle supported lipid bilayer system should be well suited for membrane anchor insertion of Ras and other proteins carrying palmitoyl groups as these

Technical Notes

anchors are shorter than half the bilayer thickness. A high mechanical stability without loss of fluidity is provided (25), and these particles are easy to handle, e.g., for changing buffers or concentrating the particles by gentle centrifugation and resuspension. A large, defined surface area can be offered with varying lipid composition. Double palmitoylated Ras was inserted into this artificial membrane by the addition of the protein amount required for covering of the desired fraction of lipid surface. A reasonable upper limit of coverage seemed to be 20%, and the required protein concentration [Ras] was calculated according to eq 1 [nanoparticle] · Ananoparticle · 20% (1) [Ras] ) F · Vnanoparticle · ARas · NA where ARas is the footprint of the Ras-protein estimated from its structure to be 9 nm2 and NA the Avogadro constant. [nanoparticle] is the concentration of nanoparticles in mg/mL, F is the density of the nanoparticles taken from the manufacturer information, which is 2 g/cm3. Vnanoparticle and Ananoparticle are the volume and the surface area of the single nanoparticle, respectively, that were determined from the radius. After incubation overnight, 25% of added Ras (1 µM) was immobilized as judged by SDS-polyacrylamide electrophoresis of samples from the separated phases. This corresponds to the amount of ca. 1000 molecules of Ras per lipidated nanoparticle. Noninserted Ras was removed by two washing cycles. Affinity of Ras/Effector Interactions at Artificial Membranes. In order to characterize the interaction of Ras immobilized on the lipidated particles with other proteins, we employed fluorescence based methods previously established for studying Ras/effector binding in solution. In order to perform the GDI assay (16, 18), Ras immobilized on lipidated nanoparticles and loaded with N-methyl-anthraniloyl-GppNHp (mantGppNHp) is incubated with effector proteins at defined concentrations. The mant-nucleotide is excited at 360 nm, and fluorescence is detected at 450 nm. The particle suspension shows no detectable sedimentation even after 1 h, and therefore, continuous stirring is not required. Intriguingly, light scattering of the milky particle suspension only weakly interferes with fluorescence excitation and emission. Compared to fluorescence measurements in solution with approximately 50% fluorescence decrease after mantGppNHp dissociation, the scattering background in the presence of lipidated particles still gives rise to ca. 20% change of the initial signal. From this, approximately equal contributions to the signal by light scattering and fluorescence can be estimated (Figure 3A). The affinity of three different effector proteins was titrated with help of the GDI assay. This assay relies on the inhibition of the dissociation of the guanine nucleotide by effector binding, and an increasing Ras/effector complex concentration is reported by a decreasing dissociation rate of the nucleotide (26). A single exponential fit (Figure 3B) yields the observed rate constant, which is plotted versus effector concentration in Figure 3C and D. From this, the KD value of the Ras/effector complex is obtained by fitting a quadratic equation. An overall error on the KD values of 30% is estimated from the experimental errors on the kinetic data and the measurements of the protein concentrations. The results are listed in Table 1 together with the data obtained in solution. Only an increase by a factor of 1.5 to 3 is observed for the KD values of all three effectors when measuring membrane bound Ras. It should be stressed here that only the Ras binding domain of the effectors was used in these studies. Other domains of the effector adjacent to the RBD as the cysteine rich region in Raf and Nore1A may contribute to membrane interaction as well (27, 28). Dynamics of Ras/Effector Interactions at Artificial Membranes. For measuring the dynamics of the interaction between effector protein and Ras attached to an artificial membrane

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Figure 2. Dynamic light scattering and particle size distribution. (A) Autocorrelation curves of small unilamellar vesicles, naked nanoparticles, and lipidated particles (from left to right). (B) The histogram shows the hydrodynamic radii of these three species where the width of the size distribution (at half-maximum value) is indicated by the vertical lines at the top of the bars.

through its C-terminal palmitoyl anchors we used a stopped flow apparatus (biologic, France). Again, the presence of lipid bilayer covered nanoparticles does not pose any technical problems. But, as in solution, the relative change of mant-fluorescence upon formation of the Ras/effector complex is much smaller compared to the mant-nucleotide dissociation measured in the GDI-assay. It is less than 10% even in the absence of particles and decreases to 3% in the presence of our nanoparticle preparations. Therefore, we used a FRET couple based on a Trp residue introduced at position 305 of Nore1A (donor) and the mant-nucleotide (acceptor) bound to Ras in order to obtain a clear fluorescence readout for complex formation. Fluorescence was excited at 290 nm and detected through a 420 nm cutoff filter. Fluorescence changes were much larger at lower temperatures, and therefore, we measured at 10 °C. The fit to the fluorescence time course by a single exponential equation did not yield satisfying residuals. Therefore, a double exponential function was used, and the observed rate constant of the faster phase was plotted against effector concentration (Figure 4A). The linear plot of the observed rate constants versus Nore1A concentration yields a value of 1.5 µM-1 s-1 for the apparent association rate constant kon (Figure 4B), which compares to 8.9 µM-1 s-1 for Nore1A wild type in solution in the absence of NaCl (29). Notably, the second phase is almost as large in amplitude as the first one, and it is 15-20-fold slower than the fast reaction. In control stopped flow experiments using the same buffer, the same Nore1A mutant and Ras loaded with mantGppNHp but without the palmitoyl modification, we observed single exponential fluorescence changes yielding a kon value of 3.3 µM-1 s-1. There is no slow phase of association of Ras/Nore1A in solution. The dissociation rate constant koff can be obtained from the intercept of the plot in Figure 4B, and it is obvious from this that the koff value is close to zero and therefore not well defined

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Figure 3. Ras/effector affinity measured by GDI-assay. The basis of the GDI assay is the dissociation of mantGppNHp from Ras achieved by the addition of an excess of nonfluorescent nucleotide and indicated by the strong decrease of fluorescence in buffer. (A) Ras-2Palm-3PPO bound to lipidated nanoparticles with a decay amplitude of 24% (upper curve) and in solution (lower curve) with a decay amplitude of 60%. (B) Fluorescence traces of the GDI-assay with the double palmitoylated Ras immobilized on lipidated nanoparticles in the presence of 0 µM, 0.5 µM, and 2.0 µM Raf-RBD (from bottom to top). (C) GDI plot for Ras-2Palm-3PPO with Nore1A (O) and RalGDS (b) as effectors, and (D) with Raf on lipidated nanoparticles each, fitted with a quadratic equation yielding the KD values given in Table 1. Table 1. Ras/Effector Affinitya effector Nore1A M305W RalGDS Raf

DISCUSSION

KD (µM) in solution

KD (µM) on lipid. part.

0.3

0.45

3.5 0.13

3.2 0.3

a KD values for Ras · mantGppNHp/effector complexes in solution (28, 30-32) and on lipidated nanoparticles, respectively, obtained from GDI experiments at 37 °C, with an overall error of 30%. The solutions in all experiments contained 100 mM NaCl and were buffered with 25 mM Tris at pH 7.9 and 5 mM MgCl2.

by this experiment. Nevertheless, this rate constant can be determined with high precision by a displacement experiment. The preformed complex of Ras · mantGppNHp and Nore1A(M305W) is mixed with an excess of nonfluorescent Ras. The overall rate of the formation of the nonfluorescent complex is controlled by the dissociation of the preformed complex. In Figure 4C, the observed fluorescence time course is shown representing the dissociation of the Ras · mantGppNHp/Nore1A M305W complex. A single exponential equation plus linear slope is fitted to this curve, which yields a value of koff ) 0.15 s-1 (0.18 s-1 in solution) (30) for the dissociation of the complex. The slower linear decrease of fluorescence can be attributed to photo bleaching as demonstrated in control experiments. Together, from these stopped flow experiments, a KD value of 0.10 µM is calculated that fits well to the GDI results above when taking the temperature difference into account.

Many biochemical studies report on the characterization of protein/protein interactions, but most of them ignore the membranous environment in the case when one of the partner proteins is usually bound to a membrane and neglect the possible implications for binding affinity and dynamics. With the present work, we provide a versatile method that allows one to take into account the effect of membranes on interactions between proteins of which one is attached to the membrane. We show here that conventional fluorescence techniques developed for studies in solution are also applicable for measurements of Ras/ effector interactions on nanoparticle supported lipid bilayers. An efficient synthesis of native-like small GTP binding proteins is the crucial requirement for investigations of protein interactions at membrane, if possible with the option to vary the composition of the hydrophobic anchorage. Here, expressed protein ligation was successfully employed in order to link a small synthetic peptide equipped with the desired hydrophobic anchor moieties to the Ras protein synthesized in bacteria. The Ras protein carrying two palmitoyl groups at the C-terminus was obtained in high yield, was easy to purify, and was shown to be intact in terms of nucleotide and effector binding, respectively. As an artificial membrane system, we selected nanoparticles covered with DOPC lipids. As shown by others, such particles are straightforward to prepare, and they are covered homogenously by a lipid double layer. Incubation with palmitoylated Ras leads to attachment of the protein to the lipid surface by insertion of the hydrophobic moieties into the bilayer. Control experiments with unmodified Ras and other proteins such as the effectors clearly prove that there is no tendency of these

Technical Notes

Figure 4. Interaction dynamics characterized by stopped flow. (A) Ras2Palm-3PPO in complex with mantGppNHp and bound to lipidated nanoparticles was mixed with varying concentrations of Nore1A M305W in a stopped flow apparatus. Double exponential equations were fitted to the fluorescence traces recorded. (B) The observed rate constants of the fast phase are plotted versus the concentration. The slope of the linear fit yields the kon value of 1.5 µM-1 s-1. (C) Dissociation of the complex between double palmitoylated Ras · mantGppNHp on nanoparticles and Nore1A M305W. For displacement, an excess of GppNHp loaded Ras was mixed with the preformed complex. The exponential part of the curve yields the koff value of 0.15 s-1.

proteins to stick to the lipidated particles. The particles can be spun down and resuspended, which facilitates separation of bilayer-bound protein from soluble proteins as well as changing buffer conditions, e.g., during nucleotide exchange in Ras. The suspension of these particles with and without Ras attached is stable for hours as no sedimentation or coagulation is observed, and moreover, dynamic light scattering demonstrates a low degree of polydispersity and indicates an average particle radius of 130 nm. Thus, a robust and versatile protein/membrane system is provided, suitable for investigations with various biophysical techniques including spectroscopy. We aimed at a quantitative characterization of Ras/effector interactions beyond their qualitative verification by experiments such as cosedimentation. Surprisingly, we found that conventional fluorescence spectroscopy is only perturbed a little by interference of light scattering originating from the nanoparticle suspension. Therefore, we were able to employ fluorescence assays previously established for studies on Ras/effector complexes in solution. For three different effector proteins, the GDI

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assay with Ras attached to lipidated particles yielded values of the equilibrium dissociation constant (KD) that are similar to the respective values obtained in solution (Table 1). These results demonstrate on the one hand that the Ras binding domains of the effectors used in these studies do not contribute to membrane binding and on the other hand that the membrane environment does not change the ability of Ras to bind to the effector. There is no significant interference and no enhancement either. The GDI assay used in this pilot study is an indirect method as it reports the increasing saturation of Ras/effector binding through a decreased rate of nucleotide exchange. This is a big advantage here since artifacts such as unspecific effector binding to the particles can be excluded. The GDI effect most clearly indicates a specific interaction between Ras and effector protein showing the same characteristics as those in solution and emphasizing the full integrity of our Ras/membrane system. We utilized stopped flow with fluorescence detection in order to determine the rate constants of Ras/effector interactions. After mixing Ras · mantGppNHp attached to nanoparticle supported lipid bilayers and the Trp mutant of Nore1A (M305W), the observed fluorescence traces were analyzed. There is a biphasic behavior with two rate constants, which are different by more than a factor of 10. The concentration dependence of the faster phase yields a kon value comparable to Ras/Nore1A association in solution where the value for nonmodified Ras is two times larger. The slow phase of the association suggests the presence of another species of Ras immobilized on the lipidated nanoparticles, which seems to be not directly accessible for Nore1A binding. Notably, the dissociation is a single exponential and indicates a homogeneous Ras/Nore1A complex population. The koff value extracted from the single exponential fit to the displacement experiment is almost identical to the value obtained for the complex in solution. Similar to the equilibrium data, we can conclude that the results on the rate constants for association and dissociation of the Ras/Nore1A complex are not significantly different when soluble and membrane bound Ras are compared apart from an unknown fraction of Ras binding Nore1A 10 times more slowly. In summary, we demonstrated the application of readily available fluorescence techniques for the investigation of protein/ protein interactions at the surface of an artificial membrane system based on nanoparticle supported lipid bilayers. Not only protein complex affinity but also binding dynamics can be quantified. The interaction of double palmitoylated, membrane bound Ras with the RBD of three different effectors does not show significant differences to the results obtained from studies in solution. In a next step, this experimental setup can be used in order to address the role of other protein domains located next to the RBD of the respective effector proteins. To this end, we provide a widely usable assay that will allow the determination of the influence of membrane composition one the one hand and lipid binding domains such as C1 and PH domains on the other hand on the overall interaction of protein complexes located at lipid membranes.

ACKNOWLEDGMENT We are grateful for financial support by Gerhard C. Starck Stiftung (grant to D.F.), Deutsche Forschungsgemeinschaft (SFB 642), and Fonds der chemischen Industrie.

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