Revealing conformational substates of lipidated N-Ras protein by pressure modulation

Abstract

Regulation of protein function is often linked to a conformational switch triggered by chemical or physical signals. To evaluate such conformational changes and to elucidate the underlying molecular mechanisms of subsequent protein function, experimental identification of conformational substates and characterization of conformational equilibria are mandatory. We apply pressure modulation in combination with FTIR spectroscopy to reveal equilibria between spectroscopically resolved substates of the lipidated signaling protein N-Ras. Pressure has the advantage that its thermodynamic conjugate is volume, a parameter that is directly related to structure. The conformational dynamics of N-Ras in its different nucleotide binding states in the absence and presence of a model biomembrane was probed by pressure perturbation. We show that not only nucleotide binding but also the presence of the membrane has a drastic effect on the conformational dynamics and selection of conformational substates of the protein, and a new substate appearing upon membrane binding could be uncovered. Population of this new substate is accompanied by structural reorientations of the G domain, as also indicated by complementary ATR-FTIR and IRRAS measurements. These findings thus illustrate that the membrane controls signaling conformations by acting as an effective interaction partner, which has consequences for the G-domain orientation of membrane-associated N-Ras, which in turn is known to be critical for its effector and modulator interactions. Finally, these results provide insights into the influence of pressure on Ras-controlled signaling events in organisms living under extreme environmental conditions as they are encountered in the deep sea where pressures reach the kbar range.

Proteins generally and intrinsically can exist in a multitude of conformational substates at ambient conditions and are endowed with conformational fluctuations; i.e., a protein molecule in a given state spans a larger number of conformational substates (CS) (1). A prominent example is myoglobin, which has been shown to exist in several functional substates important for ligand binding (2). There are two types of inherent protein motions: equilibrium fluctuations, which are motions between CS in equilibrium, and nonequilibrium transitions known as functionally important motions, which are motions that lead from one state to another state in a protein, respectively. The identification and study of functionally relevant CS of proteins is not straightforward, largely because the low fractional populations at ambient conditions are not easily detectable by spectroscopic means (3). As a result, many events in protein conformational dynamics go unnoticed that might be decisively important for protein function, such as binding, catalysis, and protein–protein or protein–nucleic acid interactions (2, 3).

In order to gain insight into the free energy landscape of proteins, temperature and chemical perturbations (including pH, ionic strength, denaturing agents) are often employed to shift the population equilibrium and characterize the otherwise rare conformational substates. Temperature produces simultaneous changes in internal energy and volume, whereas the effect of a denaturant depends on its binding properties (4, 5). Because some of the CS might be separated by small energy differences only, separation by temperature or chemical perturbation can be difficult to achieve. Conversely, pressure as the third fundamental thermodynamic variable controls the population of protein CS by affecting the equilibrium through volume differences (3, 5–11). Pressure favors states with smaller partial volumes, which are often more solvated than the native state. Electrostriction, hydration of newly exposed polar or nonpolar residues, and loss of cavities, which arise due to packing defects in the folded ensemble of proteins, are some of the effects observed upon pressurization of proteins. (3, 9, 11). According to Akasaka et al. (11), low-lying excited CS generally have a lower molar volume, such that application of pressure will populate such states that may be intimately involved in function. Thus, pressure provides an elegant means to populate excited states for spectroscopic studies.

Conformational selection has emerged as an alternative to the “induced fit model” and postulates that all CS of proteins preexist in solution in different regions of the energy landscape (featuring several minima)—not always easily detectable, however (12–14). Conformational selection has been observed for protein–ligand, protein–protein, and protein–DNA/RNA interactions, where the binding of different partners leads to a redistribution of the already existing conformations (i.e., a population shift). Conformational selection is likely to be also the critical mechanism for the functioning of signaling molecules such as Ras and Rho G-proteins (15). The conformational dynamics of these proteins is linked to signaling pathways where many functional states crucial for signaling may not be populated enough to be easily characterized. This seems to hold true also for members of the Ras superfamily. The Ras proteins act as membrane-associated molecular switches in the early steps of signal transduction pathways associated with cell growth or differentiation (16, 17). For biological activity, the Ras proteins need to be anchored to membranes, which is achieved by posttranslational lipidation. The lipid anchors are connected to the G domain by a linker region comprising a hypervariable region (HVR) (Fig. 1A). Ras shuttles between a GDP (inactive) and a GTP (active) bound state. When bound to GTP, it interacts with a variety of effectors. This cycle is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which stimulate GTP loading and hydrolysis, respectively. Both these states of the protein are distinguished by conformational differences mainly in the switch I and II regions (Fig. 1 B and C) (18–22).

Fig. 1.

(A) Schematic representation of the switch regions, α-4 and α-5-helix and the HVR domain including the lipid anchors (gray) in the N-Ras protein in the GDP (green) bound form. The structure of the G-domain was adopted from the PDB entry 4Q21. Highlighted are the switch I [30–38; blue] and switch II (residues 59–67; yellow) regions, the α-4-helix (residues 126–137; red) and the α-5-helix (residues 151–171, pink). (B and C) Representation of the flexibility of the switch regions in Ras-GTP forms (B) as compared to Ras-GDP forms (C). It can be clearly seen that in the available X-ray structures these loop regions tend to be in defined positions when fixed by the influence of the γ-phosphate of the GTP nucleotides, whereas the GDP forms feature a large number of different conformations of the switch regions (the C terminus is in the back). Images prepared with PyMOL (http://www.pymol.org) using the PDB ID codes 5P21, 1P2T, 2RGE, 3lBH, 1CTQ, 3RAP, 1U8Y, 2PMX, 3RAB, and 1YU9 for GTP and 4Q21, 1IOZ, 3CON, 1KAO, 1U8Z, 1XTQ, 1X1R, 3KKQ, 1Z0A, and 2GF9 for the GDP forms.

Ras proteins are thought to sample multiple conformations. Active Ras seems to exhibit diverse conformations of switch I and II in solution, among which mainly two conformations of switch I have been characterized so far, denoted as state 1 and state 2 (19). State 1 corresponds to a GTP-bound but inactive state and interacts with GEFs. In contrast, state 2 can interact with different effector proteins (20, 21). Notably, signaling of these lipidated proteins takes place in the presence of membranes, and the nucleotide binding state influences the partitioning behavior (23) and orientation of the protein at the lipid interface (24–28). Lipid interfaces and protein partitioning into lipid membranes are expected to have a marked effect on the CS of the membrane-associated signaling proteins, but this possibility has hardly been explored.

Here we employ hydrostatic pressure as a tool to shift the conformational equilibrium of N-Ras to sufficiently populate low-lying excited substates of Ras, which might be decisively important in signaling. To this end, we have studied the lipidated protein N-Ras HD/Far (hexadecyl/farnesyl) in its GDP and GTP analog (GppNHp) bound form (denoted as N-Ras (GDP) and N-Ras (GTP), respectively, hereafter) in the presence and absence of a canonical lipid raft membrane (29) by means of high pressure FTIR spectroscopy. Currently only few high pressure studies have been performed for probing conformational equilibria (6, 7, 11, 21). Our results show that both the nucleotide binding state of the protein and the presence of the lipid membrane have a drastic effect on the conformational dynamics and selection of conformational substates of the protein. We reveal a conformational substate appearing upon membrane binding that had not been detected before. Complementary polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) and infrared reflection absorption spectroscopy (IRRAS) data were taken to reveal orientational changes of the protein at the lipid interface.

Results

Effect of Pressure on the Conformational Substates of N-Ras Modulated by the Bound Nucleotide State.

We first measured high pressure FTIR spectra of N-Ras in its different nucleotide states in bulk solution in the pressure range from 1 bar to 10 kbar (1,000 MPa) and analyzed the pressure-induced changes in the amide I′ region of N-Ras (Fig. S1). At 1 bar, N-Ras (GTP/GDP) shows an asymmetric amide I′ band with predominant α-helices and β-sheets, in good agreement with the X-ray crystal structure data (Table S1) (30). The N-Ras protein retains its compact tertiary structure even at high pressures of 10 kbar, implying a remarkably high pressure stability of the protein, which makes it suitable for such kind of studies: Only small but significant changes in spectral shape are visible upon pressurization. The second derivative spectra of the amide I′ band allow a better visualization of the changes (Fig. 2 A and B). For N-Ras (GDP), the α-helical subband shows a splitting at around 3.5 kbar suggesting pressure-induced changes in the α-helical/turn region of the protein forming solvent exposed turns/loops and solvated α-helices (9, 31, 32), thus leading to clearly developed subbands at higher pressures. Clearly, a slight decrease in the intensity of intramolecular β-sheets with concomitant increase of a band at the lower wavenumber side, which is typical for exposed β-sheets, is observed, the effect being less pronounced for the GTP-bound form.

Fig. 2.

Pressure-induced changes in the second derivative amide I′ spectra for (A) N-Ras GDP and (B) N-Ras GTP in bulk, (C) N-Ras GDP in the presence of the lipid bilayer, and D) N-Ras GTP in the presence of the lipid bilayer.

The pressure-induced secondary structural changes can be more easily monitored by means of a quantitative fit analysis of the amide I′ band region using mixed Gaussian and Lorentzian line shapes for deconvolution (9, 31, 32). As can be deduced from Fig. 3 A and C, even at high pressures, the N-Ras (GDP) and (GTP) still retain compact structures with only minor conformational changes occurring. For N-Ras (GDP), a decrease is seen for helices by about 4%, intramolecular β-sheets by 6%, turns by 5%, and a concomitant increase for random coil (unordered) structures by 3%, and exposed β-sheets by 10%. Like the GDP-bound form, N-Ras (GTP) shows only minor changes in the secondary structures—i.e., a decrease in helices by 2%, intramolecular β-sheets by 3% and turns by 3%, accompanied by an increase of random coils by 3% and exposed β-sheets by 4%. These data indicate a pressure-induced shift toward more solvent exposed β-sheet/disordered structures. The differences (albeit small) observed between the two forms apparently connote the influence of the bound nucleotide on the packing mode of the protein, which determines the amplitude of pressure-dependent conformational changes. Assuming equilibrium between the two states, the volume change ΔV⁰ (1 bar till 10 kbar) accompanying the conformational change can be determined (see SI Text). A ΔV⁰ value of -24 ± 6 mL/mol is obtained for N-Ras (GDP) from the pressure-dependent changes of turns/loops, random and exposed β-sheet structures. The pressure-induced changes are similar but smaller in N-Ras (GTP), for which a ΔV⁰ value of about -14.7 mL/mol is obtained. Such volume changes are a factor of 3–5 smaller than those observed upon pressure-induced unfolding of proteins (9).

Fig. 3.

Secondary structural changes as a function of pressure at T = 25 °C for N-Ras GDP and N-Ras GTP (A and C) in bulk, for N-Ras GDP in the presence of lipid bilayer (B), and for N-Ras GTP in the presence of the lipid bilayer (D). Pressure-induced wavenumber shifts for the various secondary structure are also shown for N-Ras GDP (E) and GTP (G) in bulk, for N-Ras GDP in the presence of the lipid bilayer (F), and for N-Ras GTP in the presence of the lipid bilayer (H).

Because pressure generally causes compression of the chemical bonds equivalent to changes of the force constant, linear pressure-induced frequency shifts (so-called elastic effects) are frequently observed (9, 31, 32). The inactive form of N-Ras in bulk solution (Fig. 3E) shows a blue shift for turns/loops; all other structures exhibit red shifts. The α-helical subband observed at 1,658 cm^-1 remains intact up to 3.5 kbar, where it forms two shoulders, one increasing up to 1,663 cm^-1 (solvated turns and loops), the other decreasing to 1,654 cm^-1 (solvated helices). The position of the band for intramolecular β-sheets observed at 1,637 cm^-1 remains constant up to 3.5 kbar, beyond which a subband at lower wavenumbers, which is typical for solvent exposed β-sheets, starts appearing (also shown in Fig. 2A). For active N-Ras (GTP) in bulk solution (Fig. 3G), a similar scenario is observed but with the exposed β-sheets appearing at higher pressures (> 5 kbar), indicating a higher pressure stability of this form, and the extent of exposed β-sheets arising with increasing pressure is lower for the N-Ras (GTP) (Fig. 3 A and C).

Effect of Membrane Binding on the Pressure-Dependent Conformational Changes of N-Ras.

Members of the Ras superfamily are found attached to the inner side of the plasma membrane making the membrane interaction of the Ras proteins crucial for its signaling properties so that the effect of membrane binding on the pressure-induced conformational changes needs to be explored and compared with the bulk solution behavior. FTIR amide I′ spectra were recorded for N-Ras (GDP) and N-Ras (GTP) bound to a zwitterionic model raft membrane consisting of DOPC∶DPPC∶Chol 1∶2∶1 (29) (Fig. S1). As expected, no major secondary structural changes or even unfolding are observed in the protein upon membrane insertion, in agreement with previous reports (23, 33). Significant conformational changes are identified in the second derivative spectra for N-Ras (GDP) and N-Ras (GTP) inserted into the membrane upon pressurization, however (Figs. 2 C and D). Changes are observed mainly in the α-helical, loop and β-sheet region when compared with the bulk behavior. The α-helical subband is broader, suggesting existence of different spatial orientations of helices (mainly α-4 and α-5) and the hypervariable region (HVR), and their involvement in membrane binding. This finding is in accord with our previously published reports on membrane interaction of K-Ras 4B (24). Pronounced splitting for the α-helical band for N-Ras (GDP) is observed only above 7 kbar, and thereafter both the band shoulders can be fairly distinguished up to 10 kbar. For N-Ras (GTP) inserted into the membrane, definite splitting of the α-helical band is already observed at low pressures (clearly visible above 600 bar), forming a shoulder at 1,664 cm^-1 (solvated turns/loops) and at 1,653 cm^-1 (solvated helices). Likewise, for the membrane-bound N-Ras (GDP) and (GTP), the intensity of the intramolecular β-sheets changes nonlinearly (see below), and the intensity at the lower wavenumber side typical for exposed β-sheets increases significantly upon pressurization.

Fig. 3 B and D shows the variation of the secondary structural contents of membrane-bound N-Ras in the different nucleotide states with pressure. For membrane-bound N-Ras (GDP), the helix content decreases by about 3%, similar to the bulk, turns by 6.0%, and increases are observed for random coils by 2% and exposed β-sheets by 6%. Similarly, membrane-bound N-Ras (GTP) also shows minor changes in the secondary structure only, such as a decrease in helices by 3%, turns by 6%, and increases in random coils by 3% and exposed β-sheets by 6%. Most remarkably, the pressure dependence for intramolecular β-sheets is strikingly different for both membrane-bound Ras forms when compared with the bulk in that increasing pressure seems to first stabilize the intramolecular β-sheets. At higher pressures (> 4 kbar), a trend reversal is observed, and the intramolecular β-sheet contribution is slightly decreased again. The occurrence of the intensity maximum of this band indicates existence of an additional conformational equilibrium—i.e., population of a further, membrane-induced conformational substate of the protein (denoted as state 3 hereafter). Fig. 3 F and H shows the corresponding wavenumber shifts of the secondary structures of membrane-bound N-Ras (GDP) and (GTP). The behavior shown by the membrane-bound N-Ras (GDP) is similar to that of bulk N-Ras (GDP), differing only in the pressure value at which the α-helical subband displays a pronounced splitting (at approximately 7 kbar compared with 3.5 kbar in the bulk). The clearly separated wavenumber for the exposed β-sheets also appears at higher pressures when compared with the bulk, suggesting a higher conformational stability in the membrane-bound state. Differently, for membrane-bound N-Ras (GTP), the splitting of the α-helical subband starts at rather low pressures (< 600 bar) already, suggesting significant involvement of the α-helical region of the G domain upon membrane insertion in the GTP-bound state. This would be in agreement with recent findings of Gorfe et al., where it is proposed that H-Ras (GDP) predominately exhibits membrane interaction through the HVR, and GTP loading triggers structural rearrangements in its switch regions that are coupled to reorientations of helices (α-4, α-5) and their interaction with the lipid interface (26, 27).

Because N-Ras exhibits 91% sequence identity to H-Ras, this mode of interaction might apply to N-Ras as well. To corroborate our findings, complementary polarized ATR-FTIR and IRRAS experiments were carried out, allowing us to yield additional information on the orientation of the protein at the lipid interface. The dichroic ATR-FTIR spectra (Fig. S3) demonstrate distinct and different orientations of N-Ras GDP and GTP, respectively, with respect to the bilayer interface. A negative peak in the helix region (at 1648–1665 cm^-1) indicates an orientation of the majority of the helices that is essentially parallel to the membrane bilayer. The changes observed in the dichroic spectra imply minor yet significant differences in the orientation of N-Ras (GDP) and N-Ras (GTP) relative to each other and to the membrane bilayer. A more comprehensive and quantitative analysis of the orientation of the protein at the lipid interface can be achieved by using IRRAS on corresponding lipid monolayer films (for details see SI Text and Fig. S4). Clearly, different angle dependent IRRA spectra are visible for the two bound nucleotide states of N-Ras. Simulations of the spectra were performed for two representative orientations, denoted model 1 (GDP-bound) and model 2 (GTP-bound), adapted from ref. 26. It was found that the N-Ras (GDP) IRRA spectra are comparable to the simulated ones for model 1 with respect to the intensity distribution over the whole amide I' vibrational wavenumber region, and the simulated spectra for model 2 match reasonably well with the N-Ras (GTP) measured ones. These data reveal that N-Ras (GDP) is likely to adopt an orientation as depicted by Fig. S4A, Inset, which is similar to the conformations whose averaged structure is represented by model 1, where the protein mainly interacts with the membrane through the HVR region and N-terminal residues. N-Ras (GTP) shows an orientation (Fig. S4B, Inset) that would be consistent with the one described by model 2, which involves domain reorientation leading to a more extensive interaction of helices α-4 and α-5 with the membrane.

To support the conclusions drawn from the high pressure FTIR data, high pressure fluorescence spectroscopy was also carried out on N-Ras (GTP) both in bulk and bound to membrane, by mapping the intrinsic tyrosine emission maxima as a function of pressure (Fig. S5). No changes were observed for the protein in the bulk, implying high pressure stability of the protein, whereas the emission maximum exhibits a red shift upon pressurization when bound to the membrane, implying increasing solvent exposure of the tyrosine residues upon pressurization starting as low as 200 bar. These results also reveal that membrane binding has a marked effect on the pressure-induced changes of the conformational substates of N-Ras (GTP).

In order to gain also information about the structural changes occurring upon unfolding of N-Ras, temperature dependent studies were carried out as well. The temperature-induced changes are pronounced and strikingly different from the pressure-induced structural changes (Fig. S2). Above 60 °C, unfolding of N-Ras sets in, which is accompanied by a decrease of ordered structural contents and a concomitant increase in conformations originating from unordered structures and intermolecular β-sheet formation. Within the experimental error, we could not find any marked differences in the thermal behavior of N-Ras (GTP) and N-Ras (GDP), both in the bulk and in the membrane-bound forms.

Discussion and Conclusions

In an attempt to fully understand the structure, dynamics, and function of signaling proteins and their signaling networks, the vast conformational space exhibited by them needs to be explored. For this endeavor, knowledge of not only the low-lying native conformation(s) but also of their high-energy conformers is required. These additional functionally relevant states are often present as relatively minor fractions of the ensemble under normal conditions, making their detection and characterization an arduous task. The principle of conformational selection seems to be the underlying mechanism for the functioning of Ras proteins as well (15), and pressure-modulation is a rather new and efficient means for the exploration of the various conformational and functional substates responsible for the various signaling outputs of the protein. Pressure can shift the equilibrium already existing between distinct substates at ambient pressure toward low volume conformers, which are generally more solvated, thereby decreasing the total volume of the protein system (3, 6, 7, 11, 34, 35). As can be seen from the temperature dependent data on N-Ras (Fig. S2), temperature modulation to reveal low-lying excited substates is less successful and results in full unfolding and subsequent aggregation of the protein, only. On the contrary, pressure is a very mild perturbant and results in fully reversible changes, a prerequisite for determining thermodynamic properties of the system.

It is generally assumed that Ras proteins in bulk solution have a less open conformation of the switch regions I and II and hence a smaller flexibility in the GTP-bound compared to the GDP-bound form (19, 21). This is also the case for N-Ras as evidenced from the amplitude of the frequency shifts in the second derivative FT-IR spectra (Fig. 2 A and B) and from the overlay of different Ras crystal structures in the GDP and GTP-bound form (Figs. 1 B and C). Pressure application to the conformational ensemble of N-Ras (GTP) shifts the equilibrium toward a more solvent exposed and open conformation, in agreement with a previous NMR report on H-Ras (22). It is a well-known effect of high hydrostatic pressure to weaken hydrophobic interactions, thus allowing water to penetrate the protein interior, in particular at places where voids and cavities are present (11, 36). As a result, pressurization is expected to shift the conformational equilibrium toward the more open and solvent exposed conformations. Recent analysis of the protein structure database indicates that the residues of β-strands have a higher tendency to line the cavities compared to others, probably due to a lower packing efficiency (37). This could explain the marked increase of exposed β-sheets upon pressurization of both the GTP- and GDP-forms of N-Ras.

As indicated by our FTIR spectroscopic data, in N-Ras (GTP), pressure induces conformations with more solvent exposed structures that could correspond to state 1 according to ref. 21, which is known to have an enlarged protein surface due to the opening of the nucleotide binding pocket (necessary for stable interaction with GEFs). The corresponding volume change (up to 10 kbar) driving the conformational transition amounts to -14.7 mL/mol and agrees very well with the ³¹ P NMR data (up to 2 kbar) on (unlipidated) Ras (GTP) in showing that with increasing pressure the conformational equilibrium shifts from state 2 to state 1 (21).

The pressure-dependent conformational changes for bulk N-Ras (GDP) are significantly different from N-Ras (GTP), with the splitting of the subband in the α-helical region of the protein starting already at much lower pressures (Fig. 2A). Moreover, the population of exposed β-sheets increases with pressure at a higher rate and reaches a higher level (10%) compared with N-Ras (GTP) (4%) at the highest pressure measured (Fig. 3 A and C). These results imply a higher pressure sensitivity of the GDP-bound N-Ras, which can be explained on the following grounds. Both the Ras proteins typically have free volumes (i.e., cavities) of less than 1% of their total volume in the available X-ray structures, and the differences are probably not significant. If ever, the GTP-forms tend to have a slightly higher cavity volume than the GDP form, so that a (slightly) lower pressure sensitivity would be foreseen for N-Ras (GDP). As this is not observed, the higher predisposition to form open and solvent exposed conformations in N-Ras (GDP) must be due to other reasons. One very likely possibility is the easier opening of the highly flexible switch I region in N-Ras (GDP), which is clearly visible by comparing the initial slopes, Δ[exposed sheets]/Δp (up to 2 kbar) (Fig. 3 A and C), which is markedly higher in the GDP-bound form. This would also be in agreement with the ³¹P NMR data (21) where the maximum pressure of 2 kbar reached was able to completely populate state 1 in the—even less pressure sensitive—GTP-bound form. This explanation is further supported by ultrasound velocimetry studies that provide insight into the flexibility–compressibility relationship of proteins (38), suggesting higher compressibilities of the more flexible regions of the proteins. High pressure induces state 1 in both Ras proteins, but due to the higher flexibility in the GDP form, state 1 is populated more easily there.

As discussed above, pressure-dependent studies were performed for unlipidated H-Ras-GppNHp unlipidated in bulk solution and up to 2 kbar, only (21). Here, also the effects of membrane binding on the pressure-dependent conformational changes of both active (GTP form) and inactive (GDP form) fully lipidated N-Ras was investigated using FTIR spectroscopy up to about 10 kbar, a pressure range that is currently not accessible to NMR technology. As could be clearly demonstrated, not only nucleotide binding but also membrane binding modulates the conformational dynamics of N-Ras markedly, and, interestingly enough, brings about new and opposite changes in the conformational properties of N-Ras (GDP) and (GTP), respectively. Upon membrane binding, the pressure sensitivity of the two nucleotide states of N-Ras is unexpectedly reversed. These differences could arise from a different overall orientation of the G domain with respect to the membrane as originally proposed by Gorfe et al. (26). Complementary results from polarized ATR-FTIR (Fig. S3) and IRRAS (Fig. S4) provide conclusive hints for the reorientational changes in the Ras G-domain upon membrane insertion mediated by the bound nucleotide. The pressure-induced nonlinear shift of the intramolecular β-sheet band in both membrane-bound N-Ras forms (Fig. 3 B and D) indicates that membrane binding modulates the conformational equilibrium of N-Ras (GDP/GTP) by inducing a new, membrane-associated conformational substate, state 3.

A schematic representation of the pressure modulation of conformational substates of Ras is depicted in Fig. 4. For the bulk N-Ras, increasing pressure populates the more open and solvated conformer, which is state 1 in both cases, the effect being more pronounced for the inactive N-Ras (GDP). Owing to the high intrinsic flexibility of the switch loops in N-Ras (GDP) and the rapid interconversion between the different conformers, the energy funnels of these substates are expected to be broader. Upon membrane insertion, a new functional conformer, substate 3, is induced in both cases, which is increasingly populated upon pressurization. At higher pressures, the transition to state 1 is fostered since it then corresponds to the state with the lowest free energy.

Fig. 4.

Schematic representation of the modulation of conformational substates of active (GTP) and inactive (GDP) N-Ras by pressure in bulk solution and in the presence of the lipid bilayer membrane. Small arrows indicate changes at low pressure values, and longer arrows indicate changes occurring after application of higher pressures. The membrane-associated substate 3 is marked in red.

To conclude, our data clearly show that, next to the nucleotide state, the membrane also plays an important role in the conformational selection and dynamics of N-Ras. Membrane insertion itself at ambient pressure does not lead to significant secondary structural changes in the G-protein. However, pressure application induces minor and local changes of certain secondary structural elements and shifts the equilibrium toward conformations with more open and solvated structures having smaller volumes. Membrane insertion leads to a marked population of a new substate, state 3. This state is increasingly populated upon pressurization—and hence becomes clearly detectable—and is accompanied by a significant structural reorientation of the G domain at the lipid interface, in particular in the GTP-loaded form of N-Ras. Efficient interaction with GEFs and effectors requires selection of conformations like states 1 and 2, respectively, and shifts the equilibrium toward these states. Our study indicates that upon membrane binding, such conformational selection is additionally modulated, preselecting conformational substate 3 with significant structural rearrangements. By these conformational and orientational changes, the interaction with membrane-associated interaction partners, such as effectors and activity modulators (e.g., galectins), may be influenced. In fact, recent in vivo studies have shown that G-domain orientation of membrane-associated Ras proteins is important for effector/modulator recognition, such as of phosphoinositide-3-kinase and galectin-1, and G-domain orientation and nanoclustering—that has also been observed in our AFM studies on model biomembrane systems (23, 24)—generate Ras isoform specificity with respect to effector interactions (27).

Finally, these results shed also some light on the effect of pressure on membrane-associated Ras-controlled signaling events under extreme environmental conditions. Although pressure is an important environmental parameter, such as in the deep sea where organisms have to cope with pressures up to the 1 kbar range, the fundamental understanding of its effects remains largely fragmentary. The effects of pressure on gene expression or on biomolecules such as lipid membranes, proteins and DNA have been studied in recent years, the effects of pressure on the dynamics of metabolic events and signaling processes remain largely unexplored, however (39–42). Our data indicate that increased hydrostatic pressure shifts the conformational equilibrium toward the more open water exposed state 1, which is supposed to interact most effectively with GEFs. Furthermore, high pressure leads to substantial stabilization of the otherwise lowly populated conformer 3 induced by membrane interaction in both Ras isoforms, which is expected to foster the interaction with membrane-associated interaction partners and prevails up to a few kilobars.

Materials and Methods

Materials and Sample Preparation.

The phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Avanti Polar Lipids. Cholesterol (Chol), other reagents, and solvents were obtained from Sigma-Aldrich and Merck. Stock solutions of 10 mg mL^-1 lipids in chloroform were prepared and mixed to obtain the desired composition of the DOPC/DPPC/Chol (1∶2∶1 molar ratio) lipid mixture.

N-Ras Protein Synthesis of Double Lipidated N-Ras (N-Ras HD/Far).

Double lipidated N-Ras peptides were synthesized with an N-terminal maleimide group via a combination of solution and solid phase strategies. The maleimido peptides were coupled to a bacterially expressed truncated N-Ras protein (amino acid residues 1–181) carrying a cysteine at the C terminus. Coupling was achieved by conjugate addition of the only surface-accessible cysteine (181)-SH group of N-Ras to the maleimido function. Details on the nucleotide exchange on Ras proteins can be found in refs. 43 and 44.

High Pressure FTIR Spectroscopy.

The dry lipid mixture was hydrated with Tris buffer, pD 7.4. The hydrated lipid mixture was then subjected to five freeze-thaw-vortex cycles and brief sonication. Large multilamellar vesicles obtained herewith were transformed to large unilamellar vesicles of homogeneous size by use of an extruder (Avanti Polar Lipids) with polycarbonate membranes of 100 nm pore size at 65 °C. For the pressure-dependent studies, the GDP- and GTP-loaded N-Ras protein (c = 3 wt%) were lyophilized for 2 h and mixed with extruded lipid mixture yielding a molar ratio of protein to lipid of 1∶100. The pressure-dependent FTIR spectra were recorded with a Magna IR 550 spectrometer equipped with liquid nitrogen cooled MCT (HgCdTe) detector. For the measurements, the infrared light was focussed by a spectral bench onto the pin hole of a diamond anvil cell (with type IIa diamonds from Diamond Optics). For details of operation and data analysis see ref. 9.