Earlier work by others indicated that S4 accomplishes voltage sensing by relocating its position relative to the membrane in response to voltage change. S4 is cationic at neutral pH, and therefore its movement within the lipid membrane poses a significant thermodynamic challenge [20
]. Previously, Hessa et al. showed that the S4 segment of a truncated Shaker K+
channel inserted into the endoplasmic reticulum membrane via translocon-mediated integration [15
]. We designed a series of in vitro experiments using synthetic peptides and model lipid membranes, which bypasses the necessity for in vitro
translation and microsome preparations, and reexamined the inherent ability of S4 to partition in the membrane.
3.1. Peptide design
Solvent accessibility studies have suggested a direct contact of S4 with the hydrophobic core of the lipid bilayer [21
]. We examined this with fluorescence spectroscopy, using a tryptophan-labeled synthetic S4 peptide from shaker K+
channel S4. The use of the polarity-sensitive fluorophore tryptophan (W) obviated introducing non-amino acid moiety into the peptide.
Various peptides were designed () in order to probe the significance of the specific arrangement of amino-acid residues in the S4 transmembrane segment. To test for the possibly different propensities of the two termini to interact with the membrane we used peptides with tryptophan at C terminus (S4-W), N terminus (W-S4), and in the center (S4(F9W, W18F)). To test whether the regular spacing of cationic amino-acid residues at every 3rd position throughout the peptide affects the membrane interaction, one or more arginine residues were shifted in the sequence by one (S4-W (R10
−1)) or two positions (S4-W (R10
−2), (S4-W (R7,10
−2), S4-W (R4,7,10
−2), and S4-W (R4,7,10,13
−2)). Finally, to determine the role of helicity in the peptide-membrane interaction we disrupted the helix by placing proline in the center of the sequence (S4-W (F9P)).
3.2. Interaction with the membrane
Lipid-induced changes in the anisotropy of the tryptophan fluorescence were used to measure binding of the peptides to the membrane. Increasing concentrations of SUV were sequentially added to a fixed concentration of the peptide (3 μM), as shown in . Binding of fluorescent peptides to lipid vesicles decreases the fluorophore’s diffusion and thus increases its fluorescence anisotropy. Values of the apparent dissociation constant (Kd
) and the maximum change in anisotropy (Δrmax
) were extracted from data using Eq. 1
, and values of apparent dissociation constant (Kd
) thus obtained were used to estimate the apparent Gibbs free energy change (ΔG
) of the S4W interaction with the PC/PG membrane according to Eq. 2
. As shown in , altering the membrane composition had a significant effect on the interaction of the peptide with the lipid membrane. Inclusion of 30 mol% of the acidic phospholipid phosphatidylglycerol (PG) in the membrane (i) increased the peptide’s affinity (the value of ΔG
changed from −4.0 ± 0.9 kcal/mol to −5.2 ± 0.2 kcal/mol (the latter is an average of the two peptides with tryptophan on the two opposite termini, see ); and (ii) greatly increased the observed change in the peptide’s fluorescence anisotropy, indicating that S4 is more tightly immobilized in the negatively charged membrane.
(A) Fluorescence anisotropy changes upon Interaction of S4W with SUV. Squares, PC/PG SUV; circles, neutral PC SUV. (B) Comparison of anisotropy changes in all peptides. (*) p-value <0.05, and (**) p-value <0.005 with respect to S4W.
The obtained values of ΔG
were compared with the theoretical ones, calculated using the Wimley-White interfacial (IF) hydrophobicity scale [22
). Theoretical ΔG
values for transfer of S4-W from water to bilayer in the extended and α-helical conformations were +0.22 kcal/mol and −6.98 kcal/mol, respectively. Thus, the ΔG
values obtained from our data are in good agreement with the Wimley-White prediction for the peptide in the α-helix form.
To investigate possible effects of the spatial arrangement of the charged residues along the peptide sequence on the membrane interaction, fluorescence anisotropy changes were quantified for each peptide from . Because of the logarithmic relationship between ΔG and Kd, variations in ΔG (), were not conspicuous, but they were statistically significant nevertheless. The extent of anisotropy change (Δrmax), while following the same trend as for ΔG, varied more conspicuously (). The latter variations indicate that in addition to the overall affinity of the peptide helices towards the membrane (binding energetics, ΔG), also the location, orientation and/or mobility of the bound peptides with respect to the membrane (Δrmax) depend on the peptides’ sequences. Switching the tryptophan from the C terminus (S4-W) to the N terminus (W-S4) or to the center (S4(F9W, W18F)) did not result in significant changes (0.14 ± 0.02, 0.14± 0.02, and 0.16 ± 0.02, respectively). However, a change in the spatial arrangement of the cationic residues (S4-W(R10−1), S4-W(R10−2), S4-W(R7,10−2), S4-W(R4,7,10−2), S4-W(R4,7,10,13−2) and S4-W(Rregain−2)) has brought about a decrease in values of Δrmax (0.09 ± 0.01, 0.05 ± 0.02, 0.09 ± 0.02, 0.11 ± 0.01, 0.12 ± 0.02, and 0.14 ± 0.01, respectively). The most significant changes were observed in peptides where only Arg10 (S4-W(R10−1), S4-W(R10−2)) or Arg7 and Arg10 (S4-W(R7,10−2)) were shifted. When three or four arginines were shifted by two positions, changes ceased to be statistically significant. Reverting to the original amino-acid sequence brought fluorescence anisotropy back to the original value. Disruption of the peptide’s helical conformation (S4-W (F9P)) also resulted in a significant decrease in the anisotropy change (0.05 ± 0.02). These results suggest that both the spatial arrangement of cationic amino-acid residues and the helical confirmation are important for the S4-membrane interaction.
Table 2 Values of the apparent dissociation constant Kd and calculated apparent free energy of binding, ΔG. The tabulated values were obtained from fluorescence anisotropy data and Equations 1 and 2. Standard error for each value of ΔG was 0.1 (more ...)
None of the peptides breached membrane integrity even at high peptide/lipid ratios (1:30, data not shown), as evidenced by no significant dye release from the calcein-loaded SUV in a calcein-release assay [23
3.3. Localization in the membrane
Sensitivity of tryptophan fluorescence to the polarity of its environment was used to gain information on peptides’ localization in the membrane. In less polar (or more hydrophobic) environment, tryptophan emission spectrum exhibits blue shift, i.e., a shift of the spectrum to shorter wavelengths, compared to tryptophan in aqueous solvent [24
]. Fluorescence spectra of S4 peptides were recorded at increasing concentrations of PC/PG SUV () and magnitudes of the blue shift were evaluated with respect to the spectrum in the absence of the lipid. At saturation, the native peptide S4W exhibited a 14 ± 2 nm blue shift upon binding to the membrane. Replacing egg PC with sphingomyelin gave similar results (data not shown), which suggests that the chemical nature of the neutral lipid is not relevant to the interaction. Location of tryptophan at the N terminus or C terminus also appeared to be irrelevant: peptides S4-W and W-S4 showed similar blue shifts (14 ± 2 nm and 13 ± 2 nm, respectively). However, the peptide S4(F9W, W18F), with tryptophan in the center of the helix, showed a significantly greater blue shift (21 ± 3 nm). This suggests that the helix may be bent, with the center buried deeper in the lipid membrane than the ends. The mutated peptides S4-W(R10−1
) and S4-W(Rregain−2
) showed the following values of blue shift: 10 ± 1 nm, 4 ± 2 nm, 12 ± 2 nm, 12 ± 1 nm, 11 ± 2 nm, and 14 ± 1 nm, respectively. Only shifting of Arg10 produced a significantly smaller blue shift, indicating that the regular pattern of basic amino acid residues in the center of the peptide is more important than that at the termini. Also, disruption of the peptide’s helicity by placing a proline in the center (S4-W(F9P)) resulted in a significantly smaller blue shift, 3 ± 1 nm.
(A) Fluorescence emission spectra upon interaction of S4W with PC/PG SUV. Inset shows the blue shift. (B) Comparison of the blue shift values in all peptides. (*) p-value <0.05, and (**) p-value <0.005 with respect to S4W.
The above data show that the synthetic S4 peptide spontaneously binds to the lipid membrane. Furthermore, the blue shift indicates that at least a portion of the peptide is in contact with the non-polar lipid core. To determine the depth of the peptide’s penetration into the membrane we employed several experimental approaches. First, we measured exposure of the membrane-bound peptide to water by quenching its fluorescence with the water-soluble quencher acrylamide [25
]. shows that the Stern-Volmer quenching constants (KSV
, Eq. 4
) for S4-W in the absence and presence of lipid were 27 ± 3 M−1
and 10 ± 1 M−1
, respectively. The lipid partially shielded the C-terminal tryptophan against acrylamide quenching. To compare aqueous accessibility of various peptides, we calculated the ratio of quenching constants in the presence and absence of lipid (KSV(lipid)
) for each peptide. The ratios for S4-W, W-S4, and S4(F9W, W18F), were found to be 0.37 ± 0.02, 0.41 ± 0.03 and 0.28 ± 0.02, respectively (). The two termini are shielded from water to the same extent, but the center of the peptide is shielded more. This confirms the conclusion based on the blue-shift data. These results are not consistent with the transmembrane orientation of the peptide, where the two termini would not be quenched to the same extent by a quencher applied from one side of the membrane. Even if the peptides did span the membrane in a random orientation, there would always be a fraction of fluorophores that would be unquenchable (on the other side from the side with the quencher), which would result in curved Stern-Volmer plots in . Our results thus suggest that S4 lies in the membrane more or less parallel with the membrane surface.
Figure 3 (A) Acrylamide quenching of tryptophan fluorescence in S4W. Circles, in the absence of lipid; squares, in the presence of PC/PG SUV. (B) Ratios of Stern-Volmer quenching constants (KSV(lipid)/KSV(no lipid)) for all peptides. (*) p-value <0.05, (more ...)
The KSV(lipid)/KSV(no lipid) ratios for the peptides with arginine shifts, S4-W(R10−1), S4-W(R10−2), S4-W(R7,10−2), S4-W(R4,7,10−2), S4-W(R4,7,10,13−2) and S4-W(Rregain−2), were found to be 0.54 ± 0.03, 0.78 ± 0.09, 0.49 ± 0.04, 0.41 ± 0.04, 0.44 ± 0.05, and 0.39 ± 0.03, respectively. The values indicate that peptides in which only Arg10 and Arg 7 are shifted retain more exposure to water in the presence of lipid than the peptides in which the flanking arginines (4 and 13) are shifted as well. And again, the peptide with disrupted helicity (S4-W(F9P)) showed very high KSV(lipid)/KSV(no lipid) ratio (0.75 ± 0.10). These result indicate that two peptides, S4-W(R10−2) and S4-W(F9P), remain most exposed to water upon binding to the membrane; they probably lie on the membrane peripheral. The other peptides, with the quenching ratios of less than 0.5, are more protected from aqueous quenching they may be shielded or buried below the lipid head groups, at the level of phosphate or perhaps deeper.
To confirm this, we determined the relative location of different residues in the synthetic S4 peptide by RET. The membrane was labeled with the hydrophobic fluorophore DPH, which is a good acceptor of energy from tryptophan. Assuming that most of the DPH molecules lie in the bilayer core, energy transfer efficiency E is inversely proportional to the depth of the peptide’s penetration in the membrane. The native peptide S4W showed high efficiency, 88 ± 6% (). A comparison between RET efficiencies of all peptides is shown in . The observed pattern was the same as in the previous experiments described above: the peptides that exhibited the lowest RET efficiency were again those with shifted Arg10 and disrupted helicity, (S4-W(R10−2) and S4W(F9P), with efficiencies of 38 ± 14% and 34 ± 12%, respectively. This indicates that tryptophan in these peptides lies the farthest from the center of the lipid bilayer.
(A) Quenching of S4W fluorescence with di-bromo-PC upon interaction of with PC/PG SUV. (B) Comparison of fluorescence quenching by 6,7- di-bromo-PC in all peptides. (*) p-value <0.05, and (**) p-value <0.005 with respect to S4W.
Figure 4 (A) RET upon interaction of S4-W with 0.5% DPH-PC/PG SUV. 1, S4-W in aqueous solution; 2, S4-W with PC/PG SUV; 3, S4-W with 0.5% DPH- PC/PG SUV; 4, 0.5% DPH- PC/PG SUV excited at 280 nm. The difference between spectra 3 and 4 is due to RET. (B) Comparison (more ...)
Since both donor and acceptor molecules diffuse in the membrane, we did not attempt to translate efficiency values into distances. Rather, we confirmed these qualitative conclusions by quenching with lipidic quenchers that have quenching groups at defined depths in the hydrophobic core, such as brominated lipids (70% dibromo-PC and 30% PG) [26
]. Bromine is a known quencher of tryptophan fluorescence and the approximate distance of bromines from the membrane surface is 9 Å for 6,7-dibromo-PC, 12 Å for 9,10-dibromo-PC and 14 Å for 11,12-dibromo-PC. For the native peptide S4W the extents of quenching with 6,7-dibromo-PC, 9,10-dibromo-PC and 11,12-dibromo-PC were 74 ± 4%, 52 ± 4%, and 26 ± 7%, respectively (). The highest quenching with the lipid that is brominated at carbon positions 6 and 7 indicates that the peptide’s C terminus (where the tryptophan is located) lies just below the polar head groups, no more than 9 Å from the bilayer surface. Values of quenching by 6,7-dibromo-PC of all peptides are in . The familiar pattern emerged again, confirming the previous conclusions: the peptides with shifted Arg10 and with disrupted helicity showed the smallest degree of quenching, indicating that tryptophan in these peptides lies closer to the surface of the bilayer than tryptophan in the other peptides, where all, or almost all basic residues were shifted in phase. The latter peptides penetrate deeper below the phosphate, into the hydrophobic core, similarly to the native peptide S4-W.
3.4. Role of the electrostatics
The fluorescence anisotropy data have shown significant differences in interactions of the peptide with membranes of different lipid composition. We noticed a difference in S4-W interaction with addition of 30% of anionic lipids in the membrane (). To maintain the physiologic relevance we limited the amount of anionic charge in the membrane to 30%. Enhanced immobilization of the cationic peptide with increased anionic charge on the membrane, led us to investigate the role of electrostatic forces in the peptide-lipid interaction. High ionic strength can be used to attenuate electrostatic interactions [27
]. We used 2 M sodium chloride to shield the electrostatic peptide-lipid interactions and thus to probe the involvement of electrostatic interactions in the overall binding of S4 to membranes, using fluorescence spectroscopy, in particular spectral shift and anisotropy. shows the effect of increased ionic strength on the peptide-lipid interaction: the addition of salt, either before or after adding the lipid, reversed the blue shift to the original value in the absence of lipid. Subsequent quantitative analysis () has shown that electrostatics accounted for 54% and non-electrostatics accounted for 46% of the overall change. These results, along with those from varying lipid composition, indicate that in addition to the electrostatics other forces, such as hydrophobic and van der Waals, participate in the peptide binding. This gives support to the hypothesis that the anionic phosphate head groups of the lipid membrane attract a cationic peptide randomly structured in the aqueous phase. Close to the membrane surface, the peptide undergoes a conformational change to alpha helix, which is, more or less reversibly, stabilized in the lipid bilayer. Atomic structure of Kv
1.2 revealed that S4 indeed exists in an alpha helical conformation [12
]. Many amphipathic peptides also assume α-helical structure upon interaction with lipid membranes [28
Figure 6 (A) Fluorescence emission spectra of S4 with high salt added afterwards-: 1, S4-W excited at 280 nm; 2, S4-W with PC/PG SUV and 2M NaCl added after the lipid; 3, S4-W with PC/PG SUV. (B) Fluorescence emission spectra of S4-W with high salt present at (more ...)
3.5. Role of membrane fluidity
Properties of the membrane, such as, e.g., fluidity (gel state vs. liquid-crystal state), have been shown to affect membrane interactions [17
]. In the following experiments we studied the effect of membrane fluidity on S4 binding. We used chemically defined lipids DMPC and DMPG, which have a clear phase transition at around 23 °C [30
]. Vesicles prepared from these lipids are in the gel state at temperatures below 23 °C, and in the liquid-crystal state above 23 °C. Fluorescence anisotropy changes () in S4-W were significantly higher when the peptide bound to the fluid membrane than when it interacted with the membrane in the gel state (0.12 ± 0.02 and 0.03 ± 0.01, respectively). Apparent binding free energy ΔG
paralleled the trend: in the gel state it became much less negative, −2.2 ± 0.4 kcal/mol.
Figure 7 (A) Fluorescence anisotropy changes upon interaction of S4-W with DMPC/DMPG SUV; squares, 37°C; circles, 7°C; (B) Fluorescence anisotropy change upon Interaction of S4-W with PCPG SUV; squares, 0% cholesterol SUV; circles, 30% cholesterol (more ...)
To ascertain that this is solely an effect of the physical state of the membrane, we modulated the latter by means other than temperature, namely, by doping the membrane with cholesterol. Addition of 33% cholesterol, the main constituent of lipid rafts [32
], decreases the fluidity of the membrane and broadens or abolishes the phase transition. As shown in , binding of S4-W to cholesterol-containing membranes brought about a significantly lower change in anisotropy (0.029 ± 0.004) than binding to cholesterol-free membranes (0.14 ± 0.02). In addition to fluorescence anisotropy, all the previously used parameters were determined and compared for interactions of S4-W with cholesterol-free and cholesterol-containing membranes (): blue shift (14 ± 2 vs. 3.1 ± 1.2), acrylamide quenching (the KSV(lipid)
ratio of 0.37 ± 0.03 vs. 0.84 ± 0.05), efficiency of RET to DPH (88 ± 7 % vs. 24 ± 12 %), quenching with 6,7-dibromo-PC (74 ± 6% vs. 18 ± 8 %), and ΔG
(−5.2 ± 0.2 kcal/mol vs. −2.5 ± 0.3 kcal/mol). Since Kd
are measures of binding affinity or “strength”, our results indicate that liquid-crystal state of the membrane is necessary for efficient binding and membrane partitioning of the S4 peptide. The other measured parameters, such as the maximum fluorescence anisotropy change and the magnitude of blue shift, RET and quenching, do not report on thermodynamics of binding, but rather on different “modes” of binding, as for instance, the depth of membrane penetration and the microenvironment of the tryptophan as reporting moiety. We note that while all the studied peptides bind to the PC/PG membrane, they do so with various affinities and “modes”, depending on the amino acid sequence.
3.6. General discussion and conclusions
The charged segment S4 is thought to sense membrane potential by moving its charges in the electric field in the membrane. Evidence for the S4 movement, albeit quite indirect, is based on residue accessibility studies [21
], gating current measurements in the presence of pore-blocking toxins [33
], and distance determinations using FRET and LRET [35
]. More recently, S4 segment in a mutant variety of shaker has been shown to have proton channel and omega-current activity [36
]. These results were consistent with both of the competing models of S4 movement in the membrane, namely: the voltage-sensing paddle model and the helical screw model [37
]. While the paddle model predicts, or indeed requires, the direct contact between the charged S4 segment and the hydrophobic tails of the lipid molecules, the helical screw model predicts that the S4 segment moves perpendicularly to the plane of the membrane within a proteinaceous sheath of the channel wall. Despite extensive efforts, a comprehensive picture of the voltage sensor movement has not yet emerged. A major setback in elucidating the mechanism was the fact that channels with mutated arginines in the middle of S4 segment were either non-functional or did not even express on the cell membrane [38
]. Our work provides experimental evidence that the autonomous voltage-sensing peptide S4 of the potassium channel spontaneously binds and partitions in the lipid membrane, without any assistance from the rest of the channel protein. S4 was found to spontaneously penetrate into the negatively charged lipid membrane, with the termini about 9 Å below the membrane surface. The peptide’s high positive charge at the neutral pH does not seem to pose an obstacle, as long as the periodicity of 3 is maintained in the distribution of the basic amino acids along the S4 sequence. This periodicity distributes the positive charges unequally on the two sides of the alpha helix.
While our work was in progress, Doherty et al. [40
] published their results of solid-state NMR determination of Kv
AP S4 orientation in the lipid membrane. According to their model, both termini of the peptide are close to the opposing membrane surface, such that S4 spans the thinned lipid bilayer in a tilted orientation identical to that observed in the crystal structure of the whole voltage-sensing domain. Our data are consistent with this model and further support the notion that the orientation of S4 is determined by peptide-lipid interaction, with minimal, if any, influence of the rest of the channel protein.
To summarize, in the present work we characterized binding of autonomous S4 to lipid membranes. The apparent binding free energy (5.2 ± 0.2 kcal/mol for PC/PG SUV) estimated from the apparent dissociation constant, which, in turn, was determined from lipid-induced changes in anisotropy of tryptophan fluorescence, is not much different from the value calculated using the Wimley-White hydrophobicity scale for binding of an α-helical peptide to the lipid (6.98 kcal/mol). The peptide/membrane interactions have both electrostatic and non-electrostatic components. The peptide lies more or less parallel to the membrane surface, in the depth of about 9 Å. A cartoon model of the S4-membrane interaction consistent with our data is in . Important characteristics of the two ligands (i.e., the membrane and the peptide) that influence binding and penetration of S4 into the membrane include: the fluidity, surface charge, and surface pressure of the membrane, and the α-helicity and the regular spacing of the basic aminoacid residues along the S4 sequence.
A cartoon representation of the binding of the peptide S4 to the membrane.
An important question that our work has left unanswered is the movement of S4 in the membrane in response to changes in electric potential across the membrane. We note that the in vitro system employed in this study is suitable for addressing this issue [17