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Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
Correspondence: Address reprint requests to Mark S. P. Sansom, Tel.: 44-1865-2752371; Fax: 44-1865-275273; E-mail: mark{at}biop.ox.ac.uk.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). The voltage sensor (VS) domain is made of helices S1–S4, with the positively charged S4 helix (which has every third residue positively charged in its N-terminal half) moving in response to a change in TM voltage to initiate the conformational change underlying gating.
Crystallographic studies of the bacterial Kv homolog, KvAP (2), have revealed that the pore domain of KvAP has an architecture similar to that of other K+ channels, such as the bacterial K+ channel KcsA (3). However, the exact conformation and orientation of the S1–S4 domain (i.e., the VS domain) is less certain. In particular, the structure of the VS domain within the crystal structure of the full length channel construct is somewhat at odds with aspects of available physiological and biophysical data (4). Furthermore, the conformations of the VS domain in the x-ray and single particle electron microscopy structures (5) seem to be somewhat different, implying a conformational transition associated with channel gating.
In addition to the crystal structure of the full length KvAP channel, the structure of the isolated VS has also been solved (2) (Fig. 1 A). Both the full length channel and the isolated VS domain structures were solved using monoclonal antibody Fab fragments to aid protein crystallization. The isolated VS domain structure is broadly consistent with a range of biophysical and physiological data and thus may represent the conformation of the VS domain under physiological conditions. Combined with the relatively high resolution (1.9 Å) of the VS structure, this makes this domain an attractive candidate for simulation studies of its conformational dynamics in the context of possible channel gating mechanisms.
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) is partially resolved in the VS domain; however, it reveals the S1, S2, S3, and S4 helices. Superimposition of the VS of Kv1.2 with the isolated VS of KvAP reveals that the basic architectures of the two proteins are similar. It is therefore likely that the Kv1.2 structure captures the VS in a native conformation. It is also evident from the Kv1.2 structure that each VS is loosely positioned against the pore-forming helices of an adjacent subunit via the S4–S5 linker. In such an arrangement the VS will be exposed to a lipid environment. In addition to this work several studies have focused on the very nature of the VS/pore domain interaction and on determining the changes in conformation and/or orientation of the VS in response to membrane depolarization (4). For example, site-directed spin labeling data (7) have been used to determine how the VS and pore domains of KvAP may pack together within a lipid bilayer environment. A number of models of the conformational change underlying Kv channel gating have been proposed on the basis of structural and other data, including studies of VS-directed toxins and their interactions with Kv channels (8). The models that account for the conformational changes that accompany the VS upon membrane depolarization include the S4 helical screw (9), transporter (10), paddle (11), and twisted S4 (7) models. Two of the models, namely the canonical and the transporter, sequester the gating charges from a lipid environment by packing the S4 helices tightly against S1–S3 helices and the pore lining helices. This is contrary to the Kv1.2 crystal structure finding, which reveals that the first two arginines are located on a lipid-facing surface. In the paddle and twisted S4 models the positively charged S4 helices are significantly exposed to the surrounding lipid environment, in agreement with the Kv1.2 structure. Thus, it is of some interest to determine the nature of the intrinsic flexibility of the VS domain uncoupled from the pore (gating) domain. In this study, we use molecular dynamics (MD) simulations to explore the conformational dynamics of the VS domain. To enhance sampling of the intrinsic flexibility of the VS domain, we have performed simulations on the isolated VS domain in a detergent micelle environment. The simulations reveal that a modified "paddle", consisting of the C-terminal half of the S3 helix and the N-terminal half of the S4 helix, is intrinsically flexible on a nanosecond timescale. Although the gating process per se occurs on too slow a timescale (of the order of microseconds) to be addressed directly by MD simulations (which probe flexibility on a tens of ns timescale), the simulations provide valuable clues as to the nature of the voltage-gating mechanism. Furthermore, simulations offer the possibility of examining the dynamics of the VS in the in vitro environment used for purification and crystallization, namely in detergent micelles.
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METHODS |
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) combined with locally written code. All of the ionizable residues were in their default states (i.e., their ionization states were those expected at pH 7 based on the standard pKA values of the residues), except for D146, which was judged to have approximately equal probabilities of adopting either an ionized or neutral state (see below for details).
Setup of micelle systems
Each protein/detergent micelle was constructed using 94 DM (decyl maltoside) detergent molecules. The DM molecules were randomly rotated about their tail axes and arranged into semicircular planes, forming an expanded micelle-like torus around the isolated VS. Each detergent headgroup was approximately equidistant from its nearest neighbor. The terminal methyl groups of the DM tails were placed so that a minimum distance of 5 Å was maintained between each detergent molecule and the exterior of the isolated VS, so as to avoid unfavorable van der Waals overlaps. Similar initial protein/detergent micelle constructs have been generated in a number of other simulation studies (13,14) and have been shown to yield equilibrium structures similar to those generated by more extensive self-assembly simulations (15).
Each protein-micelle system was energy minimized and then solvated by superimposition of a box of preequilibrated SPC (16,17) water molecules, followed by removal of any water molecules too close to either protein or detergent molecules. The resultant systems were then subjected to further energy minimization. Potassium and chloride ions were added to each system by random replacement of water molecules, until a final concentration of 
0.1 M was achieved. The resultant systems were subjected to a further stage of energy minimization. This was followed by 1 ns of restrained MD during which all of the heavy (i.e., not H) protein atoms were positionally restrained. Restrained MD runs were performed at 300 K and 368 K for each protein-micelle system (Table 1). Finally, all positional restraints were removed and production run simulations were performed for each system.
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) (www.gromacs.org). The GROMOS96 force field (19) was used for all simulations. The parameters for DM were based on GROMOS96, supplemented with additional bond, angle, and dihedral terms to rigidify the sugar groups. Initial DM coordinates were derived from the structure of maltose when bound to a maltodextrin transport/chemosensory receptor (20) (PDB code 1ANF) combined with results from semiempirical calculations performed on the lipid chain using the CFF module in Insight II (www.accelrys.com/insight). All energy minimization procedures used <1000 steeps of steepest descent method to relax any steric conflicts generated during system setup. During restrained MD runs, harmonic restraints with force constants of 10 kJ mol–1 Å–2 were applied to all heavy atoms. Long-range electrostatic interactions were calculated using the particle mesh Ewald method (21), with a 10 Å cutoff for the real space calculation. A cutoff of 10 Å was used for the van der Waals interactions. The simulations were performed at constant temperature, pressure, and number of particles. The temperature of the protein, detergent, and solvent (waters and ions) were separately coupled using the Nose-Hoover thermostat (22,23) at 300 K or 368 K, with a coupling constant 
T = 0.1 ps. System pressures were isotropically coupled using the Parrinello-Rahman barostat (24,25) at 1 bar with a coupling constant P = 1 ps and compressibility = 4.5 x 10–5 bar–1. For all simulations the time step for integration was 2 fs, and the coordinates and velocities were saved every 5 ps. The LINCS algorithm (26) was used throughout to constrain bond lengths.
Simulations were performed on a 68 node PC cluster with dual Xeon4 processors. All analyses used GROMACS and/or locally written code. Simulations were analyzed by means of principal component analysis (27): trajectories were projected along selected eigenvectors to filter the relevant motions (28). The program SWINK has been used to measure the swivel and kink angles of the helices (29). Secondary structural alignments were performed using the secondary structure matching (SSM) server (30). Molecular graphics images were generated using VMD (31) or Pymol (www.pymol.org).
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RESULTS |
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), whereas only Kv channels contain the VS domain. From a structural viewpoint, the isolated VS domain of KvAP forms high resolution crystals (2) and also forms a stable folded domain when in detergent micelles (2,11) and when in lipid bilayers (32). Thus, simulations of the isolated VS may provide insights into the intrinsic conformational flexibility of this domain, which in turn will inform attempts to formulate atomic resolution models of the mechanism of voltage gating of Kv channels. Furthermore, the crystal structure of the isolated VS seems to be broadly consistent with molecular physiology and spectroscopic data and thus is likely to represent a major physiological conformation of the VS when within Kv channels (33,34).
Detergents mimic a biological membrane environment and as such play an important role in a number of biochemical techniques that have been applied to the study of membrane proteins. As DM was used to solubilize both the full length KvAP molecule and its isolated VS for crystallization, we have simulated the KvAP VS in a DM micelle. The exact size of the VS-detergent micellar aggregate is unknown. Using the surface of the VS as a guide, 94 detergent molecules were needed to form a complete toroidal micelle around the protein. Relative to pure detergent micelles, increases in numbers of detergents of 10–25 have been seen for peptide-containing micelles (35). The experimentally determined size of pure DM micelles is 70 molecules (www.anatrace.com). Thus, 94 DM molecules in a VS/DM micelle would seem to be a reasonable estimate. The geometry of protein/detergent micelles are not known in detail. Simulation studies, both of preformed (13,14) and of self-assembled (15) protein/detergent micelles, suggest an approximately toroidal geometry for the detergent, wrapped around the protein. Therefore we inserted the VS into a preformed DM micelle, using the membrane protein aromatic residues as a guide to the location of micelle around the 
-helical VS domain.
Charged residues in the VS play a crucial role in voltage sensing (36,37). Therefore pKA calculations (see Methods) were performed on the isolated VS to ascertain the most probable side-chain ionization states when the VS was placed in a membrane mimetic environment. All of the ionizable residues were predicted to exist in their default charge states, with the exception of an aspartic acid residue (D146) which was predicted to have an approximately equal likelihood for both its ionized and protonated states (its predicted pKA was 7.9 and 5.2 in vacuo and in a membrane mimetic, respectively). We therefore have performed simulations with residue D146 in each of these states (Table 1).
KvAP is from the thermophilic bacteria Aeropyrum pernix, which thrives at temperatures 368 K (95°C) (38). Therefore, in addition to simulations at 300 K, we have run simulations at 368 K to explore the conformational dynamics of KvAP VS at a temperature closer to its physiological operating temperature and also to explore enhanced protein flexibility at the higher temperature. In total we have performed 0.14 µs of MD simulations of the KvAP VS in a detergent micelle, a system containing 44,000 atoms.
Visual inspection of each VS/DM micelle system revealed that the initially ordered torus of detergent molecules in the micelle (Fig. 1 B) adjusted its shape during the course of each simulation so as to increase the solvent exposure of the ends of the helices (Fig. 1 C). This is comparable to the behavior observed in a number of other membrane protein/micelle simulations, e.g., of the ß-barrel outer membrane protein OmpA (13) and of the -helical membrane protein GlpF (14), both in micelles with octyl glucoside. The shape of the detergent component of the micelles was quantified by calculation of the eccentricity, defined as 
= 1 – I IMIN/IAVG where IMIN is the smallest of the three principal moments of inertia (I1, I2, I3), and IAVG is the average of all three. For a perfectly spherical object, = 0. For the four simulations the initial eccentricity was 
0.07. At the end of the simulations, the eccentricity was 0.19. Thus, the micelles become more ellipsoidal with time, as has also been observed with OmpA/DPC micelles (for which 0.2—see Bond and Sansom (13)) and with GlpF/OG micelles (14).
Conformational drift
To evaluate the conformational stability of the isolated VS in these micelle simulations, we have measured the degree of drift from the initial (crystal) structure. This may be evaluated as the root mean-square deviation (RMSD) of the C atoms from their coordinates at the start of the simulation (Fig. 2). The general pattern is the same in all simulations—an initial jump followed by a slower rise to a plateau. This has been observed in many MD simulations of membrane proteins, both in lipid bilayers and detergent micelles. At 300 K the C RMSD over the final 5 ns (for all residues, i.e., including the loops between helices) is 2 Å regardless of the ionization state of D146. This is comparable to that seen for other membrane proteins in micelles (e.g., 2 Å for GlpF (14) and a similar value for OmpA is obtained if one excludes the long flexible extracellular loops from the RMSD calculation for the latter protein (13)). Thus, even though the current simulations of the KvAP VS are 3–4 times longer than those of GlpF and OmpA, the conformational drift of the VS is about the same. We may therefore conclude that the VS is conformationally stable in DM micelles on a 40 ns timescale at 300 K.
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4 Å). (This is comparable to the figure for OmpA at 300 K if the flexible loops are included in the calculation.) However, if only the -helical regions are taken into consideration, the final C RMSDs are 3 Å. This indicates that at 368 K the greater structural drift is largely, but not entirely, due to conformational changes occurring in the loop regions. Again, the protonation state of D146 does not seem to influence the conformational drift at the higher temperature.
In summary, from consideration of conformational drift we can see that removal of the VS from its crystallographic interactions with the Fab molecule and embedding it in a detergent micelle does not lead, on a 40 ns timescale, to a substantial change in conformation even at an elevated temperature. By way of comparison, chymotrypsin inhibitor 2 has been observed to unfold on a 25 ns timescale in simulations at 373 K (39). Taken together, this suggests that the isolated VS domain is intrinsically stable in a micelle environment.
Secondary structure and flexibility
A more detailed analysis of the simulations reveals the relative flexibility of the different regions of the VS. Analysis of the secondary structural elements as defined by DSSP (40) (Fig. 3 A) showed that helices S1, S2, and S4 were stable over the course of each simulation, regardless of the temperature or protonation state of D146. In contrast, the S3 helix (which in the crystal structure is distorted to form distinct S3a and S3b helices, Fig. 3 D) and the S3/S4 loop show enhanced flexibility at the higher temperature. In particular, S3a unfolds at 368 K, regardless of the protonation state of D146. (There is also some loss of helicity from the N-terminus of S4 at 368 K, but only if D146 is ionized.) Thus, the elevated temperature simulations suggest a degree of conformational instability for S3a. Interestingly, a consensus TM helix secondary structure prediction analysis of KvAP (41) suggested that S3a may not form a canonical TM helix.
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atom from its time averaged coordinate (Fig. 3 C). The overall pattern for each simulation is as anticipated, with higher RMSFs for the loops and lower RMSFs for the cores of the -helices. However, there are some more significant details. First, the S2/S3a loop and the S3a region show a significantly higher RMSF at 368 K than at 300 K. This is consistent with the secondary structure analysis and suggests that S3a is unstable at the higher temperature. The second significant detail is that the S3b-S4 loop shows a relatively high RMSF in each of the simulations.
A comparison with the corresponding crystallographic temperature factor (B-value; Fig. 3 B) profile is informative. Of course, one should guard against overinterpretation of comparisons of crystallographic B-values and simulation RMSFs. However, a striking difference in the overall profiles is evident, namely that the crystallographic B-value for the S3b/S4 loop is low relative to the remainder of the protein, whereas the RMSF for this region is high relative to the protein as a whole. As shown in the inset in Fig. 3, the S3b-S4 loop corresponds to the primary interaction between the VS and the Fab molecule. Thus it would seem that either the intrinsic flexibility of the S3b/S4 loop is suppressed within the Fab/VS crystal and/or the local conformation of this region in the crystal may be altered relative to its equilibrium structure in the micellar environment. It is of possible functional significance that this region, the S3b-S4 loop, corresponds to the tip of the voltage-sensing paddle that is suggested to be capable of changing its conformation/orientation relative to the membrane in the paddle model of voltage gating (11).
The results of the analysis of secondary structure and flexibility may be summarized with reference to Fig. 3 D. Comparison of the crystallographic structure with those at the end of the VS300 and VS368 simulations for the S2–S3 region shows that there is a local change in conformation in S3a and a local distortion of the C-terminus of S3b. Thus, the simulations reveal that the S3a and S3b helices possess a degree of conformational flexibility. As we will see, this is important in allowing the S4 helix to move relative to the remainder of the VS domain, a process that is in turn of relevance to voltage gating in the channel.
The crystal structure of the isolated VS domain of KvAP superimposed on the corresponding VS domain of Kv1.2 reveals that the two structures are similar (RMSD 2.9 Å). However, they diverge most notably in the region of the S3 helix (Fig. 4). Thus, the Kv1.2 crystal structure has captured the S3 helix in a different conformation from that of KvAP, which may further support the notion of a flexible S3 helix.
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Simple visual examination of snapshots of S4 suggests a degree of hinge bending may occur in this helix about the central I130 residue (Fig. 5 A). To explore this aspect of S4 dynamics in more detail, we employed principal components analysis (PCA) to reveal the major motions of the helix. We combined PCA with analysis of the motions of S4 in terms of helix kink and swivel. Analyzing TM helix distortions in terms of kink and swivel angles has been employed to explore proline-induced distortions of TM helices in x-ray structures of membrane proteins (29) and more recently in analysis of M2 helix hinge bending as the basis of channel gating in simulations of inward rectifier K+ channels (42). If we take the motions of S4 represented by the first two eigenvectors (together these account for 50% of the motion observed in the simulations) and analyze these in terms of kink and swivel angle, we see clear evidence for both swivel motion and, to a lesser extent, kink motion about the center of S4 (Fig. 5, B and C). The central hinge point seems to be around residue I130, i.e., in the center of the S4 helix. Thus, the S4 helix does not behave as a rigid rod but rather as two semirigid -helical segments connected by a central molecular hinge. We note that the flexibility observed in the S3b/S4 loop may facilitate such motions of S4.
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,10). This is evidently of some importance for possible voltage sensing/gating models of Kv and related channels (43). We have used water penetration within the protein/detergent micelles as a measure of the degree of crevice formation in the VS. As can be seen (Fig. 6 A), a number of water molecules do infiltrate the VS, forming hydrogen bonds with, e.g., R117, R120, R123, and R126 of the N-terminal half of S4 (Table 2). Thus, our simulations indicate that hydrogen-bonding interactions with such waters may help to stabilize the positively charged S4 helix within a bilayer-like environment. Preliminary analysis of simulations of the VS in a lipid bilayer support this claim (Z. A. Sands and M. S. P. Sansom, unpublished results). Of course, in the intact channel the VS is packed next to the core pore-forming domain of the protein. However, it is likely that the micelle mimics the environment provided by the remainder of the protein in the intact channel.
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1.4 Å). Thus the voltage drop across the membrane must occur over a region at least 12 Å thick around S4. The central region corresponds to a salt bridge between a side chain in S2 (D62) and a side chain in S4 (R133; see Fig. 6 C), which is maintained throughout the course of each simulation. This salt bridge holds S2 and S4 in close proximity and prevents water from passing from one side to the other of the crevice. It is noteworthy that the central hinge in S4 (at residue I130) is just one turn of the helix before R133. Thus the two halves of S4 are connected by a hinge that fits within the narrow central region of the water penetrable crevice.
Detergent interactions
It is of some interest whether the gating charges on S4 are able to favorably interact with the micelle environment or whether they remain buried within the interior of the VS. In the paddle and the twisted S4 models, the charged S4 helices are significantly exposed to the surrounding lipid environment during their translocation from one side of the membrane to the other. Therefore favorable interactions between the gating charges and the lipid environment could significantly lower the energy barrier to channel activation. Analysis of the hydrogen-bonding patterns reveals that the side chains of Arg's 117, 120, 123, and 126 are able to form multiple hydrogen bonds with the uncharged headgroups of the decyl maltoside detergent molecules (Table 2).
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DISCUSSION |
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), we have taken advantage of the higher resolution of the VS domain structure, which allows for more accurate simulations. By embedding the VS within a detergent micelle, the membrane-mimetic environment is able to more fully relax around the protein on the timescale accessible to current simulations, thus facilitating possible conformational transitions in the VS. Also, by using a suitable detergent our simulations stay close to experimental (in vitro) reality. The use of high temperature simulations (possible because of the thermophilic nature of A. pernix) helped us to more fully explore the conformational flexibility of the VS.
It is important to consider the likely effects of a detergent micelle environment on the conformational dynamics of the VS domain. Detergent micelles are widely employed in biochemical studies of membrane proteins (45), for example in NMR studies of membrane proteins (46). Whilst a detergent micelle provides a mimic of the lipid bilayer environment, both NMR (47) and simulation (13) studies suggest a small increase in protein flexibility in the micelle relative to the bilayer. It is this which we have attempted to exploit to explore the intrinsic flexibility of the VS. Of course, in the intact Kv channel this flexibility will be coupled to conformational changes leading to channel gating. This is likely to occur on a significantly longer timescale (1 µs or more).
There are two key results which emerge from our simulations of the isolated VS. The first is the intrinsic flexibility/conformational instability of the S3a region. The second is the hinge bending and swiveling motion of the S4 helix about the central I130 residue (see Fig. 7). The latter result correlates well with recent results from site-directed spin labeling experiments (7), which have been interpreted in terms of a swivel-like distortion in the center of S4. The overall model that emerges is of a VS domain which exhibits a limited degree of flexibility. The S4 helix is able to kink/swivel about a central (I130) hinge and sits at the center of a crevice that can be penetrated by water at either end. The conformational instability of the S3a region facilitates the motion of the N-terminal segment of S4 (i.e., S4a) linked to S3b.
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), hanatoxin (48), and SGTX1 (49)) have been used to probe the location of the paddle relative to the membrane. It is thought that these small peptide toxins partition to the water/bilayer interface, with their hydrophobic residues pointing down toward the hydrophobic core of the bilayer ((50); D. Bemporad, Z. A. Sands, C. L. Wee, A. Grottesi, and M. S. P. Sansom, unpublished results). Mutational studies on these VS-binding toxins combined with mutational studies of Kv channels point to an interaction between polar/charged residues on the toxin surface with residues in the S3b helix of the VS (8,51,52). Together these studies suggest that the tip of the gating paddle, as defined by S3b, is located at the extracellular interfacial region of the membrane.
Furthermore, recent topological experiments suggest that despite the presence of basic side chains, an S4 helix can insert into a lipid bilayer (53). This is of particular interest given that we observe water molecules penetrating the VS/detergent micelle to solvate the charged residues on S4. This suggests that comparative simulations of S4, of the VS domain, and of the intact channel in bilayer versus micelle may reveal aspects of the stability of the S4 helix in different membrane environments. There have also been a number of simulations of KvAP and related channels, based on the crystal structure of the intact channel protein (44,54,55). Although it is difficult to evaluate the relationship of such simulations to the physiological conformations of Kv channels, they do suggest a complex interplay between VS domain, water molecules, and the membrane environment during voltage sensing.
Taken together with the current simulation studies, this body of data suggests a modified paddle model in which the intrinsic flexibility of the VS domain enables the S3b-S4a helical hairpin to move relative to the bilayer and the remainder of the channel protein in response to a change in transbilayer voltage. Such a mechanism could result in the S3b-S4a helical hairpin, changing its degree of water and bilayer exposure during the voltage-sensing transition. Further studies, both experimental and computational, are needed to unpack the exact nature of the changes in protein conformation, and in protein/water/lipid interactions, that occur during voltage sensing.
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ACKNOWLEDGEMENTS |
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This work was supported by funding from The Welcome Trust.
Submitted on August 5, 2005; accepted for publication November 9, 2005.
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90% of the simulation time are shown as blue, 
V) by the S4a region swiveling relative to the S4b region, which remains fixed relative to the remainder of the VS. The conformational flexibility of S3a may provide a restoring spring able to return the voltage-sensing paddle (formed by S3b-S4a) to its initial conformation once the resting membrane potential is restored. (B) Schematic diagram of the VS TM topology, colored on conformational flexibility (blue, low; red, high), and indicating the hinge region that divides S4 into S4a and S4b.