KvAP Channel Research Articles

Structural Characterization of the Voltage Sensor Domain of the KvAP Channel Vectorially-Oriented within a Phospholipid Bilayer Membrane

Voltage-gated cation (Na+, K+) channels are responsible for the generation and propagation of action potentials in neurological signal transmission. Kv-channels are transmembrane proteins consisting of a homo-tetramer of 4 subunits that assemble about a 4-fold axis normal to the membrane plane to form the K+ ion-selective pore. Each of the four subunits is comprised of six transmembrane helices, the S1-S4 helices forming the voltage-sensor domain (VSD) and the S5-S6 helices contributing to form the pore domain (PD). Despite several advances in the field, a complete understanding of the mechanism of electromechanical coupling interconverting the closed-to-open states is yet to be achieved. Positively charged arginine residues predominately in the S4 helix of the VSD are responsible for voltage sensing and the VSD's are arranged around the periphery of the PD in extensive contact with the lipid bilayer. This prompted us to focus initially on the structure of VSD itself within a phospholipid bilayer environment for the present study. A hydrated, phospholipid bilayer membrane environment has been reconstituted for the VSD of KvAP, vectorially oriented on the surface of inorganic multilayer substrates. This has been established by X-ray and neutron reflectivity (enhanced by interferometry), the latter employing a specifically deuterated phospholipid and water contrast variation, for the reconstituted membrane at both the solid-vapor and solid-liquid interfaces. This accomplishment now allows an investigation of the profile structure of the VSD within the lipid bilayer as a function of the applied transmembrane electric potential via x-ray reflectivity with millisecond time-resolution, employing high energy x-rays (> 20KeV) & pixel array detectors, and neutron reflectivity, employing selectively deuterium-labeled VSD proteins achieved via semi-synthesis. The same approach can be extended to the intact KvAP channel.

A potassium Channel Voltage-Sensing Domain in a Non-Phospholipid Bilayer

Non-phospholipid, cationic 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) lipid based membranes fail to support the function of a voltage-dependent K+ channel due to the lack of phosphate groups (Schmidt et al., Nature 444, 775, 2006). However, the specific effects of the presence or absence of phosphate groups in the channel membrane environment on the voltage-sensing mechanism remain unknown. To characterize the conformational dynamics and protein-lipid interactions in the absence of lipid phosphates, we performed all-atom simulations of the KvAP channel voltage-sensing domain (VSD) in a DOTAP bilayer in excess water. We generated prolonged trajectories for an up-state (Krepkiy et al., Nature 462, 473, 2009) and a down-state (Schow et al., Biophys J 98, 2857, 2010) conformation of the VSD, and contrasted the results with simulations of the VSD embedded in a POPC bilayer. Pair interaction energies reveal differences in the way each VSD conformation interacts with the different components of the membrane environment in the presence and absence of phosphate groups, which result in significant changes to the system electrostatics. This work is supported by NIH grants GM74637 and GM86685 and NSF grant CHE-0750175. This research was supported in part by NSF through Teragrid resources provided by the TACC at UT Austin.

Phospholipids as a Structural and Functional Determinant for Voltage Sensors in a Kv Channel

Recent studies proposed that phosphodiester groups in a phospholipid bilayer help stabilize a voltage sensor in its activated conformation. However, the nature of such channel-lipid interaction is unclear. We are studying the structure and function of the KvAP channel in lipid bilayers. We obtain the structural information of the channel in membranes by electron crystallographic study of two-dimensional crystals, and utilize both biochemical assays and electrical recordings to determine the conformational states of the voltage sensors and the pore domain in reconstituted channels. Our electron crystallographic studies start to reveal that the KvAP voltage sensors in a phospholipid membrane can take an alternative conformation, which is different from the expected four-helix bundle structure observed in the X-ray crystallographic studies of Kv channel proteins in detergents or mixed detergent/lipid micelles. The functional implications of such a new conformation of the voltage sensor are being investigated. Our functional studies converge to the conclusion that annular phospholipids around the channel are required for its voltage sensors to switch from the deactivated to the activated state. Our data suggest that a phospholipid bilayer may be an essential factor for the structure and function of a voltage sensor.

High-Resolution Orientation and Depth of Insertion of the Voltage-Sensing S4 Helix of a Potassium Channel in Lipid Bilayers

The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltage-sensing domain. The interaction of these positively charged Arg residues with the lipid membrane has been of intense interest for understanding how membrane proteins fold to allow charged residues to insert into lipid bilayers against free-energy barriers. Using solid-state NMR, we have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers. Two-dimensional 15N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40° and two possible rotation angles differing by 180° around the helix axis. Remarkably, the tilt angle and one of the two rotation angles are identical to those of the S4 helix in the intact voltage-sensing domain, suggesting that interactions between the S4 segment and other helices of the voltage-sensing domain are not essential for the membrane topology of the S4 helix. 13C– 31P distances between the S4 backbone and the lipid 31P indicate a ∼ 9 Å local thinning and 2 Å average thinning of the DMPC (1,2-dimyristoyl- sn-glycero-3-phosphochloline)/DMPG (1,2-dimyristoyl- sn-glycero-3-phosphatidylglycerol) bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 Å from the guanidinium C ζ of the second Arg to 31P indicates the existence of guanidinium phosphate hydrogen bonding and salt bridges. These data suggest that the structure of the Kv gating helix is mainly determined by protein–lipid interactions instead of interhelical protein–protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix.

NMR Structural and Dynamical Investigation of the Isolated Voltage-Sensing Domain of the Potassium Channel KvAP: Implications for Voltage Gating

The structure and dynamics of the isolated voltage-sensing domain (VSD) of the archaeal potassium channel KvAP was studied by high-resolution NMR. The almost complete backbone resonance assignment and partial side-chain assignment of the (2)H,(13)C,(15)N-labeled VSD were obtained for the protein domain solubilized in DPC/LDAO (2:1) mixed micelles. Secondary and tertiary structures of the VSD were characterized using secondary chemical shifts and NOE contacts. These data indicate that the spatial structure of the VSD solubilized in micelles corresponds to the structure of the domain in an open state of the channel. NOE contacts and secondary chemical shifts of amide protons indicate the presence of tightly bound water molecule as well as hydrogen bond formation involving an interhelical salt bridge (Asp62-R133) that stabilizes the overall structure of the domain. The backbone dynamics of the VSD was studied using (15)N relaxation measurements. The loop regions S1-S2 and S2-S3 were found mobile, while the S3-S4 loop (voltage-sensor paddle) was found stable at the ps-ns time scale. The moieties of S1, S2, S3, and S4 helices sharing interhelical contacts (at the level of the Asp62-R133 salt bridge) were observed in conformational exchange on the micros-ms time scale. Similar exchange-induced broadening of characteristic resonances was observed for the VSD solubilized in the membrane of lipid-protein nanodiscs composed of DMPC, DMPG, and POPC/DOPG lipids. Apparently, the observed interhelical motions represent an inherent property of the VSD of the KvAP channel and can play an important role in the voltage gating.

Lipid−Protein Nanodiscs as Reference Medium in Detergent Screening for High-Resolution NMR Studies of Integral Membrane Proteins

The choice of a suitable detergent-based membrane mimetic is of crucial importance for high-resolution NMR studies of membrane proteins. The present report describes a new approach of detergent screening. It is based on the comparison of 2D (1)H,(15)N-correlation spectra of a protein in a membrane-bilayer "reference" medium and in "trial" detergent-based environments. The proposed "reference" medium is the lipid-protein nanodisc (LPN) representing nanoscale phospholipid bilayers wrapped around by apolipoprotein A-1. The set of zwitterionic (DPC, DMPC/DHPC), anionic (SDS, LMPG, LPPG), and weakly cationic (LDAO) detergent-based media was screened for their ability to represent the native structure of the isolated voltage-sensing domain (VSD) of the archaeal potassium channel KvAP. The VSD/LPN complexes composed of saturated zwitterionic (DMPC), anionic (DMPG), or a mixture of unsaturated differently charged (POPC/DOPG, 3:1) lipids were used as reference. All assayed detergent media demonstrate similar CD spectra of the domain with a high level (approximately 60%) of overall helicity but different 2D NMR spectra. Using the reference spectrum of the VSD in LPN, we were able to choose the detergent composition in which the membrane-like structure of the VSD is preserved.

Interactions Between a Voltage Sensor and a Toxin via Multiscale Simulations

Gating-modifier toxins inhibit voltage-gated ion channels by binding the voltage sensors (VS) and altering the energetics of voltage-dependent gating. These toxins are thought to gain access to the VS via the membrane (i.e., by partitioning from water into the membrane before binding the VS). We used serial multiscale molecular-dynamics (MD) simulations, via a combination of coarse-grained (CG) and atomistic (AT) simulations, to study how the toxin VSTx1, which inhibits the archeabacterial voltage-gated potassium channel KvAP, interacts with an isolated membrane-embedded VS domain. In the CG simulations, VSTx1, which was initially located in water, partitioned into the headgroup/water interface of the lipid bilayer before binding the VS. The CG configurations were used to generate AT representations of the system, which were subjected to AT-MD to further evaluate the stability of the complex and refine the predicted VS/toxin interface. VSTx1 interacted with a binding site on the VS formed by the C-terminus of S1, the S1-S2 linker, and the N-terminus of S4. The predicted VS/toxin interactions are suggestive of toxin-mediated perturbations of the interaction between the VS and the pore domain of Kv channels, and of the membrane. Our simulations support a membrane-access mechanism of inhibition of Kv channels by VS toxins. Overall, the results show that serial multiscale MD simulations may be used to model a two-stage process of protein-bilayer and protein-protein interactions within a membrane.

Double gaps along Shaker S4 demonstrate omega currents at three different closed states

The aim of the present study was to investigate in detail how the voltage sensor in the Shaker potassium channel moves during the gating process. After the publication of the open channel structure from the crystallized KvAP channel in 2003, an alternative so-called "paddle" model was put forward in contrast to the existing helical screw model. The voltage sensor S4 contains 4 arginine residues relevant for gating, R1(362), R2(365), R3(368) and R4(371), each separated by 2 neutral residues. These charged residues coil as one of three threads on the S4-α-helix. Based on a previous finding that the mutation R1S leads to the so-called omega leak current through a "gating-pore" in the closed state, we introduced gaps systematically along the arginine thread substituting long arginines by short serines. Mutations R2S or R3S did neither create transient nor steady leaks. The fact that the native residue A359, which is located three amino acids in front of R1, is a short one, motivated us to check its role. Mutation of A359 to arginine blocked the omega current in R1S mutant indicating that the omega pore is occupied by A359 and R1. Introducing further double gaps (RR to SS) at sequential positions (0+1, 1+2, 2+3), produced clear leak currents which were remarkably stable over a wide voltage range. These leaks contradict that S4 would swing together with S3 in lipid according to the paddle hypothesis. Rather, our results show that during gating the S4 segment moves in 3 helical steps through a fixed pore formed by the channel protein.

Membrane/Toxin Interaction Energetics via Serial Multiscale Molecular Dynamics Simulations.

Computing free energies of complex biomolecular systems via atomistic (AT) molecular dynamics (MD) simulations remains a challenge due to the need for adequate sampling and convergence. Recent coarse-grained (CG) methodology allows simulations of significantly larger systems (∼10(6) to 10(8) atoms) over longer (μs/ms) time scales. Such CG models appear to be capable of making semiquantitative predictions. However, their ability to reproduce accurate thermodynamic quantities remains uncertain. We have recently used CG MD simulations to compute the potential of mean force (PMF) or free energy profile of a small peptide toxin interacting with a lipid bilayer along a 1D reaction coordinate. The toxin studied was VSTx1 (Voltage Sensor Toxin 1) from spider venom which inhibits the archeabacterial voltage-gated potassium (Kv) channel KvAP by binding to the voltage-sensor (VS) domains. Here, we re-estimate this PMF profile using (i) AT MD simulations with explicit membrane and solvent and (ii) an implicit membrane and solvent (generalized Born; GBIM) model where only the peptide was explicit. We used the CG MD free energy simulations to guide the setup of the corresponding AT MD simulations. The aim was to avoid local minima in the AT simulations which would be difficult over shorter AT time scales. A cross-comparison of the PMF profiles revealed a conserved topology, although there were differences in the magnitude of the free energies. The CG and AT simulations predicted a membrane/water interface free energy well of -27 and -23 kcal/mol, respectively (with respect to water). The GBIM model, however, gave a reduced interfacial free energy well (-12 kcal/mol). In addition, the CG and GBIM models predicted a free energy barrier of +61 and +96 kcal/mol, respectively, for positioning the toxin at the center of the bilayer, which was considerably smaller in the AT simulations (+26 kcal/mol). Thus, we present a framework for serially combining CG and AT simulations to estimate the free energy of peptide/membrane interactions. Such approaches for combining simulations at different levels of granularity will become increasingly important in future studies of complex membrane/protein systems.

A New Approach to the Structural Investigation of the Voltage-Sensitive Domain of Voltage-Gated Cation Channels as a Function of the Transmembrane Voltage

Two fundamentally different approaches, self-assembly from solution and directed-assembly at the water-gas interface, are effective in the vectorial immobilization of the expressed voltage-sensitive domain of the KvAP channel on a suitably alkylated surface of silicon. The solvation of the immobilized protein was subsequently exchanged to form a phospholipid bilayer by incubating in a phospholipid-detergent solution in the presence of BioBeads. The formation of the protein monolayer and the vectorial-orientation of the protein molecules therein were investigated via interferometric X-ray reflectivity. The electron density profile of the tethered protein monolayer is consistent with the profile computed from the crystal structure, irrespective of the preparation procedure. Formation of lipid bilayer will require confirmation via neutron reflectivity using deuterated phospholipid. This approach enables an investigation of the structure of the VSD itself, as well as within the intact Kv-channel, as a function of the applied transmembrane voltage via a number of time-resolved techniques.

A Gating Model for the Archeal Voltage-Dependent K+ Channel KvAP in DPhPC and POPE:POPG Decane Lipid Bilayers

Voltage-dependent K+ (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.

Towards The Chemical Synthesis Of A Voltage Gated K+ Channel

Understanding the relationship between the atomic structure of a protein and the biological functions requires the ability to perturb the protein structure in a precise manner. This is generally accomplished by means of traditional site-directed mutagenesis. However, the modifications that can be introduced in this manner are limited by the set of naturally occurring amino acids. Chemical synthesis on the other hand facilitates the incorporation of a wide variety of side chain and peptide backbone modifications that enables precise modifications of the structural and electronic properties of the protein. Similar modifications are not possible using conventional mutagenesis, thus making chemical synthesis an important asset in investigations of protein structure and function. The size of the protein has been a major factor limiting the use of chemical synthesis to investigate proteins. In the field of membrane proteins, chemical synthesis has so far been accomplished only for relatively small ( 150 amino acid) membrane proteins are required. We are presently developing methodology for the chemical synthesis of the voltage gated K+ channel KvAP. Voltage gated ion channels are responsible for the generation of electrical impulses by excitable cells. They have been the subject of intense research, however, a number of key questions remain about the mechanisms of ionic selectivity and gating in these channels. We believe that using chemical synthesis for protein modification in combination with functional and structural studies will enable us to provide new information on these physiologically important processes.

Voltage-Dependent Conformational Changes of the Voltage Sensor of KVAP Measured with LRET

The opening and closing of voltage-dependent ions channels depend upon conformational changes initiated in the voltage-sensors. The nature and extent of this rearrangement has been widely investigated in K+ channels, using various techniques, but so far, there is no agreement on this fundamental mechanism in K+-channel gating.We investigated the voltage-sensor operation using a LRET technique employing Tb3+ ions bound to several Lanthanide-Binding-Tags (LBT) genetically encoded at the top of the S3 and S4 segments of the KvAP channel. A fluorescently-labeled pore-blocking toxin, the Agitoxin-2, was conveniently used as an acceptor placed at a non-mobile position near the pore-axis. Three mutations, S179G, K181D and P176E, were systematically introduced in the pore region to increase toxin binding. Various fluorophores were covalently attached to cysteines individually inserted at positions N5, Q13 and D20 of the agitoxin. After purification, mutant channels were reconstituted into proteoliposomes and submitted to a Nernst-clamp procedure combining the use of valinomycin and a K+ chelator. This method allowed us to stably clamp the liposomal electrical potential at negative and positive potentials. Thus, this allowed us to stabilize the voltage-sensor in its closed state and in its open-inactivated state during the data acquisition period. Channels were then blocked with the fluorescent toxins and LRET measurements were recorded. Sensitized emissions of the acceptors were fitted with a square-base pyramidal multi-exponential model (Posson and Selvin, 2008) allowing the extraction of the four distances separating the position of the acceptor near the pore and the position of the donors located further away in the four subunits. The voltage-dependent coordinates of the LBTs indicate a rotational movement of the top of S4, with little participation of the top of the S3 segment.Support: FWF and AHA0725763Z, NIHGM068044 and GM30376.

Using Electron Spin Echo Envelope Modulation (ESEEM) to Probe the Local Environment of Residues in the KcsA Potassium Channel

Abstract: Recent studies on the voltage-dependent potassium channel KvAP have demonstrated the importance of the lipid bilayer in maintaining the correct orientation and packing of the voltage-sensor domain with respect to the pore domain [1, 2]. It has also been suggested that lipid may play an integral role in voltage-dependent gating [3, 4]. To date, the precise environment surrounding the four important S4 arginine residues on the voltage-sensing paddle is still unknown, with some models placing them in an aqueous crevice, and others a lipid environment. To learn more about the intricate role of lipid in the structure and function of potassium channels we have studied deuterium and phosphate ESEEM on spin-labeled, liposome reconstituted KcsA. By scanning the trans-membrane helices of KcsA, we show that deuterium coupling can be used to determine residue depth within a lipid bilayer. In addition, residues that interact with the phosphate head-groups of the lipid can be determined by phosphate coupling, and their precise location modeled.

How to Extend the Use of Grid‐Based Interaction Energy Maps from Chemistry to Biotopics

Many computational tools routinely used in chemistry could successfully be applied to the biosciences since protein-protein and protein-ligand interactions are governed by the laws of chemistry. This paper shows that it is possible to extend the use of existing computational tools from their traditional application field (e.g. chemistry) to culturally-related research areas by the implementation of simple but well-designed utilities. In particular, a computational strategy obtained by combining GRID (the program originally designed by Peter Goodford, and now distributed by Molecular Discovery Ltd.) and BIOCUBE4mf (an application freely available at www.casmedchem.unito.it) was used to a) characterize the surface properties of the cavity of the Bacillus anthracis protective antigen heptameric prepore; b) suggest how to design mutagenesis experiments; c) quantitatively show the selectivity of the KvAP channel for K(+) over Na(+) ions and d) rationalize the pharmacokinetic behavior of 1,4-DHP third-generation drugs.

Molecular Dynamics Simulation of Kv Channel Voltage Sensor Helix in a Lipid Membrane with Applied Electric Field

In this article, we present the results of the molecular dynamics simulations of amphiphilic helix peptides of 13 amino-acid residues, placed at the lipid-water interface of dipalmitoylphosphatidylcholine bilayers. The peptides are identical with, or are derivatives of, the N-terminal segment of the S4 helix of voltage-dependent K channel KvAP, containing four voltage-sensing arginine residues (R1–R4). Upon changing the direction of the externally applied electric field, the tilt angle of the wild-type peptide changes relative to the lipid-water interface, with the N-terminus heading up with an outward electric field. These movements were not observed using an octane membrane in place of the dipalmitoylphosphatidylcholine membrane, and were markedly suppressed by 1), substituting Phe located one residue before the first arginine (R1) with a hydrophilic residue (Ser, Thr); or 2), changing the periodicity rule of Rs from at-every-third to at-every-fourth position; or 3), replacing R1 with a lysine residue (K). These and other findings suggest that the voltage-dependent movement requires deep positioning of Rs when the resting (inward) electric field is present. Later, we performed simulations of the voltage sensor domain (S1–S4) of Kv1.2 channel. In simulations with a strong electric field (0.1 V/nm or above) and positional restraints on the S1 and S2 helices, S4 movement was observed consisting of displacement along the S4 helix axis and a screwlike axial rotation. Gating-charge-carrying Rs were observed to make serial interactions with E183 in S1 and E226 in S2, in the outer water crevice. A 30-ns-backward simulation started from the open-state model gave rise to a structure similar to the recent resting-state model, with S4 moving vertically ∼6.7 Å. The energy landscape around the movement of S4 appears very ragged due to salt bridges formed between gating-charge-carrying residues and negatively charged residues of S1, S2, and S3 helices. Overall, features of S3 and S4 movements are consistent with the recent helical-screw model. Both forward and backward simulations show the presence of at least two stable intermediate structures in which R2 and R3 form salt bridges with E183 or E226, respectively. These structures are the candidates for the states postulated in previous gating kinetic models, such as the Zagotta-Hoshi-Aldrich model, to account for more than one transition step per subunit for activation.

Coarse-Grained MD Simulations of Membrane Protein-Bilayer Self-Assembly

Complete determination of a membrane protein structure requires knowledge of the protein position within the lipid bilayer. As the number of determined structures of membrane proteins increases so does the need for computational methods which predict their position in the lipid bilayer. Here we present a coarse-grained molecular dynamics approach to lipid bilayer self-assembly around membrane proteins. We demonstrate that this method can be used to predict accurately the protein position in the bilayer for membrane proteins with a range of different sizes and architectures.

Coarse-grained simulation: a high-throughput computational approach to membrane proteins

An understanding of the interactions of membrane proteins with a lipid bilayer environment is central to relating their structure to their function and stability. A high-throughput approach to prediction of membrane protein interactions with a lipid bilayer based on coarse-grained Molecular Dynamics simulations is described. This method has been used to develop a database of CG simulations (coarse-grained simulations) of membrane proteins (http://sbcb.bioch.ox.ac.uk/cgdb). Comparison of CG simulations and AT simulations (atomistic simulations) of lactose permease reveals good agreement between the two methods in terms of predicted lipid headgroup contacts. Both CG and AT simulations predict considerable local bilayer deformation by the voltage sensor domain of the potassium channel KvAP.

CGDB: A database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.

Bilayer deformation by the Kv channel voltage sensor domain revealed by self-assembly simulations

Coarse-grained molecular dynamics simulations are used to explore the interaction with a phospholipid bilayer of the voltage sensor (VS) domain and the S4 helix from the archaebacterial voltage-gated potassium (Kv) channel KvAP. Multiple 2-mus self-assembly simulations reveal that the isolated S4 helix may adopt either interfacial or transmembrane (TM) locations with approximately equal probability. In the TM state, the insertion of the voltage-sensing region of S4 is facilitated via local bilayer deformation that, combined with side chain "snorkeling," enables its Arg side chains to interact with lipid headgroups and water. Multiple 0.2-mus self-assembly simulations of the VS domain are also performed, along with simulations of MscL and KcsA, to permit comparison with more "canonical" integral membrane protein structures. All three stably adopt a TM orientation within a bilayer. For MscL and KcsA, there is no significant bilayer deformation. In contrast, for the VS, there is considerable local deformation, which is again primarily due to the lipid-exposed S4. It is shown that for both the VS and isolated S4 helix, the positively charged side chains of S4 are accommodated within the membrane through a combination of stabilizing interactions with lipid glycerol and headgroup regions, water, and anionic side chains. Our results support the possibility that bilayer deformation around key gating charge residues in Kv channels may result in "focusing" of the electrostatic field, and indicate that, when considering competing models of voltage-sensing, it is essential to consider the dynamics and structure of not only the protein but also of the local lipid environment.