Regulation of channel function due to physical energetic coupling with a lipid bilayer

Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions. This study provides a new insight helping better understand of the underlying mechanisms of membrane protein functions in cell membrane leading to important biological implications.     

Interaction of multicomponent anionic liposomes with cationic pyridylphenylene dendrimer: Does the complex behavior depend on the liposome composition?

Dendrimers are individual macromolecular compounds having a great potential for biomedical application. The key step of the cell penetration by dendrimers is the interaction with lipid bilayer. Here, the interaction between cationic pyridylphenylene dendrimer of third generation (D₃⁵⁰⁺) and multicomponent liquid (CL/POPC), solid (CL/DPPC) and cholesterol-containing (CL/POPC/30% Chol) anionic liposomes was investigated by dynamic light scattering, fluorescence spectroscopy, conductometry, calorimetric studies and molecular dynamic (MD) simulations. Microelectrophoresis and MD simulations revealed the interaction is electrostatic and reversible with only part of pyridinium groups of dendrimers involved in binding with liposomes. The ability of dendrimer molecules to migrate between liposomes was discovered by the labeling liposomes with Rhodamine B. The phase state of the lipid membrane and the incorporation of cholesterol into the lipid bilayer were found to not affect the mechanism of the dendrimer - liposome complex formation. Rigid dendrimer adsorption on liposomal surface does not induce the formation of significant defects in the lipid membrane pave the way for possible biological application of pyridylphenylene dendrimers.     

Arginine in Membranes: The Connection Between Molecular Dynamics Simulations and Translocon-Mediated Insertion Experiments

Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths.     

Molecular Dynamics Simulations of Phospholipid Bilayers

Abstract

Molecular dynamics (MD) simulations at 37°C have been performed on three phospholipid bilayer systems composed of the lipids DLPE, DOPE, and DOPC. The model used included 24 explicit lipid molecules and explicit waters of solvation in the polar head group regions, together with constant-pressure periodic boundary conditions in three dimensions. Using this model, a MD simulation samples part of an infinite planar lipid bilayer. The lipid dynamics and packing behavior were characterized. Furthermore, using the results of the simulations, a number of diverse properties including bilayer structural parameters, hydrocarbon chain order parameters, dihedral conformations, electron density profile, hydration per lipid, and water distribution along the bilayer normal were calculated. Many of these properties are available for the three lipid systems chosen, making them well suited for evaluating the model and protocols used in these simulations by direct comparisons with experimental data. The calculated MD behavior, chain disorder, and lipid packing parameter, i.e. the ratio of the effective areas of hydrocarbon tails and head group per lipid (a_t/a_h), correctly predict the aggregation preferences of the three lipids observed experimentally at 37°C, namely: a gel bilayer for DLPE, a hexagonal tube for DOPE, and a liquid crystalline bilayer for DOPC. In addition, the model and conditions used in the MD simulations led to good agreement of the calculated properties of the bilayers with available experimental results, demonstrating the reliability of the simulations. The effects of the cis unsaturation in the hydrocarbon chains of DOPE and DOPC, compared to the fully saturated one in DLPE, as well as the effects of the different polar head groups of PC and PE with the same unsaturated chains on the lipid packing and bilayer structure have been investigated. The results of these studies indicate the ability of MD methods to provide molecular-level insights into the structure and dynamics of lipid assemblies.     

Protein shape change has a major effect on the gating energy of a mechanosensitive channel

Increasing experimental evidence has shown that membrane protein functionality depends on molecular composition of cell membranes. However, the origin of this dependence is not fully understood. It is reasonable to assume that specific lipid-protein interactions are important, yet more generic effects due to mechanical properties of lipid bilayers likely play a significant role too. Previously it has been demonstrated using models for elastic properties of membranes and lateral pressure profiles of lipid bilayers that the mechanical properties of a lipid bilayer can contribute as much as ∼10 k_BT to the free energy difference associated with a change in protein conformational state. Here, we extend those previous approaches to a more realistic model for a large mechanosensitive channel (MscL). We use molecular dynamics together with the MARTINI model to simulate the open and closed states of MscL embedded in a DOPC bilayer. We introduce a procedure to calculate the mechanical energy change in the channel gating using a three-dimensional pressure distribution inside a membrane, computed from the molecular dynamics simulations. We decompose the mechanical energy to terms associated with area dilation and shape contribution. Our results highlight that the lateral pressure profile of a lipid bilayer together with the shape change in gating can induce a contribution of ∼30 k_BT on the gating energy of MscL. This contribution arises largely from the interfacial tension between hydrophobic and hydrophilic regions in a lipid bilayer.     

Comparative Computational Study of Interaction of C60-Fullerene and Tris-Malonyl-C60-Fullerene Isomers with Lipid Bilayer: Relation to Their Antioxidant Effect

Oxidative stress induced by excessive production of reactive oxygen species (ROS) has been implicated in the etiology of many human diseases. It has been reported that fullerenes and some of their derivatives–carboxyfullerenes–exhibits a strong free radical scavenging capacity. The permeation of C₆₀-fullerene and its amphiphilic derivatives–C₃-tris-malonic-C₆₀-fullerene (C₃) and D₃-tris-malonyl-C₆₀-fullerene (D₃)–through a lipid bilayer mimicking the eukaryotic cell membrane was studied using molecular dynamics (MD) simulations. The free energy profiles along the normal to the bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) for C₆₀, C₃ and D₃ were calculated. We found that C₆₀ molecules alone or in clusters spontaneously translocate to the hydrophobic core of the membrane and stay inside the bilayer during the whole period of simulation time. The incorporation of cluster of fullerenes inside the bilayer changes properties of the bilayer and leads to its deformation. In simulations of the tris-malonic fullerenes we discovered that both isomers, C₃ and D₃, adsorb at the surface of the bilayer but only C₃ tends to be buried in the area of the lipid headgroups forming hydrophobic contacts with the lipid tails. We hypothesize that such position has implications for ROS scavenging mechanism in the specific cell compartments.     

Interaction of gabaergic ketones with model membranes: A molecular dynamics and experimental approach

γ-Aminobutyric-acid receptor (GABA_A-R), a membrane intrinsic protein, is activated by GABA and modulated by a wide variety of recognized drugs. GABA_A-R is also target for several insecticides which act by recognition of a non-competitive blocking site. Mentha oil is rich in several ketones with established activity against various insects/pests. Considering that mint ketones are highly lipophilic, their action mechanism could involve, at least in part, a non-specific receptor modulation by interacting with the surrounding lipids. In the present work, we studied in detail the effect on membranes of five cyclic ketones present in mint plants, with demonstrated insecticide and gabaergic activity. Particularly, we have explored their effect on the organization and dynamics of the membrane, by using Molecular Dynamics (MD) Simulation studies in a bilayer model of DPPC. We performed free diffusion MD and obtained spatially resolved free energy profiles of ketones partition into bilayers based on umbrella sampling. The most favored location of ketones in the membrane corresponded to the lower region of the carbonyl groups. Both hydrocarbon chains were slightly affected by the presence of ketones, presenting an ordering effect for the methylene groups closer to the carbonyl. MD simulations results were also contrasted with experimental data from fluorescence anisotropy studies which evaluate changes in membrane fluidity. In agreement, these assays indicated that the presence of ketones between lipid molecules induced an enhancement of the intermolecular interaction, increasing the molecular order throughout the bilayer thickness.     

An explorative study on Staphylococcus aureus MurE inhibitor: induced fit docking,binding free energy calculation,and molecular dynamics

Staphylococcus aureus MurE enzyme catalyzes the addition of l-lysine as third residue of the peptidoglycan peptide moiety. Due to the high substrate specificity and its ubiquitous nature among bacteria, MurE enzyme is considered as one of the potential target for the development of new therapeutic agents. In the present work, induced fit docking (IFD), binding free energy calculation, and molecular dynamics (MD) simulation were carried out to elucidate the inhibition potential of 2-thioxothiazolidin-4-one based inhibitor 1 against S. aureus MurE enzyme. The inhibitor 1 formed majority of hydrogen bonds with the central domain residues Asn151, Thr152, Ser180, Arg187, and Lys219. Binding free-energy calculation by MM-GBSA approach showed that van der Waals (ΔG_vdW, ?57.30?kcal/mol) and electrostatic solvation (ΔG_solv, ?36.86?kcal/mol) energy terms are major contributors for the inhibitor binding. Further, 30-ns MD simulation was performed to validate the stability of ligand–protein complex and also to get structural insight into mode of binding. Based on the IFD and MD simulation results, we designed four new compounds D1–D4 with promising binding affinity for the S. aureus MurE enzyme. The designed compounds were subjected to the extra-precision docking and binding free energy was calculated for complexes. Further, a 30-ns MD simulation was performed for D1/4C13 complex.     

Mechanism of Long-Chain Free Fatty Acid Protonation at the Membrane-Water Interface

Long-chain free fatty acids (FFAs) play an important role in several physiological and pathological processes such as lipid fusion, adjustments of membrane permeability and fluidity, and the regulation of enzyme and protein activities. FFA-facilitated membrane proton transport (flip-flop) and FFA-dependent proton transport by membrane proteins (e.g., mitochondrial uncoupling proteins) are governed by the difference between FFA’s intrinsic pK_a value and the pH in the immediate membrane vicinity. Thus far, a quantitative understanding of the process has been hampered, because the pK_a value shifts upon moving the FFA from the aqueous solution into the membrane. For the same FFA, pK_a values between 5 and 10.5 were reported. Here, we systematically evaluated the dependence of pK_a values on chain length and number of double bonds by measuring the ζ-potential of liposomes reconstituted with FFA at different pH values. The experimentally obtained intrinsic pK_a values (6.25, 6.93, and 7.28 for DOPC membranes) increased with FFA chain length (C16, C18, and C20), indicating that the hydrophobic energy of transfer into the bilayer is an important pK_a determinant. The observed pK_a decrease in DOPC with increasing number of FFA double bonds (7.28, 6.49, 6.16, and 6.13 for C20:0, C20:1, C20:2, and C20:4, respectively) is in line with a decrease in transfer energy. Molecular dynamic simulations revealed that the ionized carboxylic group of the FFAs occupied a fixed position in the bilayer independent of chain length, underlining the importance of Born energy. We conclude that pK_a is determined by the interplay between the energetic costs for 1) burying the charged moiety into the lipid bilayer and 2) transferring the hydrophobic protonated FFA into the bilayer.     

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.     

Effects of salinity on the CO<Subscript>2</Subscript> permeation across lipid bilayer for microalgae biofixation: a molecular dynamics study

The continuous threat of increasing CO₂ concentration in the atmosphere has altered the carbon balance of our planet causing global climate change. Biological fixation of atmospheric CO₂ by unicellular microorganisms such as microalgae is a promising technology pursued extensively by researchers as a means for carbon capture. The study aimed to provide an atomic level of study that will demonstrate the effect of the salinity on the mechanism of CO₂ absorption across microalgae lipid bilayer. Molecular dynamics simulations were utilized to calculate the free energies of CO₂ molecule as it permeates inside the microalgae cell. In thermodynamics, the transport process of a molecule can be demonstrated through its free energy gradient. Thus, calculating the free energies of CO₂ molecule across microalgae lipid bilayer can elucidate the mechanisms of permeation processes. Four microalgae lipid bilayer structures were constructed that contains 128-DPPC (dipalmitoylphosphatidylcholine) lipid bilayer with 3640 water molecules with different NaCl concentrations: 0, 3, 13, and 19 NaCl molecules which correspond to a salinity level of 0, 50, 200, and 300 mM, respectively. The cavity insertion Widom method was used to calculate the free energy of CO₂ molecule along the lipid bilayer. The results demonstrated that the salinity does not affect the free energies significantly, thus, it does not hamper CO₂ transport across microalgae lipid membrane.     

A Molecular Dynamics Study of DMPC Lipid Bilayers Interacting with Dimethylsulfoxide–Water Mixtures

The diffusion process of dimethylsulfoxide (DMSO) through zwitterionic dimyristoylphosphatidylcholine (DMPC) lipid bilayer was studied by means of molecular dynamics (MD) simulations. To account for the cryoprotectant concentration difference between the inside and the outside of the cell, dual DMPC lipid bilayers which separate two aqueous reservoirs with and without DMSO were modeled. The initial configuration of the simulation model had DMSO molecules present in one of the aqueous phases (outside the cell) at two different concentrations of ~3 and ~6?mol%. MD simulations were performed on the systems for 50?ns at 323?K and 1?bar. Although the simulation time considered in the study was insufficient for the DMSO molecules to reach the other aqueous phase and equilibrium, early stages of the diffusion process indicated that DMSO molecules had a tendency to diffuse towards the other aqueous phase. The effects of DMSO on bilayer structural characteristics during the diffusion process were investigated. Simulations were analyzed to correlate the following properties of lipid bilayers in the presence of two different aqueous phases: area per lipid, lipid thickness, mass density profiles, lipid tail order parameter and water dipole orientation. Area per lipid calculated for the leaflet facing the aqueous DMSO?Cwater mixture did not show any significant difference compared to area per lipid for the DMSO-free pure DMPC bilayer. Mass density profiles revealed that DMSO molecules had a strong tendency to diffuse toward the aqueous phase with pure water. The lipid tail order parameter calculated for the sn-1 tail of the leaflet facing the aqueous DMSO?Cwater mixture showed that the ordering of lipid tails decreased compared to the leaflet exposed to pure water. However, the ordering of lipid tails in a system where a single bilayer is hydrated by an aqueous DMSO?Cwater mixture is far lower.     

Membrane Partitioning of Anionic,Ligand-Coated Nanoparticles Is Accompanied by Ligand Snorkeling,Local Disordering,and Cholesterol Depletion

Intracellular uptake of nanoparticles (NPs) may induce phase transitions, restructuring, stretching, or even complete disruption of the cell membrane. Therefore, NP cytotoxicity assessment requires a thorough understanding of the mechanisms by which these engineered nanostructures interact with the cell membrane. In this study, extensive Coarse-Grained Molecular Dynamics (MD) simulations are performed to investigate the partitioning of an anionic, ligand-decorated NP in model membranes containing dipalmitoylphosphatidylcholine (DPPC) phospholipids and different concentrations of cholesterol. Spontaneous fusion and translocation of the anionic NP is not observed in any of the 10-µs unbiased MD simulations, indicating that longer timescales may be required for such phenomena to occur. This picture is supported by the free energy analysis, revealing a considerable free energy barrier for NP translocation across the lipid bilayer. 5-µs unbiased MD simulations with the NP inserted in the bilayer core reveal that the hydrophobic and hydrophilic ligands of the NP surface rearrange to form optimal contacts with the lipid bilayer, leading to the so-called snorkeling effect. Inside cholesterol-containing bilayers, the NP induces rearrangement of the structure of the lipid bilayer in its vicinity from the liquid-ordered to the liquid phase spanning a distance almost twice its core radius (8–10 nm). Based on the physical insights obtained in this study, we propose a mechanism of cellular anionic NPpartitioning, which requires structural rearrangements of both the NP and the bilayer, and conclude that the translocation of anionic NPs through cholesterol-rich membranes must be accompanied by formation of cholesterol-lean regions in the proximity of NPs.     

Study of curcumin behavior in two different lipid bilayer models of liposomal curcumin using molecular dynamics simulation

Liposomal formulation of curcumin is an important therapeutic agent for the treatment of various cancers. Despite extensive studies on the biological effects of this formulation in cancer treatment, much remains unknown about curcumin–liposome interactions. Understanding how different lipid bilayers respond to curcumin molecule may help us to design more effective liposomal curcumin. Here, we used molecular dynamics simulation method to investigate the behavior of curcumin in two lipid bilayers commonly used in preparation of liposomal curcumin, namely dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG). First, the free energy barriers for translocation of one curcumin molecule from water to the lipid bilayer were determined by using the potential of mean force (PMF). The computed free energy profile exhibits a global minimum at the solvent–headgroup interface (LH region) for both lipid membranes. We also evaluated the free energy difference between the equilibrium position of curcumin in the lipid bilayer and bulk water as the excess chemical potential. Our results show that curcumin has the higher affinity in DMPG compared to DPPC lipid bilayer (?8.39 vs. ?1.69 k_BT) and this is related to more hydrogen bond possibility for curcumin in DMPG lipid membrane. Next, using an unconstrained molecular dynamic simulation with curcumin initially positioned at the center of lipid bilayer, we studied various properties of each lipid bilayer system in the presence of curcumin molecule that was in full agreement with PMF and experimental data. The results of these simulation studies suggest that membrane composition could have a large effect on interaction of curcumin–lipid bilayer.     

Simulation of Nanoparticle Permeation through a Lipid Membrane

A metric of nanoparticle toxicity is the passive permeability rate through cellular membranes. To assess the influence of nanoparticle morphology on this process, the permeability of buckyball-sized molecules through a representative lipid bilayer was investigated by molecular-dynamics simulation. When C₆₀ was compared with a prototypical opened C₆₀ molecule and a representative combustion-generated particle, C₆₈H₂₉, the calculated free-energy profiles along the permeation coordinate revealed a sizable variation in form and depth. The orientation of the anisotropic molecules was determined by monitoring the principal axis corresponding to the largest moment of inertia, and free rotation was shown to be hindered in the bilayer interior. Diffusion constant values of the permeant molecules were calculated from a statistical average of seven to 10 trajectories at five locations along the permeation coordinate. A relatively minor variation of the values was observed in the bilayer interior; however, local resistance values spanned up to 24 orders of magnitude from the water layer to the bilayer center, due primarily to its exponential dependence on free energy. The permeability coefficient values calculated for the three similarly sized but structurally distinct nanoparticles showed a significant variance. The use of C₆₀ to represent similarly sized carbonaceous nanoparticles for assessments of toxicity is questioned.     

Different mechanisms of the biochemical adaptation of mycelial fungi to temperature stress: Changes in the lipid composition

A comparative study was conducted concerning the effect of temperature stress on the lipid composition of representatives of the subkingdomsEomycota andNeomycota. Changes in the composition of lipid acyl chains (such as saturation and desaturation, isomerization, and changes in the length of fatty acid carbon chains), in the phospholipid composition, and in the contents of sterols and other neutral lipids were revealed. Hyperthermia resulted in (i) an increase in the phosphatidylcholine level, (ii) a decrease in the phosphatidylethanolamine level, (iii) a rise in the content of reserve lipids (triacylglycerols), and (iv) a decline in the free fatty acid level in the neutral lipids. An inverse pattern occurred under hypothermic conditions. The peculiarities in the patterns of the temperature adaptation-related changes in the lipid bilayer composition are considered in terms of the systematic position of the fungi.     

Membrane perturbation induced by interfacially adsorbed peptides

The structural and energetic characteristics of the interaction between interfacially adsorbed (partially inserted) alpha-helical, amphipathic peptides and the lipid bilayer substrate are studied using a molecular level theory of lipid chain packing in membranes. The peptides are modeled as "amphipathic cylinders" characterized by a well-defined polar angle. Assuming two-dimensional nematic order of the adsorbed peptides, the membrane perturbation free energy is evaluated using a cell-like model; the peptide axes are parallel to the membrane plane. The elastic and interfacial contributions to the perturbation free energy of the "peptide-dressed" membrane are evaluated as a function of: the peptide penetration depth into the bilayer's hydrophobic core, the membrane thickness, the polar angle, and the lipid/peptide ratio. The structural properties calculated include the shape and extent of the distorted (stretched and bent) lipid chains surrounding the adsorbed peptide, and their orientational (C-H) bond order parameter profiles. The changes in bond order parameters attendant upon peptide adsorption are in good agreement with magnetic resonance measurements. Also consistent with experiment, our model predicts that peptide adsorption results in membrane thinning. Our calculations reveal pronounced, membrane-mediated, attractive interactions between the adsorbed peptides, suggesting a possible mechanism for lateral aggregation of membrane-bound peptides. As a special case of interest, we have also investigated completely hydrophobic peptides, for which we find a strong energetic preference for the transmembrane (inserted) orientation over the horizontal (adsorbed) orientation.     

The energetics of transmembrane helix insertion into a lipid bilayer

Free energy profiles for insertion of a hydrophobic transmembrane protein α-helix (M2 from CFTR) into a lipid bilayer have been calculated using coarse-grained molecular dynamics simulations and umbrella sampling to yield potentials of mean force along a reaction path corresponding to translation of a helix across a lipid bilayer. The calculated free energy of insertion is smaller when a bilayer with a thinner hydrophobic region is used. The free energies of insertion from the potentials of mean force are compared with those derived from a number of hydrophobicity scales and with those derived from translocon-mediated insertion. This comparison supports recent models of translocon-mediated insertion and in particular suggests that: 1), helices in an about-to-be-inserted state may be located in a hydrophobic region somewhat thinner than the core of a lipid bilayer; and/or 2), helices in a not-to-be-inserted state may experience an environment more akin (e.g., in polarity/hydrophobicity) to the bilayer/water interface than to bulk water.