In silico partitioning and transmembrane insertion of hydrophobic peptides under equilibrium conditions

Abstract

Nascent transmembrane (TM) polypeptide segments are recognized and inserted into the lipid bilayer by the cellular translocon machinery. The recognition rules, described by a biological hydrophobicity scale, correlate strongly with physical hydrophobicity scales that describe the free energy of insertion of TM helices from water. However, the exact relationship between the physical and biological scales is unknown, because solubility problems limit our ability to measure experimentally the direct partitioning of hydrophobic peptides across lipid membranes. Here we use microsecond molecular dynamics (MD) simulations in which monomeric polyleucine segments of different lengths are allowed to partition spontaneously into and out of lipid bilayers. This approach directly reveals all states populated at equilibrium. For the hydrophobic peptides studied here, only surface-bound and transmembrane-inserted helices are found. The free energy of insertion is directly obtained from the relative occupancy of these states. A water-soluble state was not observed, consistent with the general insolubility of hydrophobic peptides. The approach further allows determination of the partitioning pathways and kinetics. Surprisingly, the transfer free energy appears to be independent of temperature, which implies that surface-to-bilayer peptide insertion is a zero-entropy process. We find that the partitioning free energy of the polyleucine segments correlates strongly with values from translocon experiments but reveals a systematic shift favoring shorter peptides, suggesting that translocon-to-bilayer partitioning is not equivalent but related to spontaneous surface-to-bilayer partitioning.

Fig. 1

(A) Schematic partitioning equilibrium for peptides that are sufficiently hydrophobic to insert autonomously into membranes (ΔG_S→TM). Only two states are populated at equilibrium: an α-helix located either on the membrane surface (S) or transmembrane inserted (TM). Water soluble states (W) are not populated, consistent with the experimental fact that these peptides precipitate out of solution. (B) Schematic depiction of the translocon-bilayer partitioning equilibrium presumably probed by the translocon-mediated insertion experiments (ΔG_app). The entry of the peptide into the translocon (“Enter”), and subsequent secretion (“Exit”) are thought to be non-equilibrium processes.

Fig. 2

Illustration of the fast folding and adsorption process from the initial water solvated unfolded state (W). The insertion depth z_CM is plotted versus the peptide helicity for the pathway taken by L10 at 80 °C. Interfacial adsorption from the initial state in water occurs in ~2 ns (U). The peptide then folds (S) and subsequently inserts (TM). All other studied sequences behave exactly similar. Only the S and TM states are observed for the remainder (1–2 µs) of the simulations.

Fig. 3

Temperature dependence of the partitioning kinetics: (A) A plot of the centre of mass (z_CM) position of the peptide (here GL8) along the membrane normal shows multiple transitions between S (z = ~12 Å), and TM (z = 0 Å) states. The number of transitions rises rapidly as the temperature is increased (inset, panel C). (B) Arrhenius plots of the insertion and expulsion rates (shown are L7, L8, and GL8) all exhibit single-exponential kinetics. Flanked peptides have much larger barriers (slope of fit; c.f. L8 and GL8). Consequently, the temperature has to be raised by ~100–150 °C to observe partitioning events in the 2 µs GLn simulations. The Arrhenius plot allows to extrapolate the insertion and expulsion times to room temperature. For example, GL8 has a predicted insertion time of τ_S→TM ≈ 9 ms at 30 °C, which is ~10⁵ times slower than at 217 °C. (C) The partitioning rate increase R over ambient temperatures shows rapid growth with respect to temperature. This effect is much smaller for unflanked peptides, where the insertion barrier is weaker.

Fig. 4

Peptide location and bilayer deformation of the interfacial surface bound (S) state of Ln and Gln peptides: (A) the density cross-section profile of the bilayer shows that in the S state the peptide (here L7) is buried below the water interface. A representative conformer is shown to scale. The Leucine sidechains (green) are chiefly in contact with the acyl tails (CH₂), and there is only a small overlap with the phosphocholine headgroups and carbonyl-glycerol (C/G) groups. Other peptides behave exactly similar. (B) The peptide induced distortion to the bilayer at equilibrium can be visualized by plotting the time-averaged phosphate position from the bilayer center. This shows local thinning by 5–10% for L7 as the lipid headgroups bend over the peptide to cover the termini (phosphate is represented as an orange sphere).

Fig. 5

Thermostability of the peptide and bilayer: (A) circular dichroism spectra of the secondary structure of GL12 in POPC vesicles (peptide/lipid ratio = 1/100) over a temperature range of 45 to 85 °C. The spectra indicate predominantly helical conformers and display low sensitivity to temperature. (B) The thermostability of the peptides observed in the MD simulations is comparable (shown are L8, L12, and GL12; all other peptides behave similar). Shorter peptides are marginally less helical due to terminal fraying. (C) The effect of heating on the lipid bilayer can be visualized by plotting the equilibrium trans-bilayer density profiles in the presence of peptide (here L8) for temperatures in the range 30–120 °C (dark to light colors). Comparison of the principal structural groups (CH₃ = methyl, CH₂ = acyl tails, P/C = phosphocholine headgroups, G/C = carbonyl-glycerol linker, H₂O = water) shows temperature induced broadening of the Gaussians and a slight decrease in the total density. However, the trans-bilayer density profile and location of the principal structural groups does not change significantly.

Fig. 6

Two-dimensional free energy projection for the Ln (80 °C) and GLn (217 °C) peptides (n = 5–10) as a function of the position along the membrane normal (z) and tilt angle (θ). The surfaces reveal two distinct minima; one for TM inserted and one for surface bound peptides. For short peptides (n ≤ 7) the interfacial S state (z ≈ 12 Å, θ ≈ 90°) dominates, while longer peptides (n ≥ 8) are preferentially TM inserted (z ≈ 0 Å, θ ≈ 20°). For a given peptide length n, the surfaces of unflanked Ln and GGPG-flanked GLn peptides are very similar, despite the different temperatures, demonstrating ΔG is chiefly determined by the number of leucines n. At 80 °C, the Ln runs are not fully converged. Heating to 120 °C results in full convergence, similar to the GLn simulations at 217 °C. The values of ΔG have been shifted so that the lowest bin is set to zero.

Fig. 7

Bilayer insertion efficiency and transfer free energy as a function of peptide length n. (A) The experimental values are for translocon mediated insertion into dog pancreas rough microsomes of GGPG-(L)_n-GPGG constructs embedded into the leader peptidase carrier sequence. (B) The computed values are for spontaneous partitioning of Ln peptides into POPC lipid bilayers at 30–160 °C, and for GGPG-(L)_n-GPGG at 217 °C. (C) Both measurements display perfect two-state Boltzmann behavior (R² > 0.99), with a transition in the native state from surface bound to TM inserted upon lengthening of the peptide. (D) This is reflected in the free energy of insertion ΔG(n) as a function of peptide length n (insertion for negative ΔG – shaded). The straight lines indicate the two-state Boltzmann fit, while the data points show the computed (red, green) and experimental (blue) values for the individual peptides (^*measured ΔG, peptide IDs: 43 & 380 – 383; ^**predicted ΔG, http://dgpred.cbr.su.se/).

Fig. 8

Temperature dependence of the insertion propensities p_TM and the transfer free energies ΔG_S→TM. (A) Convergence can be visualized by plotting a running average of p_TM against simulation time (note the logarithmic time-axis). Higher temperatures accelerate convergence towards the equilibrium (dashed line), which is independent of the starting configuration (here: TM for L8, S for GL8). (B) Free energy plots of the L7 peptide for 30–160 °C. Full convergence is seen at 120 and 160 °C, while the S-state is being populated in only one interface at 30–80 °C due to incomplete sampling. (C) Overall insertion propensities for L7, L8, and GL8 as a function of temperature. Error bars are derived from block averaging (10 blocks). (D) The corresponding free energies of insertion ΔG_S→TM appear to show no systematic variation with temperature.

Fig. 9

The role of bilayer thickness and deformation on shifting the partitioning equilibrium. (A) Peptide induced bilayer deformation for GLn (n = 6–16) sequences in their TM inserted orientation. The deformation rises with increased negative hydrophobic mismatch. (Inset: GL8, the thick line indicating the average position of the phosphate groups). (B) The partitioning equilibrium is shifted towards the TM orientation upon decreasing the bilayer thickness due to a reduced hydrophobic mismatch.