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 poly-leucine 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 favouring shorter peptides, suggesting that translocon-to-bilayer partitioning is not equivalent, but related to spontaneous surface-to-bilayer partitioning.

Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps*

“…A recent study showed this exceptional thermostability by using a helical polyleucine peptide inserted into synthetic lipid vesicles. No loss of helical structure was observed by CD over a temperature range between 45 and 85°C (22). Our CD data of the 5-HT 3 R are consistent with a folding model of the protein subunit in a lipid membrane depicted in Fig.…”

“…This has resulted in the proposal of a two-stage mechanism (21): during membrane insertion the ␣-helices form first and subsequently assemble into compact functional transmembrane protein structures. Since then, this model has been further refined and extended by taking into consideration the intermediate insertion steps such as helical protein parts at the membrane interface eventually switching to a transmembrane structure, as well as membrane protein oligomerization within the lipid bilayer (17,18,22).…”

Charge Composition Features of Model Single-span Membrane Proteins That Determine Selection of YidC and SecYEG Translocase Pathways in Escherichia coli

“…Consistent with this hypothesis, Ulmschneider et al (54) have shown by microsecond scale molecular dynamics simulations that polyleucine transmembrane segments with eight or more leucines can insert spontaneously. Another possibility with the highly hydrophobic TM segment is that the protein can use either the YidC-only or the Sec pathway.…”

Structural differences between thermophilic and mesophilic membrane proteins

“…To the extent that the translocon measures the thermodynamic preference for a sequence segment to insert in the bilayer or remain in an aqueous environment, 29 it makes sense that a lower free energy well would be required at high temperature to ensure the insertion decision is made. Theoretical calculations and experimental measurements indicate that the free energy of partitioning peptides into bilayers is relatively constant with temperature, 43,44 and hence higher temperatures should decrease the probability of insertion. This imperative may explain the general depletion of strongly polar residues that is observed.…”