Tipping the Scale from Disorder to Alpha-helix: Folding of Amphiphilic Peptides in the Presence of Macroscopic and Molecular Interfaces

Abstract

Secondary amphiphilicity is inherent to the secondary structural elements of proteins. By forming energetically favorable contacts with each other these amphiphilic building blocks give rise to the formation of a tertiary structure. Small proteins and peptides, on the other hand, are usually too short to form multiple structural elements and cannot stabilize them internally. Therefore, these molecules are often found to be structurally ambiguous up to the point of a large degree of intrinsic disorder in solution. Consequently, their conformational preference is particularly susceptible to environmental conditions such as pH, salts, or presence of interfaces. In this study we use molecular dynamics simulations to analyze the conformational behavior of two synthetic peptides, LKKLLKLLKKLLKL (LK) and EAALAEALAEALAE (EALA), with built-in secondary amphiphilicity upon forming an alpha-helix. We use these model peptides to systematically study their aggregation and the influence of macroscopic and molecular interfaces on their conformational preferences. We show that the peptides are neither random coils in bulk water nor fully formed alpha helices, but adopt multiple conformations and secondary structure elements with short lifetimes. These provide a basis for conformation-selection and population-shift upon environmental changes. Differences in these peptides' response to macroscopic and molecular interfaces (presented by an aggregation partner) can be linked to their inherent alpha-helical tendencies in bulk water. We find that the peptides' aggregation behavior is also strongly affected by presence or absence of an interface, and rather subtly depends on their surface charge and hydrophobicity.

Fig 1. Helical wheel representation of LK and EALA.

Both peptides are designed to display secondary amphiphilicity when they adopt the α-helix conformation.

Fig 2. Three separate contributions govern the dynamic conformational equilibrium of an amphiphilic peptide.

The folding of the individual molecule in solution (A); the partitioning of hydrophobic/hydrophilic residues induced by macroscopic interfaces (B) or molecular interfaces upon aggregation (C). Combinations of these effects (connecting arcs D, E and F) determine the preferred secondary structure in a given environment.

Fig 3. Time evolution of secondary structure for LK (left) and EALA (right) when isolated in bulk water.

Snapshots depicting various conformations adopted by these molecules (A and B), DSSP analysis of secondary structure (C and D), SASA for hydrophobic sidechains (h-SASA), number of intra-peptide backbone hydrogen bonds (H-bond) and short-range Coulomb interaction energies (Coul-SR) between the charged groups (E and F) are given for both of the molecules. See Methods section for the color coding and representation of peptides in A and B.

Fig 4. Simulations of a single peptide at the vacuum/water interface for LK (left) and EALA (right).

Simulations are started with random conformations at the center of the water slab. Upon adsorption of peptides (after 45 ns for LK and 50 ns for EALA) partitioning of hydrophobic/hydrophilic residues and folding into the α-helix structure (after 800 ns for LK and 350 ns for EALA) is observed. Snapshots depicting the adsorption and adoption of the α-helix structure (A and B) and the DSSP analysis of the secondary structure evolution (C and D) are shown for both molecules. SASA, intra-peptide backbone hydrogen bonds and short-range Coulomb interaction energies between the charge groups (E and F) display the partitioning of hydrophobic/hydrophilic residues and secondary structure formation in the presence of the interface.

Fig 5. Time evolution of secondary structure of a single AK (in-silico mutated form of LK) peptide at the vacuum/water interface.

Initially the peptide is submerged in bulk water and moves to the interface in 80 ns and remains there for the rest of the simulation.

Fig 6. Folding and association of a pair of LK (left) and EALA (right) peptides in bulk water.

Snapshots illustrating the aggregation process (A and B), DSSP secondary structure analysis (C and D), SASA, short range Coulombic interaction energies between the charged groups and the number of intra and inter-peptide backbone hydrogen bonds (E and F) are displayed as a function of simulation time. Association of peptides take place at 180 ns for LK and 300 ns for EALA, which can be observed via the sharp drop in SASA.

Fig 7. PMF results comparing the separation of a LK dimer in bulk water for the cases where lysine residues are positively charged, lysine residues are neutral and the leucine residues are in silico mutated to alanine residues.

The curves are shifted so that the maximum points are zero. The distance refers to the distance between the center of mass of backbone atoms of the peptides. For the mutated AK dimer, when the peptides are in contact they maintain their helical structures. However when they are separated or when they make a loose contact the α-helical structure is not conserved.

Fig 8. Time evolution of the secondary structure of a dimer of LK (left) and EALA (right) at the vacuum/water interface.

Typical snapshots when the peptides are associated at the interface (A and B), DSSP structural analysis (C and D), angle between the helix axis for the peptides and center-to-center distance (E and F), the h-SASA for each peptide along with the buried SASA for the whole peptide, the inter and intra molecular short-range Coulomb energies and the number of inter- and intra-molecular hydrogen bonds (G and H) are shown in figure.