The weakly polar nature of aromatic residues leads to π…π interactions wherein the positively polarized hydrogen atoms of one ring can interact with the δ- π-electron cloud of a second aromatic ring. In 1985, Burley and Petsko observed, “that on an average about 60% of aromatic side chains in proteins are involved in aromatic pairs, 80% of which form networks of three or more interacting aromatic side chains [1-6]. Phenyl ring centroids are separated by a preferential distance of between 4.5 Å and 7 Å, and dihedral angles approaching 90° are most common”

This π…π interaction has a quadrupole - quadrupole character (distance dependence ~ 1/r⁵). Subsequently, a large number of analyses of aromatic π…π interactions in the context of peptides as well as in proteins have re-emphasized the importance of aromatic residues in the stability of structures and they have pointed out the occurrence of several potential orientations of closely packed aromatic rings (Figure 1). The π…π interactions contribute free energy between -0.6 kcal.mol^-1 and 1.3 kcal.mol^-1 towards the stability of protein structures. It has been suggested from the experiments as well as from the theoretical analysis that van der Waals and the electrostatic forces play important roles in the stability of π…π interactions.

Figure 1: Possible models of aromatic interaction in benzene dimer. Aromatic pairs calculated with a fixed centroid-centroid distance of 5.5 Å.

Aromatic Interactions in Synthetic Peptides

One of the earliest monomeric β - hairpins to be examined was derived from the B1 subunit of protein G1. The characteristic stabilizing agent of this peptide was the ‘hydrophobic core’ constituted by three aromatic residues, namely, Tyr, Phe and Trp, along with the apolar Val residue. A noticeable feature was also the strong Phe-Tyr interaction at the first non-hydrogen bonding position following the turn, stabilizing the strand segments (Figure 2). Several peptide hairpins have subsequently been designed using aromatic amino acids as stabilizing agents. One of the most investigated of the designed peptides is the ‘Trpzip’ sequences. These tryptophan zipper peptides are 16 residues in length and contain two pairs of interacting Trp residues that interact in a T-shaped manner (Figure 3).

Figure 2: Structure of residues 42 - 56 of the B1 subunit of staphylococcal protein G in the solid state, determined using X-ray crystallography, showing a strong cluster of hydrophobic residues required for stability of the hairpin. This aromatic interaction was also shown to be preserved in the isolated hairpin, using solution NMR studies. Two views of the hairpin are shown (PDB code: 1PGA).

Figure 3: (Left) Superposition of 20 best structures calculated for the tryptophan zipper peptide using NMR (PDB code: 1LE0) [13,14]. Two pairs of strong aromatic interactions at the non-hydrogen bonding position of the hair-pin were found to stabilize the structure and impart protein-like properties to the 16-residue peptide. (Right) Illustration of the T-shaped aromatic interaction in 1LE0, using the Trp residues immediately after the turn as an example.
Note that the T-shaped interaction is also present in the second aromatic pair towards the termini.

These interactions not only stabilize the peptide but also confer protein-like properties to the sequence, including a high Tm of 79°C and requiring high concentrations of urea and guanidine hydrochloride for denaturation. Other studies on peptide hairpins containing interacting Phe residues clearly indicate that the energy of interaction of Phe-Phe pairs is about -0.5 kcal/mol compared to its Cha analog [7-9]. A study comparing aromatic interactions with salt bridges, demonstrated that the removal of an aromatic ring caused significant hairpin destabilization whereas the replacement of a salt bridge did not cause any major change in peptide stability [10-12]. In all these peptides, a consistent edge-to-face geometry was observed indicating that this interaction was preferred despite the conformational flexibility allowed for the side chains of aromatic residues.

Aromatic interactions have also been demonstrated in helical peptides. In these cases, however, the interactions are largely intermolecular, as against the intramolecular stabilization provided by interacting aromatic pairs in hairpins. For example, several reports of crystal structures of peptide helices exist wherein aromatic interactions aid in three-dimensional propagation of crystals (Figure 4) [13-15]. Interestingly, such interactions are also found to largely populate the T-shaped geometries and very few examples exist wherein strong stacking interactions between side chains of Phe / Trp / Tyr are seen (Figure 5). In another study, an interaction between residues at the ‘i’ and ‘I + 4 / 5’ residues was implicated in stabilizing helical structures. Such interactions are uncommon in peptides, but are of greater incidence in proteins. In a recent study, it was shown that aromatic interactions do not contribute significantly towards peptide stability. This observation was also supported by a previous investigation, which revealed that aliphatic - aromatic interactions are more stabilizing than aromatic pairs in peptides. The stability observed was of the order (Ile / Val) – Tyr > Phe–Tyr > Tyr–Tyr > Trp-Tyr. Reports, however, do exist in which aromatic interactions serve as structure stabilizing agents and the observation of aromatic interactions might be dependent on the system under investigation, with the twist of the backbone in peptide hairpins also playing a role in the formation of favorable aromatic interactions.

Figure 4: Example of aromatic interactions in peptides. Figure adapted from Aravinda [16]. Interaction between Phe3 and Phe7 in the peptide was observed in the crystal. Centroid - centroid distances are marked.

Figure 5: Example of aromatic stacking and edge to face interactions stabilizing a dimeric helix bundle in a de novo designed peptide. Aromatic residues have been indicated as ball and stick. Solution structure of this peptide has been solved using NMR (PDB: 1QP6) [17,18].

Preferred modes of aromatic interactions have also been observed in the case of peptides, as has been noticed for proteins and benzene dimers. The possible modes of Trp - Trp interactions are illustrated in Figures 6 and 7. The ability of Trp residues to not only stabilize secondary structure but also involve in interaction with other aromatic systems has been exploited in the design of a peptide with the ability to bind ATP and FAD for specific recognition of ssDNA in solution.

Figure 6: Examples of Trp - Trp interactions in crystal structures of peptides. Distances between the centroids of the 5-membered and 6-membered rings along with their interplanar angles (γ) are indicated below each figure. Distances between the aromatic rings are largely distributed between 5 Å – 7 Å.

Figure 7: Summary of the unique aromatic-aromatic interactions observed in the crystals of peptides I-III. The parameters Rcen, ç, and Rclo are indicated: (a-c) peptide I; (d-k) peptide II; (l-o) peptide III. Figure 8 clearly shows van der waalsinteractions and molecular conformation of the peptides in crystal the benzyl group-the side chain molecular structure packing is attached with hydrogen. All the crystal was grown in water /methanol equimolar combination[19-21].

C-H…π interactions involving the aromatic groups either as donor or as an acceptor serves as a potential contributor to the overall stability of proteins. The interactions between aromatic C-H donor groups and aromatic π- acceptors and the interactions between aliphatic C-H donor groups and aromatic π- acceptors are most widely observed in proteins and in peptides. C-H…π interactions involving aromatic π-systems are generally found in the interior of proteins and the overall stabilization energy for potential C-H…π interaction of about 0.5 kcal mol^-1- 1.0 kcal mol^-1 [16-22]. This interaction can play an important role in the molecular recognition and in the binding of DNA or RNA. C-H…π interactions might also be responsible for the stabilization of secondary structures such as α or 3₁₀-helices (Figure 9).

Figure 8: Molecular crystals of peptides I-III. All the hydrogen bonds are shown as dotted lines: (a) I, Boc-Val-Ala-Phe-Aib-Val-Ala- Phe-Aib-OMe; (b) II, Boc-Val-Ala- Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe; (c) III, Boc-Aib-Ala-Phe-Aib-Phe-Ala-Val-Aib-OMe.

Figure 9: Folded proteins were stabilized by C-H…π interactions.

Burley and Petsko first described N-H…π interaction based on a data set of 33 high resolution (2 Å or better) protein crystal structures (Figure 10). They examined the interactions between side chain amino groups (lysine, arginine, asparagines, glutamine, histidine) and π-acceptors (phenylalanine, tyrosine, tryptophan) and concluded that there was a significant enthalpic contribution towards the protein stability. The N-H…π interactions play a role in the stabilization of peptide and protein structure and protein - ligand binding, which were described experimentally. The interactions between aromatic ring and NH of a nearby amide can be characterized as either aromatic - NH (side chain) or aromatic - NH (backbone). Both these interactions have a quadruple - dipole nature (distance dependence ~ 1/r⁴). The preferred geometry of N-H…π interaction is the one in which the NH group is positioned directly above the ring center. The distance between the N- atom and the ring center is ≤ 3.8 Å. The strength of the energy for N-H…π interaction is of the order of 1 kcal mol^-1 - 4 kcal mol^-1 [23].

Figure 10: Simple example for the close proximity in benzene water, and benzene and amine.

Amide…π interactions have been suggested to be an important stabilizing factor of folded structures in proteins. There have been detailed analyses of interactions between the aromatic ring and backbone amide group in proteins and peptide structures. For example, the ab initio quantum mechanical calculations using a model system consisting of benzene ring and amide plane have been used to analyze the aromatic - amide interaction. The theoretical studies suggest that aromatic - amide interaction contributes about 1 kcal mol^-1 - 4 kcal mol^{- 1} for the net stabilization (Figure 11).

Figure 11: Benzene and Amide interaction.

Cation…π interactions play an important role in the stability of protein structures. These interactions are basically an ion-quadrupole interaction (distance dependence ~ 1/r³), which occurs between a positively charged group (ammonium or guanidium group) and the electron rich π-cloud of an aromatic ring. These interactions are energetically favorable in protein structures and the electrostatic interaction energy is calculated to be ~ 3 kcal mol^-1 [24]. Several studies have been reported on the occurrence of cation…π interactions in the context of protein structures and protein-ligand interactions (Figures 12 and 13) [24].

Figure 12: Aromatic - amide interactions observed in the crystals of peptides. The van der Waals surfaces are shown. The parameters R 5_cen, R 6_cen , γ and R 5_clo , R 6_clo are indicated. Table 1 lists the relevant peptide sequences obtained from Cambridge Structural Database.

^aThe database contained 31 entries with Trp residues. Among them, only acyclic structures with short aromatic - aromatic (centroid - centroid distance between six membered rings of indole ≤ 6.5 Å) and / or aromatic - amide (centroid - centroid distance of five membered ring of indole and amide centroid ≤ 4.5 Å) distances are listed.

Table 1: List of Trp peptides from Cambridge Structural Database^a.

Figure 13: 623 amino acids protein significantly stabilized by Cation…π interactions.