The Role of Aromatic–Aromatic Interactions in Strand–Strand Stabilization of β-Sheets

Aromatic–aromatic interactions have long been believed to play key roles in protein structure, folding, and binding functions. However, we still lack full understanding of the contributions of aromatic–aromatic interactions to protein stability and the timing of their formation during folding. Here, using an aromatic ladder in the β-barrel protein, cellular retinoic acid-binding protein 1 (CRABP1), as a case study, we find that aromatic π stacking plays a greater role in the Phe65–Phe71 cross-strand pair, while in another pair, Phe50–Phe65, hydrophobic interactions are dominant. The Phe65–Phe71 pair spans β-strands 4 and 5 in the β-barrel, which lack interstrand hydrogen bonding, and we speculate that it compensates energetically for the absence of strand–strand backbone interactions. Using perturbation analysis, we find that both aromatic–aromatic pairs form after the transition state for folding of CRABP1, thus playing a role in the final stabilization of the β-sheet rather than in its nucleation as had been earlier proposed. The aromatic interaction between strands 4 and 5 in CRABP1 is highly conserved in the intracellular lipid-binding protein (iLBP) family, and several lines of evidence combine to support a model wherein it acts to maintain barrel structure while allowing the dynamic opening that is necessary for ligand entry. Lastly, we carried out a bioinformatics analysis and found 51 examples of aromatic–aromatic interactions across non-hydrogen-bonded β-strands outside the iLBPs, arguing for the generality of the role played by this structural motif.