To probe the selectivity possible in hydrophobic clusters, we have compared the cross-strand interactions of phenylalanine (Phe) and cyclohexylalanine (Cha) in a beta-hairpin peptide. We have found a preference for self-association among the aromatic residues, which provides 0.55 kcal/mol in stability relative to Cha-Cha cross-strand pair. NMR analysis of the Phe-Phe cross-strand pair indicates that it interacts in an edge-face interaction, despite the fact that it is highly solvent-exposed. The interaction geometry as well as the enthalpic and entropic values for the peptide containing the Phe-Phe cross-strand pair suggest that the preference for self-association arises from inherent differences in the nature of aromatic and aliphatic interactions in water.

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However, whether the cationic component of the interaction is necessary for binding in the aromatic cage has not been addressed. In this article, the interaction of trimethyllysine with tryptophan is compared with that of its neutral analog, tert-butylnorleucine (2-amino-7,7-dimethyloctanoic acid), within the context of a ␤-hairpin peptide model system. These two side chains have near-identical size, shape, and polarizabilities but differ in their charges. Comparison of the two peptides reveals that the neutral side chain has no preference for interacting with tryptophan, unlike trimethyllysine, which interacts strongly in a defined geometry. In vitro binding studies of the histone 3A peptide containing trimethyllysine or tert-butylnorleucine to HP1 chromodomain indicate that the cationic moiety is critical for binding in the aromatic cage. This difference in binding affinities demonstrates the necessity of the cation-interaction to binding with the chromodomain and its role in providing specificity. This article presents an excellent example of synergy between model systems and in vitro studies that allows for the investigation of the key forces that control biomolecular recognition.cation-pi interactions ͉ histone code ͉ lysine methylation ͉ posttranslational modifications ͉ protein-protein interactions W ith rapid advancements in genomics, epigenetics has become the next major challenge in understanding how genetic information is controlled (1). It is becoming clear that posttranslational modifications of proteins are a key component in controlling gene expression. These modifications include a number of subtle structural changes, including Lys and Arg methylation, Lys acylation, and Ser/Thr/Tyr phosphorylation, which act as chemical switches to induce or repress proteinprotein interactions. Among all histone modifications, lysine methylation is especially important for chromatin function because of its stability and direct contribution to heritable patterns of gene expression (for review, see ref.2). To understand how such modest structural modifications can control biomolecular recognition events, it is critical to understand the underlying noncovalent interactions involved.Methylation of Lys induces a protein-protein interaction through the binding of methyl lysine (KMe n , n ϭ 1-3) in an aromatic cage. This interaction first was described for the binding of methylated histone 3 (H3) tail to the HP1 chromodomain ( Fig. 1) (3, 4). HP1 and methylated H3 interact specifically whether lysine 9 is mono-, di-, or trimethylated. However, the binding is most effective when lysine is trimethylated (5). In addition, more recent findings have shown that phosphorylation of serine 10 prevents interaction of HP1 with methylated H3 (for review, see ref. 6). Therefore, a binary switch mechanism has been proposed for the recognition of methyllysine-containing peptides by chromodomains. Interestingly, binding of a methylated lysine in an aromatic cage is not exclusive to chromodomains. Plant homeobox domain (PHD) fingers an...

We have measured the rotational barriers of meta- and para-substituted N-benzyl-2-(2-fluorophenyl)pyridinium bromides in aqueous solution by dynamic NMR as a model system for offset-stacking interactions in proteins. Because the benzyl ring can stack with the 2-fluorophenyl ring in the offset conformation in the ground state, but not the transition state, the rotational barrier reflects the magnitude of the stacking interaction. Only a small (0.1 kcal/mol) change in rotational barrier was found for para substituents relative to hydrogen. A much larger energy difference was found for electronegative meta substituents (up to 0.66 kcal/mol for CF3). Evidence suggests that this is due at least in part to an electrostatic interaction between electron-poor hydrogens on one ring with the electronegative substituents on the other ring.

The influence of natural and unnatural i, i + 4 aromatic side chain-side chain interactions on alpha-helix stability was determined in Ala-Lys host peptides by circular dichroism (CD). All interactions investigated provided some stability to the helix; however, phenylalanine-phenylalanine (F-F) and phenylalanine-pentafluorophenylalanine (F-f5F) interactions resulted in the greatest enhancement in helicity, doubling the helical content over i, i + 5 control peptides at internal positions. Quantification of these interactions using AGADIR multistate helix-coil algorithm revealed that the F-F and F-f5F interaction energies are equivalent at internal positions in the sequence (deltaGF-F = deltaGF-f5F = -0.27 kcal/mol), despite the differences in their expected geometries. As the strength of a face-to-face stacked phenyl-pentafluorophenyl interaction should surpass an edge-to-face or offset-stacked phenyl-phenyl interaction, we believe this result reflects the inability of the side chains in F-f5F to attain a fully stacked geometry within the context of an alpha-helix. Positioning the interactions at the C-terminus led to much stronger interactions (deltaGF-F = -0.8 kcal/mol; deltaGF-f5F = -0.55 kcal/mol) likely because of favorable chi(1) rotameric preferences for aromatic residues at C-capping regions of alpha-helices, suggesting that aromatic side chain-side chain interactions are an effective alpha-helix C-capping method.