Influence ofN-Methylation on a Cation−π Interaction Produces a Remarkably Stable β-Hairpin Peptide

Abstract

The methylation of lysine in histone tails is a common posttranslational modification that functions in histone-regulated chromatin condensation, with binding of methylated lysine occurring in aromatic pockets on chromodomain proteins. We have synthesized a highly stable 12-residue beta-hairpin peptide that exploits the histone-related cation-pi interaction between a methylated lysine residue and a tryptophan residue. Thermodynamic analysis reveals significant entropic stabilization of the peptide due to methylation of the lysine residue. Chemical denaturation of the peptide demonstrates two-state behavior. In comparison to other reported, highly stable designed beta-hairpins, this peptide is the most thermally stable beta-hairpin reported to date. This study provides insight into the role of Lys methylation in histone proteins and more generally in mediating protein-protein interactions.

Recognition of trimethyllysine by a chromodomain is not driven by the hydrophobic effect

“…2) (17,20), ¶ we found that methylation of Lys enhanced its interaction with Trp significantly, but that the driving force became more entropically favorable, suggesting an increased hydrophobic component; in contrast, the enthalpic component, attributed to the charge-quadrupole interaction, decreased relative to unmodified Lys (17). This finding led to the question of whether the positive charge in KMe3 indeed is necessary for interaction with an aromatic residue or an aromatic pocket in aqueous solution or whether hydrophobic and van der Waals interactions alone will suffice.…”

“…To this end, we have synthesized the neutral analog of KMe3, tert-butyl norleucine (2-amino-7,7-dimethyloctanoic acid; tBuNle), investigated its interaction with Trp in a ␤-hairpin model system, and then compared these results to in vitro binding assays with the HP1 chromodomain. This hairpin model system has been used previously to investigate the cation-interaction between KMe3 and Trp (17). In this model system, we find striking contrasts between the behavior of the two side chains with Trp that provide insight into the role of hydrophobicity and the importance of the charge-quadrupole component of the Trp-KMe3 interaction.…”

Effect of pH, urea, peptide length, and neighboring amino acids on alanine α‐proton random coil chemical shifts

“…If a small aromatic amino acid is in the N-terminal position adjacent to alanine (9,12,18), then there is a measurable but CSI insignificant upfield shift (*0.05 ppm) of the alanine -proton due to the anisotropic effects resulting from the proximity of the aromatic amino acid. However, if the small aromatic amino acid is instead in the C-terminal position (10,13,19) then the upfield shift is chemically significant for CSI (*0.10 ppm). It is interesting to note that the upfield shifts are not additive, and when two small amino acids are incorporated into a model peptide (8,11,17,20,21) the upfield shift is still chemically significant for CSI, but the magnitude does not appreciably increase beyond that of a single small aromatic amino acid in the C-terminal position.…”

“…[6][7][8] CSI is of particular importance for those designing peptides that form discrete and stable -hairpins, because with relatively straightforward 1D and 2D NMR studies, information about protein secondary structure can be quickly obtained. [9][10][11][12][13][14][15] While CSI also applies to amide proton (HN), - 13 C, carbonyl 13 C, and amide 15 NH chemical shifts, the current study focuses on -proton chemical shifts. 8,16,17 In CSI the -proton of a particular amino acid in the protein is referenced to the random coil value for that amino acid.…”

“…19,[23][24][25] Groups studying -hairpin structure have resorted to synthesizing at least two peptides for each hairpin studied, one with -hairpin structure and a control peptide of similar sequence with no hairpin structure, to account for the shift effects of neighboring amino acids. [9][10][11][12][13][14] In the current study it is our goal to add to the collective knowledge of chemical shift effects and correction factors by systematically studying the effect of solution environments (pH, urea) and molecular environments (peptide length, free vs. capped termini, and neighboring amino acids) on -proton chemical shifts. It is important for the CSI community to understand not only the range of variables that cause significant chemical shift effects, but also the magnitude of the shift effects.…”