Lysine methylation is an important histone post-translational modification (PTM) for manipulating chromatin structure and regulating gene expression, and its dysregulation is associated with various diseases including many cancers. While characterization of Lys methylation has seen improvements over the past decade due to advances in proteomic mass spectrometry and methods involving antibodies, chemical methods for selective detection of proteins containing PTMs are still lacking. Here, we detail the development of a unique labeling method wherein a synthetic receptor probe for trimethyl lysine (Kme3), CX4-ONBD, is used to direct selective fluorescent labeling of Kme3 histone peptides. This supramolecular approach reverses the paradigm of ligand-directed affinity labeling by making the receptor the synthetic component and the ligand the component to be labeled. We show that the probe mediates a strong turn-on fluorescence response in the presence of a Kme3 histone peptide and shows >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. We also demonstrate the utility of the probe through the design of a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Our synthetic receptor-mediated affinity labeling approach broadens the scope of PTM detection by chemical means and may facilitate the development of more versatile in vitro enzymatic assays.

A network of reader proteins and enzymes precisely controls gene transcription through the dynamic addition, removal, and recognition of post-translational modifications (PTMs) of histone tails. Histone PTMs work in concert with this network to regulate gene transcription through the histone code, and the dysregulation of PTM maintenance is linked to a large number of diseases, including many types of cancer. A wealth of research aims to elucidate the functions of this code, but our understanding of the effects of PTMs, specifically the methylation of lysine (Lys) and arginine (Arg), is lacking. The development of new tools to study PTMs relies on a sophisticated understanding of the mechanisms that drive protein and small molecule recognition in water. In this review, we outline the physical organic concepts that drive the molecular recognition of Lys and Arg methylation by reader proteins and draw comparisons to the binding mechanisms of small molecule receptors for methylated Lys and Arg that have been developed recently.

Dynamic combinatorial chemistry was used to generate a set of receptors for peptides containing methylated lysine (KMen, n = 0–3) and study the contribution of electrostatic effects and pocket depth to binding affinity and selectivity. We found that changing the location of a carboxylate resulted in an increase in preference for KMe2, presumably based on ability to form a salt bridge with KMe2. The number of charged groups on either the receptor or peptide guest systematically varied the binding affinities to all guests by approximately 1–1.5 kcal mol−1, with little influence on selectivity. Lastly, formation of a deeper pocket led to both increased affinity and selectivity for KMe3 over the lower methylation states. From these studies, we identified that the tightest binder was a receptor with greater net charge, with a Kd of 0.2 μM, and the receptor with the highest selectivity was the one with the deepest pocket, providing 14-fold selectivity between KMe3 and KMe2 and a Kd for KMe3 of 0.3 μM. This work provides key insights into approaches to improve binding affinity and selectivity in water, while also demonstrating the versatility of dynamic combinatorial chemistry for rapidly exploring the impact of subtle changes in receptor functionality on molecular recognition in water.

Methylated lysine 9 on the histone 3 (H3) tail recruits heterochromatin protein 1 from Drosophila (dHP1) via its chromodomain and results in gene silencing. The dHP1 chromodomain binds H3 K9Me3 with an aromatic cage surrounding the trimethyllysine. The sequence selectivity of binding comes from insertion of the histone tail between two β-strands of the chromodomain to form a three-stranded β-sheet. Herein, we investigated the sequence selectivity provided by the β-sheet interactions and how those interactions compare to other model systems. Residue Thr6 of the histone tail forms cross-strand interactions with Ala25 and Asp62 of the chromodomain. Each of these three residues was substituted for amino acids known to have high β-sheet propensities and/or to form favorable side chain–side chain (SC–SC) interactions in β-sheets, including hydrophobic, H-bonding, and aromatic interactions. We found that about 50% of the chromodomain mutants resulted in equal or tighter binding to the histone tail and about 25% of the histone tail mutants provided tighter binding compared to that of the native histone tail sequence. These studies provide novel insights into the sequence selectivity of the dHP1 chromodomain for the histone tail and relates the information gleaned from model systems and statistical studies to β-sheet-mediated protein–protein interactions. Moreover, this work suggests that the development of designer histone–chromodomain pairs for chemical biology applications is feasible.

Artificial photosynthesis based on dye-sensitized photoelectrosynthesis cells requires the assembly of a chromophore and catalyst in close proximity on the surface of a transparent, high band gap oxide semiconductor for integrated light absorption and catalysis. While there are a number of approaches to assemble mixtures of chromophores and catalysts on a surface for use in artificial photosynthesis based on dye-sensitized photoelectrosynthesis cells, the synthesis of discrete surface-bound chromophore–catalyst conjugates is a challenging task with few examples to date. Herein, a versatile synthetic approach and electrochemical characterization of a series of oligoproline-based light-harvesting chromophore–water-oxidation catalyst assemblies is described. This approach combines solid-phase peptide synthesis for systematic variation of the backbone, copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) as an orthogonal approach to install the chromophore, and assembly of the water-oxidation catalyst in the final step. Importantly, the catalyst was found to be incompatible with the conditions both for amide bond formation and for the CuAAC reaction. The modular nature of the synthesis with late-stage assembly of the catalyst allows for systematic variation in the spatial arrangement of light-harvesting chromophore and water-oxidation catalyst and the role of intrastrand distance on chromophore–catalyst assembly properties. Controlled potential electrolysis experiments verified that the surface-bound assemblies function as water-oxidation electrocatalysts, and electrochemical kinetics data demonstrate that the assemblies exhibit greater than 10-fold rate enhancements compared to the homogeneous catalyst alone.

Solid-phase peptide synthesis has been applied to the preparation of phosphonate-derivatized oligoproline assemblies containing two different RuII polypyridyl chromophores coupled via “click” chemistry. In water or methanol the assembly adopts the polyproline II (PPII) helical structure, which brings the chromophores into close contact. Excitation of the assembly on ZrO2 at the outer RuII in 0.1 M HClO4 at 25 °C is followed by rapid, efficient intra-assembly energy transfer to the inner RuII (kEnT = 3.0 × 107 s–1, implying 96% relative efficiency). The comparable energy transfer rate constants in solution and on nanocrystalline ZrO2 suggest that the PPII structure is retained when bound to ZrO2. On nanocrystalline films of TiO2, excitation at the inner RuII is followed by rapid, efficient injection into TiO2. Excitation of the outer RuII is followed by rapid intra-assembly energy transfer and then by electron injection. The oligoproline/click chemistry approach holds great promise for the preparation of interfacial assemblies for energy conversion based on a family of assemblies having controlled compositions and distances between key functional groups.

Herein we report energy transfer studies in a series of Ru(II) and Os(II) linked coiled-coil peptides in which the supramolecular scaffold controls the functional properties of the assembly. A general and convergent method for the site-specific incorporation of bipyridyl Ru(II) and Os(II) complexes using solid-phase peptide synthesis and the copper-catalyzed azide–alkyne cycloaddition is reported. Supramolecular assembly positions the chromophores for energy transfer. Using time-resolved emission spectroscopy we measured position-dependent energy transfer that can be varied through changes in the sequence of the peptide scaffold. High level molecular dynamics simulations were used in conjunction with the spectroscopic techniques to gain molecular-level insight into the observed trends in energy transfer. The most efficient pair of Ru(II) and Os(II) linked peptides as predicted by molecular modeling also exhibited the fastest rate of energy transfer (with kEnT = 2.3 × 107 s–1 (42 ns)). Additionally, the emission quenching for the Ru(II) and Os(II) peptides can be fit to binding models that agree with the dissociation constants determined for the peptides via chemical denaturation.

Protein post-translational modifications (PTMs) are used in nature as a means of turning on or off a myriad of biological events. Methylation of lysine and phosphorylation of serine are important PTMs in the histone code found to modulate chromatin packing, which in turn affects gene expression. The design of peptides that fold into secondary structures can help to further our understanding of complex protein interactions. Here we report the design of the Trpswitch peptide sequence that folds into a moderately stable β-hairpin structure in aqueous solution and show that the stability of the structure can be tuned by incorporation of dimethyllysine or phosphoserine. Dimethylated Trpswitch results in an increase in β-hairpin stability, while phosphorylated Trpswitch is unstructured at neutral pH. When both modifications are incorporated into Trpswitch, a less stable β-hairpin structure is observed. This system provides a model to demonstrate how multiple PTMs may work in concert or against each other to influence structure.

Designing receptors that bind RNA is a challenging endeavor because of the unique and sometimes complex structure of RNA. However these structural features provide regions for ligands to bind using different types of interactions. To increase specificity and binding affinity to RNA, divalent systems have been designed which incorporate more than one binding motif into one molecule. Using this approach, we have designed a two part heteroconjugate, WKWK-Int, which contains a β-hairpin peptide covalently linked to an RNA intercalator. This heteroconjugate was designed to bind duplex RNA through intercalation and simultaneously interact with a single stranded bulge region using the side chains of the β-hairpin peptide. We have used fluorescence anisotropy experiments to show that the heteroconjugate has an increased binding affinity over either one of the individual ligands. Additionally, RNase footprinting experiments show that the structure of the peptide is necessary for the protection of one particular base in the RNA bulge region. When tested against other RNA molecules containing a stem-bulge structure, the designed heteroconjugate was found to be specific for this RNA sequence. This work provides evidence that the covalent linkage of two weak RNA ligands can greatly increase the binding affinity and also provide specificity to the binding event.

Two tryptophan residues were incorporated on one face of a β-hairpin peptide to form an aromatic pocket that interacts with a lysine or N-methylated lysine via cation−π interactions. The two tryptophan residues were found to pack against the lysine side chain forming an aromatic pocket similar to those observed in trimethylated lysine receptor proteins. Thermal analysis of methylated lysine variant hairpin peptides revealed an increase in thermal stability as the degree of methylation was increased, resulting in the most thermally stable β-hairpin reported to date.

The first CO2- and water-soluble peptide is reported, in which folding facilitates its solubility in CO2.

A carbohydrate–π interaction contributes –0.8 kcal mol–1 to the stabilization of a β-hairpin peptide.

Posttranslational modifications of histone proteins regulate gene expression via complex protein–protein and protein–DNA interactions with chromatin. One such modification, the methylation of lysine, has been shown to induce binding to chromodomains in an aromatic cage [Nielsen PR, et al. (2002) Nature 416:103–107]. The binding generally is attributed to the presence of cation–π interactions between the methylated lysine and the aromatic pocket. 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 β-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–π 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.

Dynamic combinatorial chemistry was used to generate a set of receptors for peptides containing methylated lysine (KMen, n = 0–3) and study the contribution of electrostatic effects and pocket depth to binding affinity and selectivity. We found that changing the location of a carboxylate resulted in an increase in preference for KMe2, presumably based on ability to form a salt bridge with KMe2. The number of charged groups on either the receptor or peptide guest systematically varied the binding affinities to all guests by approximately 1–1.5 kcal mol−1, with little influence on selectivity. Lastly, formation of a deeper pocket led to both increased affinity and selectivity for KMe3 over the lower methylation states. From these studies, we identified that the tightest binder was a receptor with greater net charge, with a Kd of 0.2 μM, and the receptor with the highest selectivity was the one with the deepest pocket, providing 14-fold selectivity between KMe3 and KMe2 and a Kd for KMe3 of 0.3 μM. This work provides key insights into approaches to improve binding affinity and selectivity in water, while also demonstrating the versatility of dynamic combinatorial chemistry for rapidly exploring the impact of subtle changes in receptor functionality on molecular recognition in water.

The first CO2- and water-soluble peptide is reported, in which folding facilitates its solubility in CO2.

Designing receptors that bind RNA is a challenging endeavor because of the unique and sometimes complex structure of RNA. However these structural features provide regions for ligands to bind using different types of interactions. To increase specificity and binding affinity to RNA, divalent systems have been designed which incorporate more than one binding motif into one molecule. Using this approach, we have designed a two part heteroconjugate, WKWK-Int, which contains a β-hairpin peptide covalently linked to an RNA intercalator. This heteroconjugate was designed to bind duplex RNA through intercalation and simultaneously interact with a single stranded bulge region using the side chains of the β-hairpin peptide. We have used fluorescence anisotropy experiments to show that the heteroconjugate has an increased binding affinity over either one of the individual ligands. Additionally, RNase footprinting experiments show that the structure of the peptide is necessary for the protection of one particular base in the RNA bulge region. When tested against other RNA molecules containing a stem-bulge structure, the designed heteroconjugate was found to be specific for this RNA sequence. This work provides evidence that the covalent linkage of two weak RNA ligands can greatly increase the binding affinity and also provide specificity to the binding event.

A carbohydrate–π interaction contributes –0.8 kcal mol–1 to the stabilization of a β-hairpin peptide.