Marcey L. Waters

Aromatic interactions in model systems.

A thorough knowledge of noncovalent interactions is crucial to the understanding of biological complexity. One of the less well understood but significant weak interactions in nature is the aromatic interaction. Recent studies have provided new insight into the driving force, stability and selectivity of these interactions. The contribution of solvophobic and electrostatic interactions have been shown to be inextricably linked. Moreover, the influence of electrostatic and solvophobic components on the selectivity of aromatic interactions has been demonstrated.

Carbohydrate−π Interactions: What Are They Worth?

Protein-carbohydrate interactions play an important role in many biologically important processes. The recognition is mediated by a number of noncovalent interactions, including an interaction between the alpha-face of the carbohydrate and the aromatic side chain of the protein. To elucidate this interaction, it has been studied in the context of a beta-hairpin in aqueous solution, in which the interaction can be investigated in the absence of other cooperative noncovalent interactions. In this beta-hairpin system, both the aromatic side chain and the carbohydrate were varied in an effort to gain greater insight into the driving force and magnitude of the carbohydrate-pi interaction. The magnitude of the interaction was found to vary from -0.5 to -0.8 kcal/mol, depending on the nature of the aromatic ring and the carbohydrate. Replacement of the aromatic ring with an aliphatic group resulted in a decrease in interaction energy to -0.1 kcal/mol, providing evidence for the contribution of CH-pi interactions to the driving force. These findings demonstrate the significance of carbohydrate-pi interactions within biological systems and also their utility as a molecular recognition element in designed systems.

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

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.

The geometry and efficacy of cation–π interactions in a diagonal position of a designed β-hairpin

Cation–π interactions are common in proteins, but their contribution to the stability and specificity of protein structure has not been well established. In this study, we examined the impact of cation–π interactions in a diagonal position of a β‐hairpin peptide through comparison of the interaction of Phe or Trp with Lys or Arg. The diagonal interactions ranged from −0.20 to −0.48 kcal/mole. Our experimental values for the diagonal cation–π interactions are similar to those found in α‐helical studies. Upfield shifting of the Lys and Arg side chains indicates that the geometries of cation–π interactions adopted in the 12‐residue β‐hairpin are comparable to those found in protein structures. The Lys was found to interact through the polarized Cε, and the Arg is stacked against the aromatic ring of Phe or Trp. Folding of these peptides was found to be enthalpically favorable (ΔH° ∼ −3 kcal/mole) and entropically unfavorable (ΔS°∼ −8 cal mole−1 K−1).

A small molecule receptor that selectively recognizes trimethyl lysine in a histone peptide with native protein-like affinity

A small molecule receptor that mimics the HP1 chromodomains affinity for trimethyl lysine has been identified from a dynamic combinatorial library. Discrimination for trimethyl lysine over the lower methylation states parallels that of the native protein receptor. These studies demonstrate the feasibility of small molecule receptors as potential sensors for protein post-translational modifications.

Sequence dependence of β-hairpin structure: Comparison of a salt bridge and an aromatic interaction

A comparison of the contributions and position dependence of cross‐strand electrostatic and aromatic side‐chain interactions to β‐sheet stability has been performed by using nuclear magnetic resonance in a well‐folded β‐hairpin peptide of the general sequence XRTVXVdPGOXITQX. Phe–Phe and Glu–Lys pairs were varied at the internal and terminal non–hydrogen‐bonded position, and the resulting stability was measured by the effects on α‐hydrogen and aromatic hydrogen chemical shifts. It was determined that the introduction of a Phe–Phe pair resulted in a more folded peptide, regardless of position, and a more tightly folded core. Substitution of the Glu–Lys pair at the internal position results in a less folded peptide and increased fraying at the terminal residues. Upfield shifting of the aromatic hydrogens provided evidence for an edge‐face aromatic interaction, regardless of position of the Phe–Phe pair. In peptides with two Phe–Phe pairs, substitution with Glu–Lys at either position resulted in a weakening of the aromatic interaction and a subsequent decrease in peptide stability. Thermal denaturation of the peptides containing Phe–Phe indicates that the aromatic interaction is enthalpically favored, whereas the folding of hairpins with cross‐strand Glu–Lys pairs was less enthalpically favorable but entropically more favorable.

A synthetic receptor for asymmetric dimethyl arginine.

Dynamic combinatorial chemistry was utilized to identify a novel small molecule receptor, A2D, for asymmetric dimethyl arginine (aRMe2), which is a post-translational modification (PTM) in proteins. It is known to play a role in a number of diseases, including spinal muscular atrophy, leukemia, lymphoma, and breast cancer. The receptor exhibits 2.5-7.5-fold selectivity over the isomeric symmetric dimethyl arginine, depending on the surrounding sequence, with binding affinities in the low micromolar range. The affinity and selectivity of A2D for the different methylated states of Arg parallels that of proteins that bind to these PTMs. Characterization of the receptor-PTM complex indicates that cation-π interactions provide the main driving force for binding, loosely mimicking the binding mode found in the recognition of dimethyl arginine by native protein receptors.

Investigation of the nature of the methionine–π interaction in β-hairpin peptide model systems

There are frequent contacts between aromatic rings and sulfur atoms in proteins. However, it is unclear to what degree this putative interaction is stabilizing and what the nature of the interaction is. We have investigated the aryl–sulfur interaction by placing a methionine residue diagonal to an aromatic ring on the same face of a β‐hairpin, which places the methionine side chain in close proximity to the aryl side chain. The methionine (Met)–aryl interaction was compared with an equivalent hydrophobic and cation–π interaction in the context of the β‐hairpin. The interaction between phenylalanine (Phe), tryptophan (Trp), or cyclohexylalanine (Cha) and Met stabilized the β‐hairpin by −0.3 to −0.5 kcal mole−1, as determined by double‐mutant cycles. The peptides were subjected to thermal denaturations that suggest a hydrophobic driving force for the interactions between Met and Trp or Cha. The observed interaction of Met or norleucine (Nle) with Trp or Cha are quite similar, implying a hydrophobic driving force for the Met–π interaction. However, the thermodynamic data suggest that there may be some differences between the interaction of Met with Trp and Phe and that there may be a small thermodynamic component to the Met•••Phe interaction.

Design of highly stabilized Β-hairpin peptides through cation - π interactions of lysine and N-methyllysine with an aromatic pocket

Two tryptophan residues were incorporated on one face of a beta-hairpin peptide to form an aromatic pocket that interacts with a lysine or N-methylated lysine via cation-pi 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 beta-hairpin reported to date.

Constitutionally selective amplification of multicomponent 84-membered macrocyclic hosts for (−)-cytidine•H+

Mixtures of dipeptide monomers create stereochemically and constitutionally complex dynamic libraries of potential receptors. When (−)-cytidine was utilized as guest an 84-membered cyclic host was amplified (70–175 fold) from a nearly undetectable initial concentration. Only the specified diastereomeric combination of the two chiral building blocks yielded a dynamic library from which the macrocyclic receptor could be amplified.