From supramolecular chemistry to the nucleosome: studies in biomolecular recognition

This review highlights the author’s indirect path to research at the interface of supramolecular chemistry and chemical biology

Controlling Peptide Folding with Repulsive Interactions between Phosphorylated Amino Acids and Tryptophan

Phosphorylated amino acids were incorporated into a designed β-hairpin peptide to study the effect on β-hairpin structure when the phosphate group is positioned to interact with a tryptophan residue on the neighboring strand. The three commonly phosphorylated residues in biological systems, serine, threonine, and tyrosine, were studied in same β-hairpin system. It was found that phosporylation destabilizes the hairpin structure by approximately 1.0 kcal/mol regardless of the type of phosphorylated residue. In contrast, destabilization due to glutamic acid was about 0.3 kcal/mol. Double mutant cycles and pH studies are consistent with a repulsive interaction as the source of destabilization. These findings demonstrate a novel mechanism by which phosphorylation may influence protein structure and function

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 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

Positional effects of click cyclization on β-hairpin structure, stability, and function

The use of the copper (I)-assisted azide-alkyne cycloaddition (CuAAC, or “click” reaction) as a method of β-hairpin stabilization was investigated at several different positions to determine the impact on hairpin structure and function, including hydrogen bonded sites, non-hydrogen bonded sites, and at the peptide termini. The role of the turn sequence in the peptide and the chain length of the azied were also investigated. It was determined that the CuAAC reaction was a suitable method for locking in β-hairpin structure in peptides possessing either the type I’ turn, VNGO and the type II’ turn, VpGO. Moreover, all cyclic variants exhibited improved thermal stability and resistance to proteolysis as compared to the non-cyclic peptides, regardless of the position in the strand. Additionally, the function of the CuAAC cyclized peptides was not altered as exhibited by similar binding affinities for ATP as the WKWK peptide. These studies provided a comprehensive method for CuAAC cyclization of β-hairpin peptides, which could further be utilized in the inhibition of protein-protein and protein-nucleic acid interactions

Redesign of a WW Domain Peptide for Selective Recognition of Single-Stranded DNA

A β-sheet mini-protein based on the FBP11 WW1 domain sequence has been redesigned for the molecular recognition of ssDNA. A previous report showed that a β-hairpin peptide dimer, (WKWK)2, binds ssDNA with low micromolar affinity but with little selectivity over duplex DNA. This report extends those studies to a three-stranded β-sheet mini-protein designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows about 10-fold selectivity for ssDNA over duplex DNA. The redesigned peptide no longer binds its native ligand, the polyproline helix, confirming that the peptide has been redesigned for the function of binding ssDNA. Structural studies provide evidence that this peptide consists of a well structured β-hairpin made of Strands 2&3 with a less structured first strand that provides affinity for ssDNA but does not improve the stability of the full peptide. These studies provide insight into protein-DNA interactions as well as a novel example of protein-redesign

Fluorogenic sensor platform for the histone code using receptors from dynamic combinatorial libraries

A sensor array has been developed that can differentiate multiple post-translational modifications in the same peptide and their relative positions in the sequence, including multiple methylations, providing a promising new tool for deciphering the histone code

Late stage modification of receptors identified from dynamic combinatorial libraries

Approaches for the late-stage modification of receptors discovered from dynamic combinatorial libraries and the investigation of the effects of simple modifications on receptor binding and selectivity

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 α-face of the carbohydrate and the aromatic side chain. To this end, this interaction has been studied in the context of a β-hairpin in aqueous solution, in which the interaction can be investigated in the absence of other cooperative noncovalent interactions. In this β-hairpin system both the aromatic side chain as well as the carbohydrate was varied in an effort to gain greater insight into the driving force and magnitude of the carbohydrate-π 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-π interactions to the driving force. These findings demonstrate the significance of carbohydrate-π interactions within biological systems and also demonstrate its utility as a molecular recognition element in designed system

β-Turn sequences promote stability of peptide substrates for kinases within the cytosolic environment

A strategy was developed to extend the lifetime of an peptide-based substrate for Abl kinase in the cytosolic environment. Small β-turn structures were added to the peptide’s N-terminus to block entry into peptidase catalytic sites. The influence of the size of the β-turn and two covalent cross-linking strategies on the rate of hydrolysis was assessed. The most peptidase-resistant substrate was degraded at a rate of 0.6 pmol mg−1 s−1 and possessed a half-life of 20.3 ± 1.7 min in a Baf/BCR-ABL cytosolic lysate, representing 16- and 40-fold improvements, respectively, over that of a control peptide lacking the β-turn structure. Furthermore, the kcat/KM value of this peptide was 432 μM−1 min−1, a 1.25X increase over the unmodified control, verifying that the added β-turn did not hinder the substrate properties of the peptide. This improved peptide was microinjected into single Baf/BCR-ABL cells and substrate phosphorylation measured. Zero to forty percent of the peptide was phosphorylated in the single cells. In contrast, when the control peptide without a β-turn was loaded into cells, the peptide was too rapidly degraded to detect phosphorylation. This work demonstrates that small β-turn structures can render peptides more resistant to hydrolysis while retaining substrate efficacy and shows that these stabilized peptides have the potential to be of high utility in single-cell enzyme assays

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS have provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequences fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes (DUBs). This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, recent work is presented highlighting the development of novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples