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The methylglyoxal-derived hydroimidazolone isomer, MGH-1, is an abundant advanced glycation end-product (AGE) associated with disease and age-related disorders. As AGE formation occurs spontaneously and without an enzyme, it remains unknown why certain sites on distinct proteins become modified with specific AGEs. Here, we use a combinatorial peptide library to determine the chemical features that favor MGH-1. When properly positioned, tyrosine is found to play an active mechanistic role that facilitates MGH-1 formation. This work offers mechanistic insight connecting multiple AGEs, including MGH-1 and carboxyethylarginine (CEA), and reconciles the role of negative charge in influencing glycation outcomes. Further, this study provides clear evidence that glycation outcomes can be influenced through long- or medium-range cooperative interactions. This work demonstrates that these chemical features also predictably template selective glycation on full-length protein targets expressed in mammalian cells. This information is vital for developing methods that control glycation in living cells and will enable the study of glycation as a functional post-translational modification., Advanced glycation end-products (AGEs), such as methylglyoxal-derived hydroimidazolone isomer (MGH-1), are associated with disease and age-related disorders, and occur spontaneously, so it is unclear why specific protein sites become modified with specific AGEs. Here, the authors use a combinatorial peptide library to determine the chemical features that favour MGH-1 formation for short peptides and demonstrate a key role of tyrosine in this process.

Glycation is a non-enzymatic post-translational modification (PTM) that remains poorly understood, largely because it is unknown how it occurs selectively. Using mass spectrometry, it was possible to evaluate total glycation levels, identify distinct glycated products, assign unique glycation sites, and correlate these data with chemical and structural features for a panel of proteins glycated in vitro. It was determined that the extent of glycation does not correlate with pKa or surface exposure at reactive sites. Rather, the data reveal that primary sequence dictates the overall likelihood that a site will become glycated, while surrounding structure further sculpts the glycation outcome. Clustered acidic residues were found to prevent glycation, whereas a combination of tyrosine and polar residues appear to promote glycation. This work contributes important new knowledge about the molecular features that govern selective glycation.

Bacterial phosphothreonine lyases, or phospholyases, catalyze a unique post-translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha-Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb-containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb-modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme-mediated strategies for bioorthogonal labeling in living cells.

Exploration across the fields of biology, chemical biology, and medicine has led to an increasingly complex, albeit incomplete, view of the interactions that drive life's processes. The ability to monitor and track the movement, activity, and interactions of biomolecules in living cells is an essential part of this investigation. In our laboratory, we have endeavored to develop tools that are capable not only of monitoring protein localization but also reporting on protein structure and function. Central to our efforts is a new strategy, bipartite tetracysteine display, that relies on the specific and high-affinity interaction between a fluorogenic, bis-arsenical small molecule and a unique protein sequence, conformation, or assembly. In 1998, a small-molecule analogue of fluorescein with two arsenic atoms, FlAsH, was shown by Tsien and coworkers to fluoresce upon binding to a linear amino acid sequence, Cys-Cys-Arg-Glu-Cys-Cys. Later work demonstrated that substituting Pro-Gly for Arg-Glu optimized both binding and fluorescence yield. Our strategy of bipartite tetracysteine display emanated from the idea that it would be possible to replace the intervening Pro-Gly dipeptide in this sequence with a protein or protein partnership, provided the assembled protein fold successfully reproduced the approximate placement of the two Cys-Cys pairs. In this Account, we describe our recent progress in this area, with an emphasis on the fundamental concepts that underlie the successful use of bis-arsenicals such as FlAsH and the related ReAsH for bipartite display experiments. In particular, we highlight studies that have explored how broadly bipartite tetracysteine display can be employed and that have navigated the conformational boundary conditions favoring success. To emphasize the utility of these principles, we outline two recently reported applications of bipartite tetracysteine display. The first is a novel, encodable, selective, Src kinase sensor that lacks fluorescent proteins but possesses a fluorescent readout exceeding that of most sensors based on Förster resonance energy transfer (FRET). The second is a unique method, called complex-edited electron microscopy (CE-EM), that facilitates visualization of protein-protein complexes with electron microscopy. Exciting as these applications may be, the continued development of small-molecule tools with improved utility in living cells, let alone in vivo, will demand a more nuanced understanding of the fundamental photophysics that lead to fluorogenicity, as well as creative approaches toward the synthesis and identification of new and orthogonal dye-tag pairs that can be applied facilely in tandem. We describe one example of a dye-sequence tag pair that is chemically distinct from bis-arsenical chemistry. Through further effort, we expect that that bipartite tetracysteine display will find successful use in the study of sophisticated biological questions that are essential to the fields of biochemistry and biology as well as to our progressive understanding of human disease.

Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5'-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.

For a range of protein substrates, N-terminal transamination offers a convenient way to install a reactive ketone or aldehyde functional group at a single location. We report herein the effects of the identity of N-terminal residues on the product distribution generated upon reaction with pyridoxal 5'-phosphate (PLP). This study was accomplished through the combination of solid-phase peptide synthesis with detailed liquid chromatography-mass spectrometry analysis. Many N-terminal amino acids provided high yields of the desired transaminated products, but some residues (His, Trp, Lys, and Pro) generated adducts with PLP itself. N-terminal Cys and Ser residues were observed to undergo beta-elimination in addition to transamination, and the transamination product of N-terminal Gln was resistant to subsequent oxime formation attempts. The information generated through the screening of peptide substrates was successfully applied to a protein target, changing an initially unreactive terminus into one that could be modified in high (70%) yield. Thus, these studies have increased our predictive power for the reaction, both in terms of improving conversion and suppressing reaction byproducts. An initial set of guidelines that may be used to increase the applicability of this reaction to specific proteins of interest is provided.