Trapping Transient Protein Species by Genetic Code Expansion

Abstract

Nature employs a limited number of genetically encoded amino acids for the construction of functional proteins. By engineering components of the cellular translation machinery, however, it is now possible to genetically encode noncanonical building blocks with tailored electronic and structural properties. The ability to incorporate unique chemical functionality into proteins provides a powerful tool to probe mechanism and create novel function. In this minireview, we highlight several recent studies that illustrate how noncanonical amino acids have been used to capture and characterize reactive intermediates, fine-tune the catalytic properties of enzymes, and stabilize short-lived protein-protein complexes.

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

“…The ability of site-specific introduction of ncAAs with various functional groups into enzymes has improved our understanding in their structures, dynamics, and catalytic mechnisms. ,, This diverse array of ncAAs includes redox-active residues for mechanistic studies, isotopic labels for infrared (IR) and NMR studies, photo-cross-linkers for mapping interactions with other molecules, heavy atoms for X-ray crystallography, spin labels for EPR, and fluorescent side probes for optical applications. In this section, we highlight instructive examples among numerous studies using ncAAs probes.…”

“…Access to abiological chemical groups, provided by the expanded amino acid alphabet, has enlarged the palette for protein engineers and organic chemists. − In principle, the complementary use of traditional approaches (rational design and directed evolution) and the expanded amino acid alphabet have provided a larger toolbox that enables the creation of novel enzymes from protein scaffolds of interest with desired functionality (Figure ). − Most of the recent reviews on chemical modification and GCE approaches are focused on the methodological development itself or the bioconjugation strategies to modify peptides and antibodies for pharmaceutical use. − Although there are some recent reviews on the use of ncAAs for biocatalyst engineering, ,− a comprehensive literature review describing chemical modifications and the incorporation of ncAAs, advancing our understanding of biocatalysis, has rarely been discussed. Therefore, the present review provides a closer look into the recent advances in biocatalysis through the chemical modification and expanded amino acid alphabet, with a focus on the enhancement of enzyme functionality and mechanistic studies.…”

Capturing Covalent Catalytic Intermediates by Enzyme Mutants: Recent Advances in Methodologies and Applications

“…Therefore, these enzyme mutants are enzyme substrate traps. Recently, with the development of the genetic code expansion (GCE) technology, [23] site‐directed mutagenesis is expanded to introduce non‐proteogenic amino acids that resemble the catalytic residues into enzymes for better intermediate trapping [24] . Compared to substrate mimic‐based trapping strategies, enzyme mutant‐based substrate traps focus on manipulating the enzyme property, exhibiting more potentials to reveal the physiological function of the enzyme of interest.…”

Cysteine-based protein folding modulators for trapping intermediates and misfolded forms

“…19 Inspired by the enzymatic process, a variety of compounds such as thiols with increased acidity, glutathione derivatives, and selenols have been developed for promotion of protein folding. [20][21][22][23][24][25] On the other hand, methodologies that can trap folding intermediates and misfolded forms are limited to genetic mutations [26][27][28] and chemical treatments using iodoacetic acid and maleimideappending compounds. 29,30 Here, we demonstrate that newly developed cysteine-based dipeptides, Cys-Dap and Cys-Tamp (Fig.…”