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Tryptophan (Trp) plays a critical role in the regulation of protein structure, interactions and functions through its π system and indole N-H group. A generalizable method for blocking and rescuing Trp interactions would enable the gain-of-function manipulation of various Trp-containing proteins in vivo, but generating such a platform remains challenging. Here we develop a genetically encoded N 1 -vinyl-caged Trp capable of rapid and bioorthogonal decaging through an optimized inverse electron-demand Diels-Alder reaction, allowing site-specific activation of Trp on a protein of interest in living cells. This chemical activation of a genetically encoded caged-tryptophan (Trp-CAGE) strategy enables precise activation of the Trp of interest underlying diverse important molecular interactions. We demonstrate the utility of Trp-CAGE across various protein families, such as catalase-peroxidases and kinases, as translation initiators and posttranslational modification readers, allowing the modulation of epigenetic signalling in a temporally controlled manner. Coupled with computer-aided prediction, our strategy paves the way for bioorthogonal Trp activation on more than 28,000 candidate proteins within their native cellular settings., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Temporal activation of proteins of interest (POIs) offers a gain-of-function approach to investigate protein functions in dynamic biological processes. Fusion of photo/chemical-switchable proteins to a POI, or site-specific blockage/decaging of catalytic residue(s) on a POI, are the most widely utilized strategies for selective protein activation. These methods, however, either lack generality (e.g., active site decaging) or would modify the POI with a bulky tag (e.g., genetic fusion). Recently, a computationally aided and genetically encoded proximal decaging strategy (CAGE-prox) has been developed for time-resolved photoactivation of a broad range of proteins in living systems. In contrast to the direct decaging of the active site of a POI, CAGE-prox relies on a unified caged amino acid that can be anchored in proximity to a protein's functional site for temporal blockage of its activity until rescued by photo/chemical decaging. In order to identify the optimal site for photo-caged unnatural amino acid insertion, which is key for the effective blockade and re-activation of the POI, a computational algorithm was developed to screen all possible positions in close proximity to the functional site that would enable turning off/on protein activity via caging/decaging operations. Here, we describe the CAGE-prox strategy, from in silico design to experimental validation, and provide various examples of its application. © 2021 Wiley Periodicals LLC Basic Protocol 1: In silico design and experimental validation of CAGE-prox Basic Protocol 2: Orthogonal activation of a POI by CAGE-prox while minimizing the activity from the endogenous protein Basic Protocol 3: CAGE-prox-enabled, time-resolved proteomics for the identification of substrates of a proteolytic enzyme Basic Protocol 4: Controlled activation of protein-based prodrugs for tumor therapy., (© 2021 Wiley Periodicals LLC.)

Biological macromolecules (proteins, nucleic acids, polysaccharides, etc.) are the building blocks of life, which constantly undergo chemical modifications that are often reversible and spatial-temporally regulated. These dynamic properties of chemical modifications play fundamental roles in physiological processes as well as pathological changes of living systems. The Major Research Project (MRP) funded by the National Natural Science Foundation of China (NSFC)-"Dynamic modifications of biomacromolecules: mechanism and chemical interventions" aims to integrate cross-disciplinary approaches at the interface of chemistry, life sciences, medicine, mathematics, material science and information science with the following goals: (i) developing specific labeling techniques and detection methods for dynamic chemical modifications of biomacromolecules, (ii) analyzing the molecular mechanisms and functional relationships of dynamic chemical modifications of biomacromolecules, and (iii) exploring biomacromolecules and small molecule probes as potential drug targets and lead compounds.

A universal gain-of-function approach for selective and temporal control of protein activity in living systems is crucial to understanding dynamic cellular processes. Here we report development of a computationally aided and genetically encoded proximal decaging (hereafter, CAGE-prox) strategy that enables time-resolved activation of a broad range of proteins in living cells and mice. Temporal blockage of protein activity was computationally designed and realized by genetic incorporation of a photo-caged amino acid in proximity to the functional site of the protein, which can be rapidly removed upon decaging, resulting in protein re-activation. We demonstrate the wide applicability of our method on diverse protein families, which enabled orthogonal tuning of cell signalling and immune responses, temporal profiling of proteolytic substrates upon caspase activation as well as the development of protein-based pro-drug therapy. We envision that CAGE-prox will open opportunities for the gain-of-function study of proteins and dynamic biological processes with high precision and temporal resolution.

Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein-protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey-bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein-protein interactions and enables simultaneous mapping of the binding interface under living conditions.

Coupling genetically encoded peptide tags or unnatural amino acids (UAAs) with bioorthogonal reactions allows for precise control over the protein-labeling sites as well as the wide choice of labeling dyes. However, the value of these site-specific protein labeling strategies in a real biology setting, particularly their advantages over conventional labeling methods including fluorescent proteins (FPs), remains to be fully demonstrated. In this tutorial review, we first introduce various strategies for site-specific protein labeling that utilize artificial peptide sequences or genetically encoded UAAs as the labeling handle. Emphasis will be placed on introducing the advantages of protein site-specific labeling techniques as well as their applications in solving biological problems, particularly as to why a site-specific protein labeling approach is needed. Finally, beyond the widely used single site-specific labeling methods, the recently emerged dual site-specific protein labeling strategies will be introduced together with their fast-growing potential in illustrating biological processes.

Considerable attention has been focused on improving the biocompatibility of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), a hallmark of bioorthogonal reaction, in living cells. Besides creating copper-free versions of click chemistry such as strain promoted azide-alkyne cycloaddition (SPAAC), a central effort has also been made to develop various Cu(I) ligands that can prevent the cytotoxicity of Cu(I) ions while accelerating the CuAAC reaction. Meanwhile, additional transition metals such as palladium have been explored as alternative sources to promote a bioorthogonal conjugation reaction on cell surface, as well as within an intracellular environment. Furthermore, transition metal mediated chemical conversions beyond conjugation have also been utilized to manipulate protein activity within living systems. We highlight these emerging examples that significantly enriched our protein chemistry toolkit, which will likely expand our view on the definition and applications of bioorthogonal chemistry.

We have developed a genetically encoded, selenium-based cleavable photo-cross-linker that allows for the separation of bait and prey proteins after protein photo-cross-linking. We have further demonstrated the efficient capture of the in situ generated selenenic acid on the cleaved prey proteins. Our strategy involves tagging the selenenic acid with an alkyne-containing dimethoxyaniline molecule and subsequently labeling with an azide-bearing fluorophore or biotin probe. This cleavage-and-capture after protein photo-cross-linking strategy allows for the efficient capture of prey proteins that are readily accessible by two-dimensional gel-based proteomics and mass spectrometry analysis.

Employing small molecules or chemical reagents to modulate the function of an intracellular protein, particularly in a gain-of-function fashion, remains a challenge. In contrast to inhibitor-based loss-of-function approaches, methods based on a gain of function enable specific signalling pathways to be activated inside a cell. Here we report a chemical rescue strategy that uses a palladium-mediated deprotection reaction to activate a protein within living cells. We identify biocompatible and efficient palladium catalysts that cleave the propargyl carbamate group of a protected lysine analogue to generate a free lysine. The lysine analogue can be genetically and site-specifically incorporated into a protein, which enables control over the reaction site. This deprotection strategy is shown to work with a range of different cell lines and proteins. We further applied this biocompatible protection group/catalyst pair for caging and subsequent release of a crucial lysine residue in a bacterial Type III effector protein within host cells, which reveals details of its virulence mechanism.

Live-cell pH measurements: An environment-sensitive fluorophore (green) was site-specifically introduced on HdeA, an acid-resistant chaperone showing pH-mediated conformational changes under low pH conditions. A survey of the attachment sites led to the discovery of one position on HdeA at which the attached fluorophore showed a strong fluorescence increase upon acidification., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Proteins are the workhorses of the cell, playing crucial roles in virtually every biological process. The revolutionary ability to visualize and monitor proteins in living systems, which is largely the result of the development of green fluorescence protein (GFP) and its derivatives, has dramatically expanded our understanding of protein dynamics and function. Still, GFPs are ill suited in many circumstances; one major drawback is their relatively large size, which can significantly perturb the functions of the native proteins to which they are fused. To bridge this gap, scientists working at the chemistry-biology interface have developed methods to install bioorthogonal functional groups into proteins in living cells. The bioorthogonal group is, by definition, a non-native and nonperturbing chemical group. But more importantly, the installed bioorthogonal handle is able to react with a probe bearing a complementary functionality in a highly selective fashion and with the cell operating in its physiological state. Although extensive efforts have been directed toward the development of bioorthogonal chemical reactions, introducing chemical functionalities into proteins in living systems remains an ongoing challenge. In this Account, we survey recent progress in this area, focusing on a genetic code expansion approach. In nature, a cell uses posttranslational modifications to append the necessary functional groups into proteins that are beyond those contained in the canonical 20 amino acids. Taking lessons from nature, scientists have chosen or engineered certain enzymes to modify target proteins with chemical handles. Alternatively, one can use the cell's translational machinery to genetically encode bioorthogonal functionalities, typically in the form of unnatural amino acids (UAAs), into proteins; this can be done in a residue-specific or a site-specific manner. For studying protein dynamics and function in living cells, site-specific modification by means of genetic code expansion is usually favored. A variety of UAAs bearing bioorthogonal groups as well as other functionalities have been genetically encoded into proteins of interest. Although this approach is well established in bacteria, tagging proteins in mammalian cells is challenging. A facile pyrrolysine-based system, which might potentially become the "one-stop shop" for protein modification in both prokaryotic and eukaryotic cells, has recently emerged. This technology can effectively introduce a series of bioorthogonal handles into proteins in mammalian cells for subsequent chemical conjugation with small-molecule probes. Moreover, the method may provide more precise protein labeling than GFP tagging. These advancements build the foundation for studying more complex cellular processes, such as the dynamics of important receptors on living mammalian cell surfaces., (© 2011 American Chemical Society)

A concise route was developed for the facile synthesis of a cyclic pyrrolysine analogue bearing an azide handle. Directed evolution enabled the encoding of this non-natural amino acid in both prokaryotic and eukaryotic cells, which offers a highly efficient approach for the site-specific protein labeling using click chemistry., (© The Royal Society of Chemistry 2011)

A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl-tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl-tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA-aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.