Incorporation Of Amino Acids Research Articles

A guide to designing photocontrol in proteins: methods, strategies and applications.

Light is essential for various biochemical processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and molecular level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chemical engineering of small-molecule photosensitive effectors (photopharmacology), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biological modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.

Linkage-Specific Synthesis of Di-ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK.

Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) Nϵ -L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination.

NMR assignment of non-modified tRNAIle from Escherichia coli

tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.

Directed Evolution of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Generates a Hyperactive and Highly Selective Variant

Pyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of Methanomethylophilus alvus PylRS (MaPylRS) has been rationally engineered to expand its substrate compatibility, enabling the incorporation of difficult ncAAs. However, mutations beyond the active site that enhance the enzymatic properties of MaPylRS have not been reported. We utilized phage-assisted non-continuous evolution (PANCE) to evolve MaPylRS to efficiently incorporate N ε-Boc-l-lysine (BocK). Directed evolution yielded several mutations outside of the active site that greatly improve the activity of the enzyme. We combined the most effective mutations to generate a new PylRS variant (PylRSopt) that is highly active and selective towards several lysine and phenylalanine derivatives. The mutations in PylRSopt can be used to enhance previously engineered PylRS constructs such as MaPylRSN166S, and PylRSopt is compatible in applications requiring dual ncAA incorporation and substantially improves the yield of these target proteins.

Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1–S4 bundle rather than a quiescent state

Transient receptor potential vanilloid1 (TRPV1) channel plays an important role in a wide range of physiological and pathological processes, and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention. Recent incredible advances in cryo-electron microscopy (cryo-EM) have yielded high-resolution structures of all TRPV subtypes (TRPV1–6) and all of them share highly conserved six transmembrane (TM) domains (S1–S6). As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist (capsaicin or resiniferatoxin), TM helicesS1 to S4 form a bundle that remains quiescent during channel activation, highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels. Here, however, we argue that the structural dynamics rather than quiescence of S1–S4 domains is necessary for capsaicin-mediated activation of TRPV1. Using fluorescent unnatural amino acid (flUAA) incorporation and voltage-clamp fluorometry (VCF) analysis, we directly observed allostery of the S1–S4 bundle upon capsaicin binding. Covalent occupation of VCF-identified sites, single-channel recording, cell apoptosis analysis, and exploration of the role of PSFL828, a novel non-vanilloid agonist we identified, have collectively confirmed the essential role of this coordinated S1–S4 motility in capsaicin-mediated activation of TRPV1. This study concludes that, in contrast to cryo-EM structural studies, vanilloid agonists are also required for S1–S4 movement during TRPV1 activation. Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.

Genetic code expansion and its application in characterization and imaging of proteins

Genetic code expansion (GCE) allows the incorporation of unnatural amino acids into proteins via using stop codons. GCE may achieve site-specific labeling of proteins in combination with the click reaction. Compared with other labeling tools such as fluorescent proteins and tagged antibodies, the compound molecules used in protein labeling by GCE technology are smaller, and therefore, may less interfere the conformational structure of proteins. In addition, through click reaction, GCE allows a 1:1 stoichiometric ratio of the target protein molecule and the fluorescent dye, and the protein can be quantified based on the fluorescence intensity. Thus, GCE technology has great advantages in the researches that require the exposition of living cells under high laser power for longer time, for example, in the context of single molecule tracing and super-resolution microscopic imaging. Meanwhile, this technology lays the foundation for improving the accuracy of positioning and molecule counting in the imaging process of living cells. This review summarized the GCE technology and its recent applications in functionally characterizing, labeling and imaging of proteins.

Drop-off-reinitiation triggered by EF-G-driven mistranslocation and its alleviation by EF-P.

In ribosomal translation, peptidyl transfer occurs between P-site peptidyl-tRNA and A-site aminoacyl-tRNA, followed by translocation of the resulting P-site deacylated-tRNA and A-site peptidyl-tRNA to E and P site, respectively, mediated by EF-G. Here, we report that mistranslocation of P-site peptidyl-tRNA and A-site aminoacyl-tRNA toward E and A site occurs when high concentration of EF-G triggers the migration of two tRNAs prior to completion of peptidyl transfer. Consecutive incorporation of less reactive amino acids, such as Pro and d-Ala, makes peptidyl transfer inefficient and thus induces the mistranslocation event. Consequently, the E-site peptidyl-tRNA drops off from ribosome to give a truncated peptide lacking the C-terminal region. The P-site aminoacyl-tRNA allows for reinitiation of translation upon accommodation of a new aminoacyl-tRNA at A site, leading to synthesis of a truncated peptide lacking the N-terminal region, which we call the ‘reinitiated peptide’. We also revealed that such a drop-off-reinitiation event can be alleviated by EF-P that promotes peptidyl transfer of Pro. Moreover, this event takes place both in vitro and in cell, showing that reinitiated peptides during protein synthesis could be accumulated in this pathway in cells.

Incorporating amino acids functionalized graphene oxide nanosheets into Pebax membranes for CO2 separation

Graphene oxide (GO) nanosheets, as versatile nanofillers, can create two-dimensional passageways in mixed matrixed membranes (MMMs), yet the flexible manipulation of GO nanosheets is hampered by the lack of appropriate chemical functional groups. In this study, we chose amino acids（arginine, histidine and cysteine）to modify GO nanosheets by direct crosslinking and the loading content of amino acids on GO nanosheets was more than 20%. The modified GO nanosheets were then filled into the Pebax matrix to prepare MMMs. The CO2 separation performance of MMMs with different nanofillers was compared and different amino acids modified GO nanosheets could fortify the solution mechanism for CO2 in the order of arginine@GO ＞ histidine@GO ＞ cysteine@GO. Density functional theory (DFT) calculations were conducted to further elucidate the interaction mechanism between gases molecules and amino acids. Under dry state, the complexation energy (Ec) between CO2 and amine groups was similar to the Ec between CO2 and carboxylic groups in amino acids. In particular, 0.4 wt% arg@GO nanosheets rendered the membranes with promising CO2 permeance of 169 Barrer and CO2/N2 selectivity of 70, and surpassed the 2008 upper bound. The amino acids modified GO nanosheets may enlighten an alternative way to explore CO2 transfer mechanism within MMMs and fabricate high separation performance membranes.

Genetic Incorporation of Fluorescent Amino Acid into Fatty Acid Binding Protein for Fatty Acid Detection

Fatty acids play critical roles in biological processes, such as energy storage, metabolism, signal transduction, and immune regulation. Therefore, it is necessary to develop in-vitro fluorescent sensors to detect free fatty acids. By genetically incorporating a synthetic fluorescent amino acid (L-(7-hydroxycoumarin-4-yl) ethylglycine, Cou) into fatty acid-binding protein (FABP), we obtained a fluorescent sensor that has a turn-on signal in the presence of the fatty acids. Its response to medium-chain and long-chain fatty acids can be increased by 5.8-fold within several minutes, highlighting its potential applications in fatty acids-related biological processes. Our newly developed fatty acid detection system based on genetic expansion technology has extended the molecular toolboxes available for important biological molecular analysis.

A Chemical Biology Primer for NMR Spectroscopists

Among structural biology techniques, NMR spectroscopy offers unique capabilities that enable the atomic resolution studies of dynamic and heterogeneous biological systems under physiological and native conditions. Complex biological systems, however, often challenge NMR spectroscopists with their low sensitivity, crowded spectra or large linewidths that reflect their intricate interaction patterns and dynamics. While some of these challenges can be overcome with the development of new spectroscopic approaches, chemical biology can also offer elegant and efficient solutions at the sample preparation stage. In this tutorial, we aim to present several chemical biology tools that enable the preparation of selectively and segmentally labeled protein samples, as well as the introduction of site-specific spectroscopic probes and post-translational modifications. The four tools covered here, namely cysteine chemistry, inteins, native chemical ligation, and unnatural amino acid incorporation, have been developed and optimized in recent years to be more efficient and applicable to a wider range of proteins than ever before. We briefly introduce each tool, describe its advantages and disadvantages in the context of NMR experiments, and offer practical advice for sample preparation and analysis. We hope that this tutorial will introduce beginning researchers in the field to the possibilities chemical biology can offer to NMR spectroscopists, and that it will inspire new and exciting applications in the quest to understand protein function in health and disease.

A Strategy to Select Macrocyclic Peptides Featuring Asymmetric Molecular Scaffolds as Cyclization Units by Phage Display.

Macrocyclic peptides (MPs) have positioned themselves as a privileged class of compounds for the discovery of therapeutics and development of chemical probes. Aided by the development of powerful selection strategies, high-affinity binders against biomolecular targets can readily be elicited from massive, genetically encoded libraries by affinity selection. For example, in phage display, MPs are accessed on the surface of whole bacteriophages via disulfide formation, the use of (symmetric) crosslinkers, or the incorporation of non-canonical amino acids. To facilitate a straightforward cyclization of linear precursors with asymmetric molecular scaffolds, which are often found at the core of naturally occurring MPs, we report an efficient two-step strategy to access MPs via the programmed modification of a unique cysteine residue and an N-terminal amine. We demonstrate that this approach yields MPs featuring asymmetric cyclization units from both synthetic peptides and when linear precursors are appended onto a phage-coat protein. Finally, we showcase that our cyclization strategy is compatible with traditional phage-display protocols and enables the selection of MP binders against a model target protein from naïve libraries. By enabling the incorporation of non-peptidic moieties that (1) can serve as cyclization units, (2) provide interactions for binding, and/or (3) tailor pharmacological properties, our head-to-side-chain cyclization strategy provides access to a currently under-explored chemical space for the development of chemical probes and therapeutics.

Structural Basis for the Peptidoglycan-Editing Activity of YfiH.

ABSTRACTBacterial cells are encased in peptidoglycan (PG), a polymer of disaccharide N-acetylglucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) cross-linked by peptide stems. PG is synthesized in the cytoplasm as UDP-MurNAc-peptide precursors, of which the amino acid composition of the peptide is unique, with l-Ala added at the first position in most bacteria but with l-Ser or Gly in some bacteria. YfiH is a PG-editing factor whose absence causes misincorporation of l-Ser instead of l-Ala into peptide stems, but its mechanistic function is unknown. Here, we report the crystal structures of substrate-bound and product-bound YfiH, showing that YfiH is a cytoplasmic amidase that controls the incorporation of the correct amino acid to the nucleotide precursors by preferentially cleaving the nucleotide precursor by-product UDP–MurNAc–l-Ser. This work reveals an editing mechanism in the cytoplasmic steps of peptidoglycan biosynthesis.

Metabolic labeling of secreted matrix to investigate cell-material interactions in tissue engineering and mechanobiology.

Re-creating features of the native extracellular matrix (ECM) with engineered biomaterials has become a valuable tool to probe the influence of ECM properties on cellular functions (e.g., differentiation) and toward the engineering of tissues. However, characterization of newly secreted (nascent) matrix and turnover, which are important in the context of cells interacting with these biomaterials, has been limited by a lack of tools. We developed a protocol to visualize and quantify the spatiotemporal evolution of newly synthesized and deposited matrix by cells that are either cultured atop (2D) or embedded within (3D) biomaterial systems (e.g., hydrogels, fibrous matrices). This technique relies on the incorporation of a noncanonical amino acid (azidohomoalanine) into proteins as they are synthesized. Deposited nascent ECM components are then visualized with fluorescent cyclooctynes via copper-free cycloaddition for spatiotemporal analysis or modified with cleavable biotin probes for identification. Here we describe the preparation of hyaluronic acid hydrogels through ultraviolet or visible light induced cross-linking for 2D and 3D cell culture, as well as the fluorescent labeling of nascent ECM deposited by cells during culture. We also provide protocols for secondary immunofluorescence of specific ECM components and ImageJ-based ECM quantification methods. Hyaluronic acid polymer synthesis takes 2 weeks to complete, and hydrogel formation for 2D or 3D cell culture is performed in 2-3 h. Lastly, we detail the identification of nascent proteins, including enrichment, preparation and analysis with mass spectrometry, which can be completed in 10 d.

Enhancing Peptide Nucleic Acid–Nanomaterial Interaction and Performance Improvement of Peptide Nucleic Acid-Based Nucleic Acid Detection by Using Electrostatic Effects

Single-stranded peptide nucleic acid (PNA) probes interact strongly with several nanomaterials, and the interaction was diminished in the presence of complementary nucleic acid targets which forms the basis of many nucleic acid sensing platforms. As opposed to the negatively charged DNA probes, the charges on the PNA probes may be fine-tuned by incorporating amino acids with charged side chains. The contribution of electrostatic effects to the interaction between PNA probes and nanomaterials has been largely overlooked. This work reveals that electrostatic effects substantially enhanced the quenching of dye-labeled conformationally constrained pyrrolidinyl PNA probes by several nanomaterials including graphene oxide (GO), reduced graphene oxide, gold nanoparticles (AuNPs), and silver nanoparticles. The fluorescence quenching and the color change from red to purple in the case of AuNPs because of aggregation were inhibited in the presence of complementary nucleic acid targets. Thus, fluorescence and colorimetric assays for DNA and RNA that can distinguish even single-base-mismatched nucleic acids with improved sensitivity over conventional DNA probes were established. Both the GO- and AuNP-based sensing platforms have been successfully applied for the detection of real DNA and RNA samples in vitro and in living cells. This study emphasizes the active roles of electrostatic effects in the PNA-nanomaterial interactions, which paves the way toward improving the performance of PNA-nanomaterial based assays of nucleic acids.

Incorporation of metal-chelating unnatural amino acids into halotag for allylic deamination

The potential of artificial metalloenzymes has led to an increase in interest for the design of novel metal-binding sites in proteins. Metal-chelating unnatural amino acids offer an auspicious solution to engineer active metal sites in a defined way. Herein, we describe the introduction of four metal-chelating unnatural amino acids into HaloTag, an attractive scaffold for the assembly of functional artificial metalloenzymes. HaloTag, engineered with 2-amino-3-(8-hydroxyquinolin-5-yl)propanoic acid (HQ-Ala-1) was used to assemble an artificial metalloenzyme for improved allylic deamination upon complementation with [(η5-C5H5)Ru(MeCN)3]+.

The Pyrrolysyl-tRNA Synthetase Activity can be Improved by a P188 Mutation that Stabilizes the Full-Length Enzyme

The amber suppression-based noncanonical amino acid (ncAA) mutagenesis technique has been widely used in both basic and applied research. So far more than two hundred ncAAs have been genetically encoded by amber codon in both prokaryotes and eukaryotes using wild-type and engineered pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl (PylT) pairs. Methanosarcina mazei PylRS (MmPylRS) is arguably one of two most used PylRS variants. However, it contains an unstable N-terminal domain that is usually cleaved from the full-length protein during expression and therefore leads to a low enzyme activity. We discovered that the cleavage takes place after A189 and this cleavage is inhibited when MmPylRS is co-expressed with Ca. Methanomethylophilus alvus tRNAPyl (CmaPylT). In the presence of CmaPylT, MmPylRS is cleaved after an alternative site K110. MmPylRS is active toward CmaPylT. Its combined use with CmaPylT leads to enhanced incorporation of Nε-Boc-lysine (BocK) at amber codon. To prevent MmPylRS from cleavage after A189 in the presence of its cognate M. mazei tRNAPyl (MmPylT), we introduced mutations at P188. Our results indicated that the P188G mutation stabilizes MmPylRS. We showed that the P188G mutation in wild-type MmPylRS or its engineered variants allows enhanced incorporation of BocK and other noncanonical amino acids including Nε-acetyl-lysine when they are co-expressed with MmPylT.

Impact of the Plastein Reaction of Casein Hydrolysates in the Presence of Exogenous Amino Acids on Their Anti-Inflammatory Effect in the Lipopolysaccharide-Stimulated Macrophages.

In this study, papain-generated casein hydrolysates (CH) with a degree of hydrolysis of 13.7% were subjected to a papain-mediated plastein reaction in the absence or presence of one of the exogenous amino acids—Gly, Pro, and Hyp—to prepare four plastein modifiers, or mixed with one of three amino acids to prepare three mixtures. The assay results confirmed that the reaction reduced free NH2 for the modifiers and caused amino acid incorporation and peptide condensation. When RAW264.7 macrophages were exposed to the CH, modifiers, and mixtures, these samples promoted macrophage growth and phagocytosis in a dose-dependent manner. In addition, the CH shared similar activity in the cells as the mixtures, while the modifiers (especially the PCH-Hyp prepared with Hyp addition) exerted higher potential than CH, the mixtures, and PCH (the modifier prepared without amino acid addition). The plastein reaction thus enhanced CH bioactivity in the cells. When RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS), the inflammatory cells produced more lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) formation, and caused more four inflammatory mediators (NO, PGE2, TNF-α, and IL-6) and two anti-inflammatory mediators (TGF-β1 and IL-10). However, the PCH-Hyp, PCH, and CH at dose levels of 100 μg/mL could combat against the LPS-induced inflammation. Overall, the PCH-Hyp was more active than the CH and PCH in reducing LDH release, ROS formation, and the secretion of these inflammatory mediators, or in increasing the secretion of the anti-inflammatory mediators. The qPCR and Western blot analysis results further confirmed that these samples had anti-inflammatory effects on the stimulated cells by suppressing the LPS-induced activation of the NF-κB signaling pathway, via regulating the mRNA/miRNA expression of iNOS, IL-6, TNF-α, IL-1β, COX-2, TLR4, IL-10, TGF-β1, miR-181a, miR-30d, miR-155, and miR-148, as well as the protein expression of MyD88, p-IKKα, p-IκBα, p-NF-κB p65, and iNOS, involved in this signaling pathway. In addition, the immunofluorescence assay results revealed that these samples could block the LPS-mediated nuclear translocation of the p65 protein and displayed the same function as the NF-κB inhibitor BAY 11-7082. It was concluded that CH could be endowed with higher anti-inflammatory activity to the macrophages by performing a plastein reaction, particularly that in the presence of exogenous Hyp.

Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism.

Nonribosomal peptide synthetases (NRPSs) are complex multi-modular enzymes containing catalytic domains responsible for the loading and incorporation of amino acids into natural products. These unique molecular factories can produce peptides with nonproteinogenic d-amino acids in which the epimerization (E) domain catalyzes the conversion of l-amino acids to d-amino acids, but its mechanism remains not fully understood. Here, we describe the development of pantetheine crosslinking probes that mimic the natural substrate l-Phe of the initiation module of tyrocidine synthetase, TycA, to elucidate and study the catalytic residues of the E domain. Mechanism-based crosslinking assays and MALDI-TOF MS were used to identify both H743 and E882 as the crosslinking site residues, demonstrating their roles as catalytic bases. Mutagenesis studies further validated these results and allowed the comparison of reactivity between the catalytic residues, concluding that glutamate acts as the dominant nucleophile in the crosslinking reaction, resembling the deprotonation of the Cα-H of amino acids in the epimerization reaction. The crosslinking probes employed in these studies provide new tools for studying the molecular details of E domains, as well as the potential to study C domains. In particular, they would elucidate key information for how these domains function and interact with their substrates in nature, further enhancing the knowledge needed to assist combinatorial biosynthetic efforts of NRPS systems to produce novel compounds.

A Simplified Protocol to Incorporate the Fluorescent Unnatural Amino Acid ANAP into Xenopus laevis Oocyte-Expressed P2X7 Receptors.

The long intracellular P2X7 C-terminus accounts for diverse downstream effects of P2X7 activation. Although the recent determination of the cryo-EM structure of the full-length P2X7 receptor finally revealed the structure and several unexpected features of the large cytoplasmic domain, its molecular function remains enigmatic. Incorporation of unnatural amino acids (UAA) via an amber Stop codon has been a powerful tool for structure-function analysis of proteins. Voltage clamp fluorometry (VCF) with the fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) provides a means to study intracellular domain movements of ion channel receptors. In the Xenopus laevis oocyte expression system, site-specific introduction of this environment-sensitive fluorophore can be achieved by the nuclear injection of cDNA encoding an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair and subsequent cytoplasmic injection of ANAP together with the respective cRNA containing the amber Stop codon. Here, we describe this protocol for expression of ANAP-labeled P2X7. In addition, we provide a simplified alternative protocol, in which we coinject cRNAs encoding the tRNA synthetase and mutant P2X7 together with the synthesized amber suppressor tRNA and ANAP in one step into the cytosol. We found that the new protocol yielded more reproducible results and was less harmful for the oocytes. By selective fluorescence labeling of the ANAP-labeled P2X7 protein in the oocyte plasma membrane and VCF recordings, we show that this method results in comparable levels of functional ANAP-labeled P2X7 protein.

Expanding the functionality of proteins with genetically encoded dibenzo[b,f][1,4,5]thiadiazepine: a photo-transducer for photo-click decoration.

Genetic incorporation of novel noncanonical amino acids (ncAAs) that are specialized for the photo-click reaction allows the precisely orthogonal and site-specific functionalization of proteins in living cells under photo-control. However, the development of a r̲ing-strain i̲n situ l̲oadable d̲ipolarophile (RILD) as a genetically encodable reporter for photo-click bioconjugation with spatiotemporal controllability is quite rare. Herein, we report the design and synthesis of a photo-switchable d̲ib̲enzo[b,f][1,4,5]t̲hiad̲iazepine-based a̲lanine (DBTDA) ncAA, together with the directed evolution of a pyrrolysyl-tRNA synthetase/tRNACUA pair (PylRS/tRNACUA), to encode the DBTDA into recombinant proteins as a RILD in living E. coli cells. The fast-responsive photo-isomerization of the DBTDA residue can be utilized as a converter of photon energy into ring-strain energy to oscillate the conformational changes of the parent proteins. Due to the photo-activation of RILD, the photo-switching of the DBTDA residue on sfGFP and OmpC is capable of promoting the photo-click ligation with diarylsydnone (DASyd) derived probes with high efficiency and selectivity. We demonstrate that the genetic code expansion (GCE) with DBTDA benefits the studies on the distribution of decorated OmpC-DBTD on specific E. coli cells under a spatiotemporal resolved photo-stimulation. The GCE for encoding DBTDA enables further functional diversity of artificial proteins in living systems.