Biosynthesis and genetic encoding of activated nitriles for fast protein conjugation and tunable fluorogenic labeling

Chem, Journal Year: 2025, Volume and Issue: unknown, P. 102385 - 102385

Published: Jan. 1, 2025

Language: Английский

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Strategies to Expand the Genetic Code of Mammalian Cells

Chemical Reviews, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 12, 2025

Genetic code expansion (GCE) in mammalian cells has emerged as a powerful technology for investigating and engineering protein function. This method allows the precise incorporation of rapidly growing toolbox noncanonical amino acids (ncAAs) into predefined sites target proteins expressed living cells. Due to minimal size these genetically encoded ncAAs, wide range functionalities they provide, ability introduce them freely at virtually any site by simple mutagenesis, this holds immense potential probing complex biology next-generation biotherapeutics. In review, we provide an overview underlying machinery that enables ncAA mutagenesis how are developed. We have also compiled updated list ncAAs been successfully incorporated Finally, our perspectives on current challenges need be addressed fully harness technology.

Language: Английский

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Expanding the diversity of nitroxide‐based paramagnetic probes conjugated to non‐canonical amino acids for SDSL‐EPR applications

ChemBioChem, Journal Year: 2025, Volume and Issue: unknown

Published: March 11, 2025

Abstract Understanding protein structure requires studying its dynamics, which is critical to elucidating functional role. Biophysical techniques have revolutionized this field over time, providing remarkable insights into structure‐function relationships. Among these, Site‐Directed Spin Labelling (SDSL) combined with Electron Paramagnetic Resonance (EPR) a powerful method delivering structural data at the residue level, irrespective of size or environment. Traditional nitroxide labels targeting cysteine residues face limitations when these are essential for function. To address this, alternatives been proposed as use non‐canonical amino acids (ncaa) coupled specific labels. This study introduces 14 N‐HO‐5223, novel label p AzPhe ncaa, along 15 N‐derivative. These were grafted two sites model protein, diflavin cytochrome P450 reductase. For comparative purpose, already reported also used. Continuous wave (cw) EPR spectroscopy confirmed HO‐5223 an effective reporter dynamics. Additionally, Double Electron‐Electron (DEER) measurements provided distance distributions between semi‐quinone FMNH⋅ state CPR and all results expand toolkit ncaa‐nitroxide pairs, enabling EPR‐based studies proteins where modification impractical, further advancing our ability decode dynamics

Language: Английский

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Use and dual use of synthetic biology

Comptes Rendus Biologies, Journal Year: 2025, Volume and Issue: 348(G1), P. 71 - 88

Published: March 7, 2025

Language: Английский

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Noncanonical Amino Acids Dictate Peptide Assembly in Living Cells

Accounts of Chemical Research, Journal Year: 2025, Volume and Issue: unknown

Published: March 19, 2025

ConspectusEmulating the structural features or functions of natural systems has been demonstrated as a state-of-the-art strategy to create artificial functional materials. Inspired by assembly and bioactivity proteins, self-assembly peptides into nanostructures represents promising approach for creating biomaterials. Conventional assembled peptide biomaterials are typically formulated in solution delivered pathological sites implementing theranostic objectives. However, this translocation entails switch from formulation conditions physiological environment raises concerns about material performance. In addition, precise efficient accumulation administered at target remains significant challenge, leading potential biosafety issues associated with off-target effects. These limitations significantly hinder progress advanced To address these concerns, past few years have witnessed development situ living new endeavor optimizing biomaterial performance benefiting advances stimuli-responsive reactions regulating noncovalent interactions. refers processes via sites. Due advantages precisely forming well-defined lesions, situ-formed assemblies integrated interesting next-generation biomedical agents.Despite great developing agents, research area still suffers limited toolkit operating under complicated conditions. Considering amino acids being incorporated backbones modified units, an acid is concern. Therefore, our laboratory intensively engaged designing discovering noncanonical (ncAAs) expand manipulating various biological Thus far, we synthesized containing ncAAs 4-aminoproline, 2-nitroimidazole alanine, Se-methionine, sulfated tyrosine, glycosylated serine, which allow us develop acid-responsive, redox-responsive, enzyme-responsive systems. Based on ncAAs, established complex self-sorting assembly, self-amplified dissipative cells optimize peptides. The resulting exhibit morphological adaptability microenvironment, contributes overcoming delivery barriers improvement targeting accumulation. utilizing developed toolkit, further created supramolecular PROTACs, antagonists, probes cancer treatment diagnosis highlight implications usage. Account, summarize journey emphasis mechanism Eventually, also provide forward conceiving prospects challenges clinical translation situ-formulated

Language: Английский

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Genomically recoded organisms: redefining and safeguarding biological systems

Trends in biotechnology, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

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Reversible Protein Labeling via Genetically Encoded Dithiolane-Containing Amino Acid and Organoarsenic Probes

Bioconjugate Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: April 11, 2025

Conventional protein labeling techniques often rely on irreversible covalent bonds, limiting dynamic control over modifications. Here, we present a reversible strategy using genetically encoded dithiolane-containing amino acid (dtF) and organoarsenic conjugation chemistry. Using dithiarsolane dicarboxylic probe A2, achieved near-quantitative ethanedithiol-mediated removal within 1 h at room temperature. A2 exhibited reduced toxicity with 7-fold higher IC50 compared to arsenoxide, its fluorescent derivative A2-FB showed no cytotoxicity up 100 μM, enabling live-cell applications. This is the first demonstration of dithiol-arsenic chemistry single residue, providing structural alternative dicysteine motifs. Reversible was validated in purified proteins (sfGFP-Y151dtF MYO-K99dtF) live Escherichia coli, offering versatile tool for modifications molecular tracking biological systems.

Language: Английский

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Resolving Conformational Plasticity in Mammalian Cells with High-Resolution Fluorescence Tools

Annual Review of Physical Chemistry, Journal Year: 2025, Volume and Issue: 76(1), P. 103 - 128

Published: April 21, 2025

Investigating protein dynamic structural changes is fundamental for understanding function, drug discovery, and disease mechanisms. Traditional studies of dynamics often rely on investigations purified systems, which fail to capture the complexity cellular environment. The intracellular milieu imposes distinct physicochemical constraints that affect macromolecular interactions in ways not easily replicated isolated experimental setups. We discuss use fluorescence resonance energy transfer, anisotropy, minimal photon flux imaging technologies address these challenges directly investigate conformational mammalian cells. Key findings from application techniques demonstrate their potential reveal intricate details plasticity. By overcoming limitations traditional vitro methods, approaches offer a more accurate comprehensive function behavior within complex environment

Language: Английский

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"Cold" Orthogonal Translation by Psychrophilic Pyrrolysyl-tRNA Synthetase Boosts Genetic Code Expansion

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: May 23, 2023

Abstract Using orthogonal translation systems (OTSs) is one of the most efficient strategies for producing unnatural proteins through incorporation non-canonical amino acids (ncAAs) into genetic code. Traditionally, efforts to expand substrate specificity start with a (hyper-)stable enzyme capable withstanding structural changes induced by necessary mutations. In contrast, we propose radically different approach PylRS system: starting enzymes that evolved cope instability in order adapt cold conditions, potentially offering greater resilience mutational changes. By finding and further engineering psychrophilic (“cold”) OTS from Methanococcoides burtonii , developed an alternative widely used mesophilic thermophilic systems. This novel demonstrated exceptional vivo efficiency broad range substrates, even at very low ncAA concentrations cultivation temperatures. The general versatility system across wide applicable host organisms suggests Cold-OTS has potential also improve protein yields these hosts help drive transformation expanded code academic pursuit high-value, chemistry-driven biotechnology.

Language: Английский

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Non-canonical amino acids for site-directed spin labeling of membrane proteins

Current Opinion in Structural Biology, Journal Year: 2024, Volume and Issue: 89, P. 102936 - 102936

Published: Oct. 24, 2024

Membrane proteins remain challenging targets for conventional structural biology techniques because they need to reside within complex hydrophobic lipid environments maintain proper structure and function. Magnetic resonance combined with site-directed spin labeling is an alternative method that provides atomic-level dynamical information from effects introduced by electron- or nuclear-based label. With the advent of bioorthogonal click chemistries genetically engineered non-canonical amino acids (ncAAs), options linking probes biomolecules have substantially broadened outside cysteine-based scheme. Here, we highlight current strategies spin-label membrane through ncAAs nuclear electron paramagnetic applications. Such advances are critical developing schemes achieve in-cell measurements protein conformational dynamics.

Language: Английский

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