Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal Labeling and Crosslinking of Proteins on Live Mammalian Cells

Meineke, Birthe; Eirich, Jürgen; Landreh, Michael; Elsässer, Simon J.

doi:10.1016/j.celrep.2020.107811

Cited by 54 publications

(108 citation statements)

References 42 publications

(68 reference statements)

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“…This has given more choices of subsequent bioorthogonal reactions. Genetically encoded tyrosine analogues offer ketone handles for fluorophores containing hydrazine or hydroxylamine groups, 65,66 and azide handles for fluorophores with alkyne or cycloalkyne groups 67 (through copper-catalyzed 68 or strain-promoted azide-alkyne [3 + 2] cycloaddition, 69 CuAAC, or SpAAC). Pyrrolysine analogues could also offer cycloalkyne handles for tetrazine-derivatized fluorophores 68,70 (through strain-promoted inverse-electron-demand diels-alder cycloaddition, SPIEDAC) (Fig.…”

Section: Experimental Designmentioning

confidence: 99%

Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Yang

Wang

et al. 2021

Appl Spectrosc

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Fluorescence-based single molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labelling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labelling sites, experimental setup and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

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Section: Experimental Designmentioning

confidence: 99%

Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Yang

Wang

et al. 2021

Appl Spectrosc

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“…This is a special tRNA that recognizes AMBER-type stop codons, allowing the introduction of the residue into the desired position. The evolved tRNA synthetase technique can be used for the incorporation of non-canonical amino acids into proteins being produced in both prokaryotic and eukaryotic cells [57][58][59].…”

Section: Phosphorylationmentioning

confidence: 99%

Post-Translational Modifications of Cytochrome c in Cell Life and Disease

Guerra‐Castellano

Márquez

Pérez‐Mejías

et al. 2020

IJMS

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Mitochondria are the powerhouses of the cell, whilst their malfunction is related to several human pathologies, including neurodegenerative diseases, cardiovascular diseases, and various types of cancer. In mitochondrial metabolism, cytochrome c is a small soluble heme protein that acts as an essential redox carrier in the respiratory electron transport chain. However, cytochrome c is likewise an essential protein in the cytoplasm acting as an activator of programmed cell death. Such a dual role of cytochrome c in cell life and death is indeed fine-regulated by a wide variety of protein post-translational modifications. In this work, we show how these modifications can alter cytochrome c structure and functionality, thus emerging as a control mechanism of cell metabolism but also as a key element in development and prevention of pathologies.

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“…The copyright holder for this preprint (which this version posted April 12, 2021. ; https://doi.org/10.1101/2021.04.12.439361 doi: bioRxiv preprint Bednar et al 3 While density functional theory predicts that SPAAC and IEDDA reactions should be mutually orthogonal and the in vivo compatibility of these reactions has been shown, site-specific encoding of the handles into the same protein and dual labeling in vivo has not yet been demonstrated [16][17][18] . A recent attempt succeeded at dual encoding two different handles into a protein using GCE, however, the functional groups required the use of copper catalyst, thereby relegating labeling to the cell surface due to copper toxicity 19 . These examples highlight the challenge of developing efficient and orthogonal dual encoding and labeling approaches.…”

Section: Introductionmentioning

confidence: 99%

Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

Bednar

Jana

Kuppa

et al. 2021

Preprint

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The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encoded tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically-defined protein-protein crosslinking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.

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Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal Labeling and Crosslinking of Proteins on Live Mammalian Cells

Cited by 54 publications

References 42 publications

Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Post-Translational Modifications of Cytochrome c in Cell Life and Disease

Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

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