A chemical biology route to site-specific authentic protein modifications

Yang, Aerin; Ha, Sura; Ahn, Jihye; Kim, Rira; Kim, Sungyoon; Lee, Younghoon; Kim, Jae-Hoon; Söll, Dieter; Lee, Hee‐Yoon; Park, Hee-Sung

doi:10.1126/science.aah4428

Cited by 216 publications

(215 citation statements)

References 29 publications

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“…eGFP in the peGFP-C1 vector (Clontech) was converted to mGFP3435 by mutating alanine 207 of eGFP to lysine using Quik-Change mutagenesis Kit XL from Agilent technologies (Primer: Fw: 5′-TAC CTG AGC ACC CAG TCC AAA CTG AGC AAA GAC CCC AAC-3′, rev: 5′-GTT GGG GTC TTT GCT CAG TTT GGA CTG GGT GCT CAG GTA-3′). cDNAs of WT human SERT and the mutated version SERT-K352A–K460A (ref.…”

Section: Methodsmentioning

confidence: 99%

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

et al. 2017

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The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by singlemolecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization. After trafficking to the plasma membrane, however, the SERT stoichiometry is fixed. Stabilization of the oligomeric SERT complexes is mediated by the direct binding to phosphoinositide phosphatidylinositol-4,5-biphosphate (PIP 2 ). The observed spatial decoupling of oligomer formation from the site of oligomer operation provides cells with the ability to define protein quaternary structures independent of protein density at the cell surface.

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Section: Methodsmentioning

confidence: 99%

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

et al. 2017

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“…[5] Alternatively, native chemical ligation/expressed protein ligation (NCL/EPL), a cysteine-based alkylation approach, and a recently developed dehydroalanine-based coupling approach may be applied for the synthesis of proteins with lysine acylations. [6] However, ECL/EPL requires a cysteine for ligation and the structures generated by the later two methods are slightly different from the natural modifications. Here we report the synthesis of proteins with genuine lysine acylations by coupling the amber suppression based mutagenesis approach [7] with traceless Staudinger ligation.…”

mentioning

confidence: 99%

A Versatile Approach for Site‐Specific Lysine Acylation in Proteins

Wang

Kurra

Wang³

et al. 2017

Angew Chem Int Ed

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Using amber suppression in coordination with a mutant pyrrolysyl-tRNA synthetase-tRNAPyl pair, azidonorleucine is genetically encoded in E. coli. Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows convenient synthesis of a protein with a site-specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su-H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified novel posttranslational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.

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“…These systems have been reviewed extensively elsewhere [12, 55, 56]. Recently, this site-specific approach has provided important insights into the dynamics of post-translational modifications, such as lysine acetylation and serine phosphorylation [16, 57]. Another intriguing application of site-specific incorporation of synthetic amino acids is biocontainment.…”

Section: Selection Of Amino Acids By Aminoacyl-trna Synthetasesmentioning

confidence: 99%

Rewiring protein synthesis: From natural to synthetic amino acids

Fan

Evans

Ling

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. Scope of review This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. Major conclusions The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. General significance Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms.

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A chemical biology route to site-specific authentic protein modifications

Cited by 216 publications

References 29 publications

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

A Versatile Approach for Site‐Specific Lysine Acylation in Proteins

Rewiring protein synthesis: From natural to synthetic amino acids

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