scholarly journals Visualizing the Complexity of Proteins in Living Cells With Genetic Code Expansion

2021 ◽  
Author(s):  
Chayasith Uttamapinant ◽  
Kanokpol Aphicho ◽  
Narongyot Kittipanukul
Keyword(s):  
Genetic Code ◽  
Protein Species ◽  
Small Proteins ◽  
Cellular Context ◽  
Technical Advances ◽  
Genetic Code Expansion ◽  
Protein Biophysics ◽  
Insight Into ◽  
Mammalian Protein ◽  
Proteins And Peptides

Genetic code expansion has emerged as an enabling tool to provide insight into functions of understudied proteinogenic species such as small proteins and peptides, and to probe protein biophysics in the cellular context. Here we discuss recent technical advances and applications of genetic code expansion in cellular imaging of complex mammalian protein species, along with considerations and challenges upon using the method.

scholarly journals Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Moirangthem Kiran Singh ◽  
Parisa Zangoui ◽  
Yuki Yamanaka ◽  
Linda J Kenney
Keyword(s):  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Click Reaction ◽  
Host Cells ◽  
Secretion Systems ◽  
Cellular Functions ◽  
Small Proteins ◽  
Type Three Secretion System ◽  
Genetic Code Expansion ◽  
Type Three Secretion

Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.

Structural Basis for Genetic-Code Expansion with Various Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

2018 ◽  
Author(s):  
Tatsuo Yanagisawa ◽  
Mitsuo Kuratani ◽  
Eiko Seki ◽  
Nobumasa Hino ◽  
Kensaku Sakamoto ◽  
...  
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Structural Basis ◽  
Genetic Code Expansion

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

scholarly journals ASIC Activation Mechanisms Delineated through Genetic Code Expansion

2021 ◽  
Vol 120 (3) ◽  
pp. 338a
Author(s):  
Matthew L. Rook ◽  
Tyler A. Couch ◽  
Jackson Hernandez ◽  
Alison J. Frontier ◽  
David M. MacLean
Keyword(s):  
Genetic Code ◽  
Activation Mechanisms ◽  
Genetic Code Expansion

Context effects of genetic code expansion by stop codon suppression

2018 ◽  
Vol 46 ◽  
pp. 146-155 ◽  
Author(s):  
Yonatan Chemla ◽  
Eden Ozer ◽  
Itay Algov ◽  
Lital Alfonta
Keyword(s):  
Genetic Code ◽  
Context Effects ◽  
Stop Codon ◽  
Genetic Code Expansion

Break the template boundary: Test of different protein scaffolds by genetic code expansion resulted in NADPH-dependent secondary amine catalysis

2021 ◽  
Author(s):  
Thomas Williams ◽  
Yu-Hsuan Tsai ◽  
Louis Luk
Keyword(s):  
Genetic Code ◽  
Secondary Amine ◽  
Transfer Hydrogenation ◽  
Protein Scaffolds ◽  
Artificial Enzyme ◽  
Enzyme Design ◽  
Catalytically Active ◽  
Amine Catalysis ◽  
Genetic Code Expansion ◽  
Distinctive Role

Abstract Here, incorporation of secondary amine by genetic code expansion was used to expand the potential protein templates for artificial enzyme design. Pyrrolysine analogue containing a D-proline could be stably incorporated into proteins, including the multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Both modified scaffolds were catalytically active, mediating transfer hydrogenation with a relaxed substrate scope. The protein templates played a distinctive role in that, while the LmrR variants were confined to the biomimetic BNAH as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for reactions. Due to the cofactor compatibility, the DHFR secondary amine catalysis could also be coupled to an enzymatic recycling scheme. This work has illustrated the unique advantages of using proteins as hosts, and thus the presented concept is expected to find uses in enabling tailored secondary amine catalysis.

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