scholarly journals Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

2021 ◽  
Vol 16 (4) ◽  
pp. 766-774
Author(s):  
Jeffery M. Tharp ◽  
Oscar Vargas-Rodriguez ◽  
Alanna Schepartz ◽  
Dieter Söll
Keyword(s):  
Amino Acids ◽  
Noncanonical Amino Acids ◽  
Genetic Encoding ◽  
Nonsense Codons

scholarly journals Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

2020 ◽  
Author(s):  
Jeffery M. Tharp ◽  
Oscar Vargas-Rodriguez ◽  
Alanna Schepartz ◽  
Dieter Söll
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Trna Synthetase ◽  
Dual Use ◽  
Noncanonical Amino Acids ◽  
Trna Synthetases ◽  
Genetic Encoding ◽  
Nonsense Codon ◽  
N Terminus ◽  
Nonsense Codons

ABSTRACTWe recently described an orthogonal initiator tRNA (itRNATy2) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here we report that a mutant of itRNATy2 (itRNATy2AUA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing itRNATy2AUA, UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and tyrosine at elongating positions. Using itRNATy2AUA, in conjunction with its cognate tyrosyl-tRNA synthetase and two mutually orthogonal pyrrolysyl-tRNA synthetases, we demonstrate that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. Furthermore, by engineering the substrate specificity of one of the pyrrolysyl-tRNA synthetases, we developed a triply orthogonal system that enables simultaneous reassignment of UAU, UAG, and UAA to produce proteins containing three distinct ncAAs at precisely defined sites. To showcase the utility of this system, we produced proteins containing two or three ncAAs, with unique bioorthogonal functional groups, and demonstrate that these proteins can be separately modified with multiple fluorescent probes.TOC Image

scholarly journals Enhancing protein stability with extended disulfide bonds

2016 ◽  
Vol 113 (21) ◽  
pp. 5910-5915 ◽  
Author(s):  
Tao Liu ◽  
Yan Wang ◽  
Xiaozhou Luo ◽  
Jack Li ◽  
Sean A. Reed ◽  
...  
Keyword(s):  
Amino Acids ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Building Blocks ◽  
Side Chain ◽  
Cross Linking ◽  
Noncanonical Amino Acids ◽  
Genetic Encoding ◽  
Selection Experiments ◽  
Significant Distance

Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.

Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-Assembly and Fluorescence

2021 ◽  
Author(s):  
Binbin Hu ◽  
Na Song ◽  
Yawei Cao ◽  
Mingming Li ◽  
Xin Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Self Assembly ◽  
Noncanonical Amino Acids

scholarly journals A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling

2021 ◽  
Vol 9 ◽  
Author(s):  
Birthe Meineke ◽  
Johannes Heimgärtner ◽  
Alexander J. Craig ◽  
Michael Landreh ◽  
Lindon W. K. Moodie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Bioorthogonal Chemistry ◽  
Bioorthogonal Reaction ◽  
Noncanonical Amino Acids ◽  
Selective Reactivity ◽  
Direct Incorporation ◽  
Chelating Groups ◽  
Amber Suppression

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

scholarly journals Genetic encoding of non-natural amino acids in Drosophila melanogaster Schneider 2 cells

2010 ◽  
Vol 19 (3) ◽  
pp. 440-448 ◽  
Author(s):  
Takahito Mukai ◽  
Motoaki Wakiyama ◽  
Kensaku Sakamoto ◽  
Shigeyuki Yokoyama
Keyword(s):  
Amino Acids ◽  
Drosophila Melanogaster ◽  
Genetic Encoding ◽  
Natural Amino Acids
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