Incorporation of Unnatural Amino Acids into Cytochrome c3 and Specific Viologen Binding to the Unnatural Amino Acid

ChemBioChem ◽  
2006 ◽  
Vol 7 (12) ◽  
pp. 1853-1855 ◽  
Author(s):  
Shin Iida ◽  
Noriyuki Asakura ◽  
Kenji Tabata ◽  
Ichiro Okura ◽  
Toshiaki Kamachi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
Cytochrome C3

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 High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

2013 ◽  
Vol 80 (5) ◽  
pp. 1718-1725 ◽  
Author(s):  
Masaomi Minaba ◽  
Yusuke Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Expression System ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Translational Efficiency ◽  
Site Specific ◽  
Content Type

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

scholarly journals Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis

2007 ◽  
Vol 283 (3) ◽  
pp. 1525-1533 ◽  
Author(s):  
Shixin Ye ◽  
Caroline Köhrer ◽  
Thomas Huber ◽  
Manija Kazmi ◽  
Pallavi Sachdev ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
G Protein ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
G Protein Coupled Receptors ◽  
Calcium Flux ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-l-phenylalanine (Acp) and p-benzoyl-l-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to ∼50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5–2 μg/107 cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Gregory M. Olenginski ◽  
Juliana Piacentini ◽  
Darcy R. Harris ◽  
Nicolette A. Runko ◽  
Brianna M. Papoutsis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Photophysical Properties ◽  
Unnatural Amino Acid ◽  
Green Fluorescent ◽  
Absorbance Band ◽  
Protein Constructs

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

scholarly journals Development and Testing of Force Field Parameters for Phenylalanine and Tyrosine Derivatives

2020 ◽  
Vol 7 ◽  
Author(s):  
Xiaowen Wang ◽  
Wenjin Li
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Force Field ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
Tyrosine Derivatives ◽  
Force Field Parameters ◽  
Enzyme Functions ◽  
Theoretical Analyses ◽  
Good Agreement

Theoretical analyses are valuable for the exploration of the effects of unnatural amino acids on enzyme functions; however, many necessary parameters for unnatural amino acids remain lacking. In this study, we developed and tested force field parameters compatible with Amber ff14SB for 18 phenylalanine and tyrosine derivatives. The charge parameters were derived from ab initio calculations using the RESP fitting approach and then adjusted to reproduce the benchmark relative energies (at the MP2/TZ level) of the α- and β-backbones for each unnatural amino acid dipeptide. The structures optimized under the proposed force field parameters for the 18 unnatural amino acid dipeptides in both the α- and β-backbone forms were in good agreement with their QM structures, as the average RMSD was as small as 0.1 Å. The force field parameters were then tested in their application to seven proteins containing unnatural amino acids. The RMSDs of the simulated configurations of these unnatural amino acids were approximately 1.0 Å compared with those of the crystal structures. The vital interactions between proteins and unnatural amino acids in five protein–ligand complexes were also predicted using MM/PBSA analysis, and they were largely consistent with experimental observations. This work will provide theoretical aid for drug design involving unnatural amino acids.

scholarly journals Optimization of Expanded Genetic Codes via Genetic Algorithms

2018 ◽  
Author(s):  
Maísa de Carvalho Silva ◽  
Lariza Laura De Oliveira ◽  
Renato Tinós
Keyword(s):  
Amino Acids ◽  
Genetic Algorithms ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Genetically Modified Organisms ◽  
Fitness Function ◽  
Unnatural Amino Acid ◽  
Standard Genetic Code ◽  
Genetic Codes

In the last decades, researchers have proposed the use of genetically modified organisms that utilize unnatural amino acids, i.e., amino acids other than the 20 amino acids encoded in the standard genetic code. Unnatural amino acids have been incorporated into genetically engineered organisms for the development of new drugs, fuels and chemicals. When new amino acids are incorporated, it is necessary to modify the standard genetic code. Expanded genetic codes have been created without considering the robustness of the code. The objective of this work is the use of genetic algorithms (GAs) for the optimization of expanded genetic codes. The GA indicates which codons of the standard genetic code should be used to encode a new unnatural amino acid. The fitness function has two terms; one for robustness of the new code and another that takes into account the frequency of use of amino acids. Experiments show that, by controlling the weighting between the two terms, it is possible to obtain more or less amino acid substitutions at the same time that the robustness is minimized.

Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

Activity of yeast d-amino acid oxidase on aromatic unnatural amino acids

2008 ◽  
Vol 50 (2-4) ◽  
pp. 93-98 ◽  
Author(s):  
Antonio Caligiuri ◽  
Paola D’Arrigo ◽  
Elena Rosini ◽  
Giuseppe Pedrocchi-Fantoni ◽  
Davide Tessaro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Acid Oxidase ◽  
Amino Acid Oxidase

scholarly journals Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

2019 ◽  
Author(s):  
Xinxin Zheng ◽  
Yinglu Cui ◽  
Tao Li ◽  
Ruifeng Li ◽  
Lu Guo ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Catalytic Mechanism ◽  
Chiral Amines ◽  
Branched Chain Amino Acid ◽  
Chiral Amino Acids ◽  
Branched Chain ◽  
Substrate Spectrum

AbstractAminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity and no requirement of external addition of cofactor. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can reversibly catalyse branched-chain amino acids (BCAAs), including L-valine, L-leucine, and L-isoleucine, with α-ketoglutaric acid to form the corresponding ketonic acids and L-glutamic acid. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 °C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum, and showed remarkably high activity with L-leucine, L-valine, L-isoleucine and L-methionine with activities of 105 U/mg, 127 U/mg, 115 U/mg and 98 U/mg, respectively. Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. To analyse the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Finally, conjugated with the ornithine aminotransferase (OrnAT) from Bacillus subtilis, the coupled system was applied to the preparation of L-tert-leucine with 83% conversion, which provided an approximately 2.7-fold higher yield than the single BCAT reaction.IMPORTANCEDespite the enormous potential of BCATs, the vast majority of enzymes still lack suitably broad substrate scope and activity, thus new sources and novel enzymes are currently being investigated. Here, we described a previously uncharacterized PsBCAT, which showed a surprisingly wide substrate range and was more active towards BCAAs. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. Based on the determined crystal structure, we found some differences in the organization of the substrate binding cavity, which may influence the substrate specificity of the enzyme. Moreover, we demonstrated efficient biocatalytic asymmetric synthesis of L-tert-leucine using a coupling system, which can be used to remove the inhibitory by-product, and to shift the reaction equilibrium towards the product formation. In summary, the structural and functional characteristics of PsBCAT were analysed in detail, and this information will play an important role in the synthesis of chiral amino acids and will be conducive to industrial production of enantiopure chiral amines by aminotransferase.

scholarly journals Development of natural and unnatural amino acid delivery systems against hookworm infection

2020 ◽  
Vol 3 (1) ◽  
pp. 471-482 ◽  
Author(s):  
Stacey Bartlett ◽  
Mariusz Skwarczynski ◽  
Xin Xie ◽  
Istvan Toth ◽  
Alex Loukas ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Cell Epitope ◽  
Unnatural Amino Acid ◽  
Nippostrongylus Brasiliensis ◽  
Peptide Fragments ◽  
Specific Pathogen ◽  
Hydrophobic Amino Acids ◽  
Natural Amino Acids

Peptide-based vaccines consist of short antigen fragments derived from a specific pathogen. Alone, these peptide fragments are poorly or non-immunogenic; however, when incorporated into a proper delivery system, they can trigger strong immune responses. To eliminate the need for toxic and often ineffective oral adjuvants, we designed single molecule-based self-adjuvating vaccines against hookworms using natural and unnatural hydrophobic amino acids. Two vaccine conjugates were synthesized, consisting of B-cell epitope p3, derived from the hookworm Na-APR-1 protein; universal T-helper peptide P25; and either double copies of unnatural lipoamino acid (2-amino-D,L-eicosanoic acid), or ten copies of the natural amino acid leucine. After challenge with the model hookworm, Nippostrongylus brasiliensis, mice orally immunized with the conjugates, but without adjuvant, generated antibody responses against the hookworm epitope, resulting in significantly reduced worm and egg burdens compared to control mice. We have demonstrated that vaccine nanoparticles composed exclusively of natural amino acids can be effective even when administered orally.

Sign in / Sign up

Export Citation Format

Share Document