scholarly journals Fluorescence Enhancement of Fluorescent Unnatural Streptavidin by Binding of a Biotin Analogue with Spacer Tail and Its Application to Biotin Sensing

2014 ◽  
Vol 2014 ◽  
pp. 1-6
Author(s):  
Xianwei Zhu ◽  
Hiroaki Shinohara
Keyword(s):  
Amino Acid ◽  
Fluorescence Quenching ◽  
Fluorescence Intensity ◽  
Fluorescence Enhancement ◽  
Binding Protein ◽  
Unnatural Amino Acid ◽  
Competitive Binding ◽  
Binding Reaction ◽  
Biosensing System

We designed a novel molecular biosensing system for the detection of biotin, an important vitamin by the combination of fluorescent unnatural streptavidin with a commercialized biotin-(AC5)2-hydrazide. A fluorescent unnatural amino acid, BODIPY-FL-aminophenylalanine (BFLAF), was position-specifically incorporated into Trp120 of streptavidin by four-base codon method. Fluorescence of the Trp120BFLAF mutant streptavidin was enhanced by the addition of biotin-(AC5)2-hydrazide with the concentration dependent, whereas fluorescence enhancement was not observed at all by the addition of natural biotin. It was considered that the spacer tail of biotin-(AC5)2-hydrazide may disturb the fluorescence quenching of the Trp120BFLAF by Trp79 and Trp108 of the neighbor subunit. Therefore, biotin sensing was carried out by the competitive binding reaction of biotin-(AC5)2-hydrazide and natural biotin to the fluorescent mutant streptavidin. The fluorescence intensity decreased by increasing free biotin concentration. The result suggested that molecular biosensor for small ligand could be successfully designed by the pair of fluorescent mutant binding protein and ligand analogue.

scholarly journals Development of a Novel Q-body Using an In Vivo Site-Specific Unnatural Amino Acid Incorporation System

Sensors ◽  
2018 ◽  
Vol 18 (8) ◽  
pp. 2519
Author(s):  
Yoichi Kurumida ◽  
Nobuhiro Hayashi
Keyword(s):  
Amino Acid ◽  
Fluorescence Intensity ◽  
Unnatural Amino Acid ◽  
Fluorescent Dye ◽  
Amino Acid Residues ◽  
Amino Acid Incorporation ◽  
System A ◽  
Acid Incorporation ◽  
Tryptophan Residues

A Q-body capable of detecting target molecules in solutions could serve as a simple molecular detection tool. The position of the fluorescent dye in a Q-body affects sensitivity and therefore must be optimized. This report describes the development of Nef Q-bodies that recognize Nef protein, one of the human immunodeficiency virus (HIV)’s gene products, in which fluorescent dye molecules were placed at various positions using an in vivo unnatural amino acid incorporation system. A maximum change in fluorescence intensity of 2-fold was observed after optimization of the dye position. During the process, some tryptophan residues of the antibody were found to quench the fluorescence. Moreover, analysis of the epitope indicated that some amino acid residues of the antigen located near the epitope affected the fluorescence intensity.

An Unnatural Amino Acid that Mimics a Tripeptide β-Strand and Forms β-Sheetlike Hydrogen-Bonded Dimers [J. Am. Chem. Soc.2000,122, 7654-7661]. 

2001 ◽  
Vol 123 (7) ◽  
pp. 1545-1546
Author(s):  
James S. Nowick ◽  
De Michael Chung ◽  
Kalyani Maitra ◽  
Santanu Maitra ◽  
Kimberly D. Stigers ◽  
...  
Keyword(s):  
Amino Acid ◽  
Unnatural Amino Acid ◽  
Hydrogen Bonded

Nγ‐Hydroxyasparagine: a Multifunctional Unnatural Amino Acid That is a Good P1 Substrate of Asparaginyl Peptide Ligases

2021 ◽  
Author(s):  
Chuan-Fa Liu ◽  
Yiyin Xia ◽  
Janet To ◽  
Ning-Yu Chan ◽  
Side Hu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Unnatural Amino Acid

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.

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