Fluorescent Non-natural Amino Acid Research Articles

Double-Fluorescent-Labeled Single-Chain Antibodies Showing Antigen-Dependent Fluorescence Ratio Change

Abstract Fluorescence ratio probes are useful tools for quantitative detection of target molecules even if the concentration of probe molecules is unknown. However, a general and widely applicable method for design and synthesis of fluorescence ratio probes has never been established. Here, we developed double-labeled single-chain antibody fragment (scFv) derivatives showing antigen-dependent fluorescence ratio change based on fluorescence resonance energy transfer (FRET) and antigen-dependent fluorescence quenching. Double-labeled scFvs were synthesized by incorporating fluorescent nonnatural amino acids labeled with TAMRA and Rhodamine Green (RhG) as a FRET donor and acceptor pair into N- and C-termini of scFv, respectively, using four-base and amber codons in a cell-free translation system. The resulting double-labeled antibody fragments showed significant fluorescence ratio change upon the antigen-binding. This result was explained by FRET occurring from RhG to TAMRA but TAMRA being quenched by Trp residues in the absence of antigen. The binding of the antigen canceled the quenching of TAMRA without changing FRET efficiency. Double-labeled antibody fragments developed here will be useful as diagnostic and imaging tools.

Incorporation of Fluorescent Nonnatural Amino Acid into Sialic Acid-Binding Lectin for Fluorescence Detection of Ligand-Binding

Abstract Lectins are important not only as biological functional molecules but as useful probes for glycan analysis. Here, we developed fluorescent-labeled lectins showing fluorescence changes upon ligand-binding. BODIPYFL-aminophenylalanine was incorporated at eight respective positions near the ligand-binding site of a sialic acid-binding lectin in response to UAG codon in a cell-free translation system. Fluorescence imaging of SDS-PAGE gel indicated the successful expression of the fluorescent-labeled lectins containing BODIPYFL-linked amino acid. Fluorescence spectral measurements revealed that the fluorescent-labeled lectins showed significant increases in the fluorescence intensity upon the binding of sialyllactose for all the incorporation positions. Particularly, the fluorescence intensity increased 3.7-fold for the labeled lectin at Thr38 position. The increase in the fluorescence intensity could occur because the fluorescence of BODIPYFL is quenched by amino acid residues located near the ligand-binding site in the absence of ligand, but the ligand-binding reduces the fluorescence quenching by removing the BODIPYFL moiety from the ligand-binding site. The W31F substitution decreased the fluorescence change of the labeled lectin, suggesting that the Trp31 residue may partially contribute to the ligand-dependent quenching. The present strategy will be utilized for producing various fluorescent-labeled lectin probes showing ligand-dependent fluorescence change.

Synthesis of Novel BRET/FRET Protein Probes Containing Light-Emitting Proteins and Fluorescent Nonnatural Amino Acids

Abstract Novel light-emitting protein probes were designed and synthesized by incorporating fluorescent nonnatural amino acids and fusing light-emitting proteins. Gaussia luciferase was fused as a BRET donor to the C-terminus of maltose-binding protein, and BODIPY558–aminophenylalanine was incorporated into Tyr210, which is located near the maltose-binding site of maltose-binding protein in response to a four-base codon (CGGG). In addition to the luminescence of luciferase, the double-labeled protein showed the emission of BODIPY558, which was strongly quenched by a neighboring tryptophan in the absence of maltose but was recovered in the presence of maltose. The emission intensity ratio of luciferase and BODIPY558 increased 4.0-fold upon the addition of maltose. Similar ligand-dependent emission changes were observed for a translation reaction mixture expressing the double-labeled protein, indicating that the BRET protein probe was detectable in the presence of excess fluorescent molecules. BODIPY558-containing maltose-binding protein fused with GFP in place of luciferase was also synthesized and showed the FRET and maltose-dependent fluorescence of BODIPY558. Novel BRET/FRET protein probes synthesized by fusing light-emitting proteins and incorporating fluorescent nonnatural amino acids will become valuable tools for the bioimaging and diagnostic detection of specific ligands.

Biosynthesis of a Fluorescent Protein with Extreme Pseudo-Stokes Shift by Introducing a Genetically Encoded Non-Natural Amino Acid outside the Fluorophore

A novel kind of fluorescent protein relying on the intramolecular interplay between two different fluorophores, one of chemical origin and one of biological origin, was developed. The fluorescent non-natural amino acid l-(7-hydroxycoumarin-4-yl)ethylglycine was site-specifically incorporated into the recombinant enhanced cyan fluorescent protein (eCFP) at a permissible surface position ∼20 Å away from the protein fluorophore using amber suppression in Escherichia coli with an engineered cognate Methanococcus jannaschii tRNA synthetase. The resulting eCFP(Cou) exhibited almost quantitative intramolecular Förster resonance energy transfer (FRET) between its two fluorophores, showing brilliant cyan emission at 476 nm upon excitation in the near-UV at 365 nm (a wavelength easily accessible via conventional laboratory UV sources), in contrast to its natural counterpart. Thus, this fluorescent protein with unprecedented spectroscopic properties reveals an extreme apparent Stokes shift of ∼110 nm between the absorption wavelength of the coumaryl group and the emission wavelength of eCFP.

Biosynthesis of proteins containing modified lysines and fluorescent labels using non-natural amino acid mutagenesis

The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins.

Photophysics of a fluorescent non-natural amino acid: p-Cyanophenylalanine

The non-natural amino acid p-cyanophenylalanine (Phe CN) has recently emerged as a useful fluorescent probe of proteins; however, its photophysical properties have not been systematically examined. Herein, we measure the fluorescence quantum yield and the fluorescence lifetime of Phe CN in a series of solvents. It is found that the fluorescence lifetime of Phe CN shows a linear dependence on the Kamlet–Taft parameter α of the protic solvents used, indicating that the solute-solvent hydrogen bonding interactions mediate the non-radiative decay rate. Thus, results of this study provide a basis for quantitative application of Phe CN fluorescence in protein conformational studies.

Incorporation of fluorescent non-natural amino acids into N-terminal tag of proteins in cell-free translation and its dependence on position and neighboring codons

Fluorescence labeling is a useful technique for structural and functional analyses of proteins. In a previous study, we developed position-specific incorporation of visible wavelength fluorescent non-natural amino acids carrying relatively small BODIPY fluorophores into proteins, in response to a four-base codon CGGG. Here, we have expanded this position-specific fluorescence labeling method to include relatively large non-natural amino acids carrying photostable rhodamine dyes. TAMRA-linked aminophenylalanine was synthesized and attached to a tRNA having a four-base anticodon, and its incorporation into proteins was examined in an Escherichia coli cell-free translation system. TAMRA-labeled amino acids were successfully incorporated into proteins, although incorporation was allowed only at the N-terminal region. Insertion of two codons encoding a stop codon in the +1 frame before four-base codon suppressed the expression of non-labeled proteins that may have been produced by spontaneous +1 frameshift upstream of the four-base codon. Alternation of the incorporation position affected the expression level of the TAMRA-labeled protein. In addition, alternation of upstream and downstream codons affected the efficiency and accuracy of TAMRA-labeled amino acid incorporation. Based on these results, a novel tag peptide was developed; it contained the four-base codon at the 9th position with optimized upstream and downstream codons. This tag peptide was effective for producing proteins with various fluorescent labels at the N-terminal region.

Screening of amber suppressor tRNAs suitable to introduce nonnatural amino acids into proteins by real-time monitoring of cell-free translation

Incorporation of nonnatural amino acids into proteins is a useful technique to analyze protein structure and function. We have reported that amber suppressor tRNAs suitable for efficient and specific incorporation of nonnatural amino acids into proteins can be obtained by screening a wide variety of naturally occurring tRNAs in an E. coli. cell-free translation system. The amber suppressor activity of the tRNAs was evaluated by incorporation of a fluorescent nonnatural amino acid and fluorescent SDS-PAGE analysis of cell-free translation products, though the SDS-PAGE was troublesome and time-consuming. In this research, we developed an alternative method for the screening of amber suppressor tRNAs by real-time measurement of fluorescence resonance energy transfer (FRET) from GFP to BODIPY558-linked nonnatural amino acid, which was incorporated into the N-terminus of GFP by amber suppressor tRNAs. Using this method, we demonstrated that the screening of the amber suppressor activity of various prokaryotic Trp tRNAs was performed in a high-throughput manner.

Comprehensive screening of amber suppressor tRNAs suitable for incorporation of non-natural amino acids in a cell-free translation system

Incorporation of non-natural amino acids into proteins in response to amber or four-base codons is a useful technology for protein research. In the case of the amber codon, however, release factor 1 can competitively decode the same codon, and consequently inhibit the incorporation of non-natural amino acids. To improve amber codon-mediated incorporation, we carried out a comprehensive screening of amber suppressor tRNAs derived from all tRNAs encoded in the genomes of Escherichia coli K12 and Mycoplasma capricolum. The amber suppressor tRNAs were synthesized from synthetic genes, aminoacylated with a fluorescent non-natural amino acid, and added to an E. coli cell-free translation system. Fluorescent SDS–PAGE analysis indicated that Trp tRNAs showed high suppressor activity in both organisms. Further mutagenesis and screening revealed that M. capricolum Trp 1 tRNA with G1C72A73 mutation is the most suitable for efficient and specific incorporation of non-natural amino acids into proteins in response to the amber codon.

Stereoselective Syntheses of Fluorescent Non-Natural Aromatic Amino Acids Based on Asymmetric Michael Additions

Four fluorescent non-natural aromatic amino acids have been synthesized based on a key stereoselective Michael addition reaction. S-1-phenylethylamine was employed as both the source of amine and the stereoselectivity controller. The overall yields were moderate (30-50%). Fluorescent properties of some of the fluorophores were also investigated. It was found that compounds with a dimethylamino group bonded to the aromatic ring display intramolecular charge transfer fluorescence.

A novel fluorescent nonnatural amino acid that can be incorporated into a specific position of streptavidin.

A novel highly fluorescent nonnatural amino acid (beta-(N-methylanthraniloyl)-L-alpha,beta-diaminopropionic acid; nmaDap) was incorporated at the 120th position of streptavidin. The position was directed by a CGGG/CCCG four-base codon/anticodon pair. The incorporation efficiency and the biotin-binding ability of the nmaDap mutant were compared to those of beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap).

Highly Sensitive Fluorescent Nonnatural Amino Acids That Can Be Incorporated into Specific Positions of Streptavidin

Highly Sensitive Fluorescent Nonnatural Amino Acids That Can Be Incorporated into Specific Positions of Streptavidin

A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe

A small and highly fluorescent non-natural amino acid that contains an anthraniloyl group (atnDap) was incorporated into various positions of streptavidin. The positions were directed by a CGGG/CCCG four-base codon/anticodon pair. The non-natural mutants were obtained in excellent yields and some of them retained strong biotin-binding activity. The fluorescence wavelength as well as the intensity of the anthraniloyl group at position 120 were sensitive to biotin binding. These unique properties indicate that the atnDap is the most suitable non-natural amino acid for a position-specific fluorescent labeling of proteins that is highly sensitive to microenvironmental changes.

Site-Directed Incorporation of Fluorescent Nonnatural Amino Acids into Streptavidin for Highly Sensitive Detection of Biotin

Fluorescent nonnatural amino acids were incorporated into specific positions of streptavidin. The positions of the nonnatural amino acids were directed by a CGGG/CCCG four-base codon/anticodon pair. The nonnatural mutants with a single 2-anthrylalanine at the 22nd, 43rd, 54th, and 120th positions, respectively, were found to bind biotin, indicating that the mutants retained active conformation. The fluorescence intensities of the anthryl groups were relatively insensitive to the positions and the biotin binding when excited at 265 nm. When the anthryl group at the 120th position was excited through energy transfer from tryptophan units, the fluorescence intensity markedly decreased with biotin binding, because of a suppression of the energy transfer. Amino acids carrying 7-methoxycoumarine fluorophore were also incorporated at the 120th position. Their fluorescence quantum yields were very sensitive to the biotin binding. The high sensitivity of the coumarine-labeled streptavidin exemplifies potential applications of fluorescent nonnatural mutants for detecting specific molecules at very low concentrations.