Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing

A new fluorescent amino acid, L-2-acridonylalanine, was incorporated into proteins at specific positions using 4-base codon/anticodon strategy. The efficiency of the incorporation was high enough to obtain enough quantities of the mutants. The acridonyl group was highly fluorescent when it was excited at the wavelengths of blue-lasers and was highly photodurable compared with conventional fluorophores often used for biological analyses. The fluorescence intensity was sensitive to small changes in the polarity of the environment. When the nonnatural amino acid was incorporated into specific positions of streptavidin, the mutant protein worked as a fluorescent sensor to biotin. Similarly, when the amino acid was incorporated into camel single-chain antibody, the mutant protein sensitively responded to the antigen molecule. The high incorporation efficiency, the high photodurability, the excitability with blue-lasers, and high sensitivity to the environment make the acridonylalanine as the promising fluorescent amino acid for sensing small molecules when incorporated into specific positions of various antibodies, receptors, and enzymes.     

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.     

Application of photoactivatable fluorescent active-site directed probes to serine-containing enzymes

A photoactivatable fluorescent anthraniloyl group has been directed to the active-site serine group of alpha-chymotrypsin and trypsin. The acylated derivatives are nonfluorescent until irradiated. When activated by light a highly reactive nitrene is generated which is capable of covalent insertion into the protein matrix. The resultant insertion product of this photolysis is a highly fluorescent reporter group which has little rotational mobility and is cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked enzyme and deacylated enzyme were determined. These measurements show that little relaxation occurs on the nanosecond time scale for the cross-linked enzyme, but upon deacylation of the serine increased dipolar relaxation of the protein with the attached reporter group is observed. The use of these active-site directed photoactivatable fluorescent probes can be extended to probe the active-site structure of complex enzymes and conformational dynamics of active-site regions in proteins and to serve as potential functional site labels in fluorescence resonance energy transfer measurements.     

Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids

Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro.     

N-anthraniloylation converts peptide p-nitroanilides into fluorogenic substrates of proteases without loss of their chromogenic properties

By simple substitution of an N-acyl group for the anthraniloyl(o-aminobenzoyl) group, chromogenic p-nitroanilide substrates are converted into highly sensitive fluorogenic substrates of proteases. The fluorescence of the anthraniloyl group is completely quenched by the p-nitroanilide moiety in the intact substrates and is released during their enzymatic hydrolysis. The approach is exemplified by the synthesis of anthraniloyl-Phe p-nitroanilide, anthraniloyl-Lys p-nitroanilide, and anthraniloyl-Gly-Gly-Phe p-nitroanilide as substrates for chymotrypsin, trypsin, and alkaline mesentericopeptidase, respectively. The kinetic parameters of these substrates are slightly better than those of similar derivatives bearing other acyl groups, suggesting that the enhanced sensitivity is completely due to the method of measurement. Since the conversion does not affect the chromogenic properties of the substrates, the same compounds can be used as usual p-nitroanilide substrates as well.     

FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids

We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.     

Incorporation of non-natural amino acids into proteins

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.     

An on-bead assay for the identification of non-natural peptides targeting the androgen receptor-cofactor interaction

An efficient and rapid on-bead screening method was established to identify non-natural peptides that target the Androgen Receptor-cofactor interaction. Binding of the Androgen Receptor ligand binding domain to peptide sequences displayed on beads in a One-Bead-One-Compound format could be screened using fluorescence microscopy. The method was applied to generate and screen both a focussed and a random peptide library. Resynthesis of the peptide hits allowed for the verification of the affinity of the selected peptides for the Androgen Receptor in a competitive fluorescence polarization assay. For both libraries strong Androgen Receptor binding peptides were found, both with non-natural and natural amino acids. The peptides identified with natural amino acids showed great similarity in terms of preferred amino acid sequence with peptides previously isolated from biological screens, thus validating the screening approach. The non-natural peptides featured important novel chemical transformations on the relevant hydrophobic amino acid positions interacting with the Androgen Receptor. This screening approach expands the molecular diversity of peptide inhibitors for nuclear receptors.     

Fluorescent and photoactivatable fluorescent derivatives of tetrodotoxin to probe the sodium channel of excitable membranes

Fluorescent and photoactivatable fluorescent derivatives of tetrodotoxin (TTX) have been synthesized. N-Methylanthraniloylglycine hydrazide, anthraniloyl hydrazide, and 2-azidoanthraniloylglycine hydrazide were coupled to the carbonyl at C6 of oxidized tetrodotoxin to form stable fluorescent hydrazones. The C6 ketone can be reductively aminated with either ammonium or methylammonium acetate to form 6-amino- or 6-(methylamino)tetrodotoxin, which can then be acylated by a variety of fluorescent reagents. The biological activity, competitive binding with [3H]tetrodotoxin for the receptor on rat axonal membranes, and equilibrium binding isotherms obtained by fluorescence enhancement or anisotropy indicate that the derivatives are only about 2-5 times less active then tetrodotoxin itself. The 2-azidoanthraniloylglycine hydrazone of oxidized tetrodotoxin, when activated by light, generates a reactive nitrene which is capable of covalent insertion into the toxin receptor. The product of the photolysis is a highly fluorescent tetrodotoxin derivative which is irreversibly linked to the receptor site. The excitation and emission spectra of the fluorescent tetrodotoxin derivatives vary with solvent polarity, and this sensitivity has been used to determine the immediate environmental characteristics of the toxin binding site of the sodium channel. It is concluded that the toxin binding site is highly polar. Emission and excitation spectra reveal that radiationless energy is transferred from tryptophan residues of the receptor to the anthraniloyl group of the TTX derivatives.     

Multiple incorporation of non-natural amino acids into a single protein using tRNAs with non-standard structures

The ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein.     

Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin

The preparations of N alpha-Fmoc-3-nitro-L-tyrosine and N-Boc-anthranilic acid Dhbt ester and their application to parallel multiple column solid-phase peptide synthesis is described. A series of peptide substrates containing an anthraniloyl group at the amino terminus and a 3-nitrotyrosyl residue close to the carboxyl terminus have been synthesized. The fluorescence of the anthraniloyl group, intramolecularly quenched by the 3-nitrotyrosine, increases with cleavage of peptide bonds situated between the two groups. The quenching mechanism is of the long-range resonance energy transfer type and long peptide substrates were constructed and used for kinetic measurement on subtilisin Carlsberg and pepsin. Complete quenching was observed even with more than 20 A between the centers of the chromophores, and substrates with up to 50 A between the chromophores were synthesized. The importance of long substrates for optimal enzymatic activity was demonstrated.     

N-anthraniloyl-Ala-Ala-Phe-4-nitroanilide, a highly sensitive substrate for subtilisins.

A new substrate for subtilisins, anthraniloyl-Ala-Ala-Phe-4-nitroanilide, has been synthesized and characterized. The peptide is a fluorogenic substrate that is intramolecularly quenched without loss of its chromogenic properties and offers a possibility for double-assay kinetic analysis. The kinetic parameters determined for subtilisin Carlsberg are Km = 0.004 mM, kcat = 104 s-1, and those for subtilisin BPN' are Km = 0.020 mM, kcat = 49 s-1. The substrate is extremely sensitive for subtilisins; the specificity constants are 10-fold higher than the corresponding values for the widely used substrate, succinyl-Ala-Ala-Pro-Phe-4-nitroanilide, and 200- to 1000-fold higher than the values obtained with succinyl-Ala-Ala-Phe-4-nitroanilide. The favorable effect of the anthraniloyl group as a P4 residue in the substrate sequence Ala-Ala-Phe-4-nitroanilide was assumed to be due to an ability to stiffen S4-P4 interactions. The mechanism proposed is hydrogen bond formation between the phenol group of tyrosine-104 and the amino group of the anthraniloyl moiety. In the spectrophotometric assay with the new substrate, the lower detection limit for subtilisin Carlsberg was 1 nM.     

New indolicidin analogues with potent antibacterial activity.

Indolicidin is a 13-residue antimicrobial peptide amide, ILPWKWPWWPWRR-NH2, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two-fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR-NH2 in which Trp-residues 4,6,8,9,11 were replaced in all positions with X = a single non-natural building block; N-substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non-natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N-terminus with a hydrophobic non-natural building block. We prepared 22 analogues of indolicidin and [Phe(4,6,8,9,11)] indolicidin, 11 of each, carrying a hydrophobic non-natural building block attached to the N-terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non-natural building blocks, are promising lead structures for developing future therapeutics.     

Engineering of an Orthogonal Aminoacyl-tRNA Synthetase for Efficient Incorporation of the Non-natural Amino Acid O-Methyl-L-tyrosine using Fluorescence-based Bacterial Cell Sorting

We describe a strategy for the rapid selection of mutant aminoacyl-tRNA synthetases (aaRS) with specificity for a novel amino acid based on fluorescence-activated cell sorting of transformed Escherichia coli using as reporter the enhanced green fluorescent protein (eGFP) whose gene carries an amber stop codon (TAG) at a permissive site upstream of the fluorophore. To this end, a one-plasmid expression system was developed encoding an inducible modified Methanocaldococcus jannaschii (Mj) tyrosyl-tRNA synthetase, the orthogonal cognate suppressor tRNA, and eGFP_UAG in an individually regulatable fashion. Using this system a previously described aaRS with specificity for O-methyl-L-tyrosine (MeTyr) was engineered for 10-fold improved incorporation of the foreign amino acid by selection from a mutant library, prepared by error-prone as well as focused random mutagenesis, for MeTyr-dependent eGFP fluorescence. Applying alternating cycles of positive and negative fluorescence-activated bacterial cell sorting in the presence or in the absence, respectively, of the foreign amino acid was crucial to select for high specificity of MeTyr incorporation. The optimized synthetase was used for the preparative expression of a modified uvGFP carrying MeTyr at position 66 as part of its fluorophore. This biosynthetic protein showed quantitative incorporation of the non-natural amino acid, as determined by mass spectrometry, and it revealed a unique emission spectrum due to the altered chemical structure of its fluorophore. Our combined genetic/selection system offers advantages over earlier approaches that relied wholly or in part on antibiotic selection schemes, and it should be generally useful for the engineering and optimization of orthogonal aaRS/tRNA pairs to incorporate non-natural amino acids into recombinant proteins.     

Facile characterization of translation initiation via nonsense codon suppression.

A new strategy for studying the mechanism of translation initiation in eukaryotes has been developed. The strategy involves the use of an in vitro translation system to incorporate a non-natural fluorescent amino acid into a protein from a suppressor tRNAPheCUA misacylated with that amino acid. It is thereby possible to monitor translation initiation efficiency at an AUG codon in different contexts; this is illustrated for three constructs encoding Escherichia coli dihydrofolate reductase mRNA with different translation initiation regions. Fluorescence measurements after in vitro translation of the mRNAs in rabbit reticulocyte lysate reflected differences in the position and efficiency of translation initiation and, therefore, can be used for characterization of the translation initiation process.     

In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC.The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG₁ antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities.In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody.We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.     

A novel fluorescent probe for protein binding and folding studies: p-cyano-phenylalanine

Recently, it is has been shown that the C=N stretching vibration of a non-natural amino acid, p-cyano-phenylalanine (PheCN), could be used as an infrared reporter of local environment. Here, we further showed that the fluorescence emission of PheCN is also sensitive to solvent and, therefore, could be used as a novel optical probe for protein binding and folding studies. Moreover, we found that the fluorescence quantum yield of PheCN is nearly five times larger than that of phenylalanine and, more importantly, can be selectively excited even when other aromatic amino acids are present, thus making it a more versatile fluorophore. To test the feasibility of using PheCN as a practical fluorescent probe, we studied the binding of calmodulin (CaM) to a peptide derived from the CaM-binding domain of skeletal muscle myosin light chain kinase (MLCK). The peptide (MLCK3CN) contains a single PheCN residue and has been shown to bind to CaM with high affinity. As expected, addition of CaM into a MLCK3CN solution resulted in quenching of the PheCN fluorescence. A series of stochiometric titrations further allowed us to determine the binding affinity (Kd) of this peptide to CaM. Taken together, these results indicated that the PheCN fluorescence is sensitive to environment and could be applicable to a wide variety of biological problems.     

In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system

Position-specific incorporation of non-natural amino acids into proteins is a useful technique in protein engineering. In this study, we established a novel selection system to obtain tRNAs that show high decoding activity, from a tRNA library in a cell-free translation system to improve the efficiency of incorporation of non-natural amino acids into proteins. In this system, a puromycin-tRNA conjugate, in which the 3'-terminal A unit was replaced by puromycin, was used. The puromycin-tRNA conjugate was fused to a C-terminus of streptavidin through the puromycin moiety in the ribosome. The streptavidin-puromycin-tRNA fusion molecule was collected and brought to the next round after amplification of the tRNA sequence. We applied this system to select efficient frameshift suppressor tRNAs from a tRNA library with a randomly mutated anticodon loop derived from yeast tRNA CCCG Phe. After three rounds of the selection, we obtained novel frameshift suppressor tRNAs which had high decoding activity and good orthogonality against endogenous aminoacyl-tRNA synthetases. These results demonstrate that the in vitro selection system developed here is useful to obtain highly active tRNAs for the incorporation of non-natural amino acid from a tRNA library.     

Analysis of recognition elements for mitochondrial processing peptidase using artificial amino acids: roles of the intervening portion and proximal arginine.

We recently demonstrated, using synthetic peptides modeled on the extension peptide of malate dehydrogenase, that amino acid residues present at the proximal and distal positions relative to the cleavage site are critical determinants for the recognition of substrates by mitochondrial processing peptidase [Niidome et al. (1994) J. Biol. Chem. 269, 24719-24722). While the proximal arginine is unexceptionally located at the -2 position, the position of the distal residue varies among mitochondrial precursor proteins. Between the proximal and distal residues, proline and/or glycine are present in most mitochondrial precursor proteins, and they are considered to play a role in the specific recognition of a substrate by the peptidase. To elucidate the role of the intervening portion, we introduced a non-natural amino acid [2-(2-aminoethoxy)acetic acid] between the distal and proximal residues. We also analyzed the functional elements in the proximal arginine by replacing the residue with various arginine or lysine analogs. The results of kinetic studies indicated that the intervening portion should be flexible for efficient processing, and that the guanidino group of the proximal arginine is recognized by the peptidase through hydrogen and ionic bonds.