Efficient Asymmetric Synthesis of Tryptophan Analogues Having Useful Photophysical Properties

Two new fluorescent probes of protein structure and dynamics have been prepared by concise asymmetric syntheses using the Schöllkopf chiral auxiliary. The site-specific incorporation of one probe into dihydrofolate reductase is reported. The utility of these tryptophan derivatives lies in their absorption and emission maxima which differ from those of tryptophan, as well as in their large Stokes shifts and high molar absorptivities

Detection of Dihydrofolate Reductase Conformational Change by FRET Using Two Fluorescent Amino Acids

Two fluorescent amino acids, including the novel fluorescent species 4-biphenyl-l-phenylalanine (<b>1</b>), have been incorporated at positions 17 and 115 of dihydrofolate reductase (DHFR) to enable a study of conformational changes associated with inhibitor binding. Unlike most studies involving fluorescently labeled proteins, the fluorophores were incorporated into the amino acid side chains, and both probes [<b>1</b> and l-(7-hydroxycoumarin-4-yl)­ethylglycine (<b>2</b>)] were smaller than fluorophores typically used for such studies. The DHFR positions were chosen as potentially useful for Förster resonance energy transfer (FRET) measurements on the basis of their estimated separation (17–18 Å) and the expected change in distance along the reaction coordinate. Also of interest was the steric accessibility of the two sites: Glu17 is on the surface of DHFR, while Ile115 is within a folded region of the protein. Modified DHFR I (<b>1</b> at position 17; <b>2</b> at position 115) and DHFR II (<b>2</b> at position 17; <b>1</b> at position 115) were both catalytically competent. However, DHFR II containing the potentially rotatable biphenylphenylalanine moiety at sterically encumbered position 115 was significantly more active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected excitation of <b>1</b>, energy transfer to <b>2</b>, and emission by <b>2</b> at 450 nm. However, the energy transfer was substantially more efficient in DHFR II. The effect of inhibitor binding was also measured. Trimethoprim mediated concentration-dependent diminution of the emission observed at 450 nm for DHFR II but not for DHFR I. These findings demonstrate that amino acids containing small fluorophores can be introduced into DHFR with minimal disruption of function and in a fashion that enables sensitive monitoring of changes in DHFR conformation

Incorporation of Phosphorylated Tyrosine into Proteins: <i>In Vitro</i> Translation and Study of Phosphorylated IκB‑α and Its Interaction with NF-κB

Phosphorylated proteins play important roles in the regulation of many different cell networks. However, unlike the preparation of proteins containing unmodified proteinogenic amino acids, which can be altered readily by site-directed mutagenesis and expressed <i>in vitro</i> and <i>in vivo</i>, the preparation of proteins phosphorylated at predetermined sites cannot be done easily and in acceptable yields. To enable the synthesis of phosphorylated proteins for <i>in vitro</i> studies, we have explored the use of phosphorylated amino acids in which the phosphate moiety bears a chemical protecting group, thus eliminating the negative charges that have been shown to have a negative effect on protein translation. Bis-<i>o</i>-nitrobenzyl protection of tyrosine phosphate enabled its incorporation into DHFR and IκB-α using wild-type ribosomes, and the elaborated proteins could subsequently be deprotected by photolysis. Also investigated in parallel was the re-engineering of the 23S rRNA of Escherichia coli, guided by the use of a phosphorylated puromycin, to identify modified ribosomes capable of incorporating unprotected phosphotyrosine into proteins from a phosphotyrosyl-tRNA_CUA by UAG codon suppression during <i>in vitro</i> translation. Selection of a library of modified ribosomal clones with phosphorylated puromycin identified six modified ribosome variants having mutations in nucleotides 2600–2605 of 23S rRNA; these had enhanced sensitivity to the phosphorylated puromycin. The six clones demonstrated some sequence homology in the region 2600–2605 and incorporated unprotected phosphotyrosine into IκB-α using a modified gene having a TAG codon in the position corresponding to amino acid 42 of the protein. The purified phosphorylated protein bound to a phosphotyrosine specific antibody and permitted NF-κB binding to a DNA duplex sequence corresponding to its binding site in the IL-2 gene promoter. Unexpectedly, phosphorylated IκB-α also mediated the exchange of exogenous DNA into an NF-κB–cellular DNA complex isolated from the nucleus of activated Jurkat cells

Fluorescent Biphenyl Derivatives of Phenylalanine Suitable for Protein Modification

In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (<i>ec</i>DHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of <i>ec</i>DHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of <i>ec</i>DHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of <i>ec</i>DHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational flexibility about the biphenyl bonds

Fluorescent Biphenyl Derivatives of Phenylalanine Suitable for Protein Modification

In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (<i>ec</i>DHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of <i>ec</i>DHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of <i>ec</i>DHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of <i>ec</i>DHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational flexibility about the biphenyl bonds

Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro

Plasmids containing 23S rRNA randomized at positions 2057–2063 and 2502–2507 were introduced into <i>Escherichia coli</i>, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puro­mycin, a natural product which acts as an analogue of the 3′-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puro­­mycin derivative in this study contained the dipeptide <i>p</i>-methoxy­phenyl­alanyl­glycine, implying the ability of the modified ribosomes in clones sensitive to this puro­mycin analogue to recognize dipeptides. Several clones inhibited by the puro­mycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (<b>2</b>) and Phe-Gly (<b>3</b>)), as well as a thiolated dipeptide analogue (<b>4</b>) and a fluorescent oxazole (<b>5</b>) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide <b>4</b> and a 7-methoxy­coumarin fluoro­phore was found to undergo fluorescence quenching. Introduction of the oxazole fluoro­phore <b>5</b> into dihydro­folate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues

Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes

Described herein are the synthesis and photophysical characterization of a library of aryl-substituted oxazole- and thiazole-based dipeptidomimetic analogues, and their incorporation into position 66 of green fluorescent protein (GFP) in lieu of the natural fluorophore. These fluorescent analogues resemble the fluorophore formed naturally by GFP. As anticipated, the photophysical properties of the analogues varied as a function of the substituents at the <i>para</i> position of the phenyl ring. The fluorescence emission wavelength maxima of compounds in the library varied from ∼365 nm (near-UV region) to ∼490 nm (visible region). The compounds also exhibited a large range of quantum yields (0.01–0.92). The analogues were used to activate a suppressor tRNA_CUA and were incorporated into position 66 of GFP using an <i>in vitro</i> protein biosynthesizing system that employed engineered ribosomes selected for their ability to incorporate dipeptides. Four analogues with interesting photophysical properties and reasonable suppression yields were chosen, and the fluorescent proteins (FPs) containing these fluorophores were prepared on a larger scale for more detailed study. When the FPs were compared with the respective aminoacyl-tRNAs and the actual dipeptide analogues, the FPs exhibited significantly enhanced fluorescence intensities at the same concentrations. Part of this was shown to be due to the presence of the fluorophores as an intrinsic element of the protein backbone. There were also characteristic shifts in the emission maxima, indicating the environmental sensitivity of these probes. Acridon-2-ylalanine and oxazole <b>1a</b> were incorporated into positions 39 and 66 of GFP, respectively, and were shown to form an efficient Förster resonance energy transfer (FRET) pair, demonstrating that the analogues can be used as FRET probes

Enhanced Binding Affinity for an i‑Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids

Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the <i>BCL2</i> i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase <b>2</b> into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson–Crick interaction of <b>2</b> and G14. In comparison, the introduction of uracil nucleobase <b>3</b> had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (<b>1</b> and <b>4</b>) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing <b>1</b> exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing <b>4</b> bound less tightly to the DNA substrate. Finally, the modified protein containing <b>1</b> in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing <b>1</b> both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA–protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1–DNA interaction, as well as the ability of nucleobase amino acid <b>1</b> to stabilize RRM1–DNA interaction

Protein Synthesis with Ribosomes Selected for the Incorporation of β‑Amino Acids

In an earlier study, β³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into <i>Escherichia coli</i> dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate β-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the β³-puromycin employed for ribosome selection influences the regio- and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNA_CUA activated with each of four methyl-β-alanine isomers (<b>1–4</b>). The modified ribosomes were found to incorporate each of the four isomeric methyl-β-alanines into DHFR but exhibited a preference for incorporation of 3­(<i>S</i>)-methyl-β-alanine (β-mAla; <b>4</b>), i.e., the isomer having the same regio- and stereochemistry as the O-methylated β-tyrosine moiety of β³-puromycin. Also conducted were a selection of clones that are responsive to β²-puromycin and a demonstration of reversal of the regio- and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included <i>in silico</i> prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3­(<i>S</i>)-methyl-β-alanine (β-mAla; <b>4</b>) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties

Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA–Protein Interaction

Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of <i>Escherichia coli</i> dihydrofolate reductase (<i>ec</i>DHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (<b>A</b>) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)­ethylglycine (HCO, <b>D</b>) at position 17. Further, 6-CNTrp (<b>A</b>) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving <b>A</b> as a fluorescence donor and benzo­[<i>g</i>]­quinazoline-2,4-(1<i>H</i>,3<i>H</i>)-dione 2′-deoxyriboside (T_f) or 4-aminobenzo­[<i>g</i>]­quinazoline-2-one 2′-deoxyriboside (C_f) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with T_f), or the domain-specific association between hnRNP LL and the <i>BCL2</i> i-motif DNA (labeled with C_f). Thus, the non-natural amino acid could be used as a FRET partner for studying protein–nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (<b>A</b>) as a fluorescence donor for the study of protein conformational events