Proteins and peptides occur ubiquitously in organisms and play key functional roles, as structural elements and catalysts. Their major natural source is ribosomal synthesis, which produces polypeptides from 20 amino acid building blocks. Peptides containing noncanonical amino acids have long been prepared by chemical synthesis, which has provided a wealth of physiologically active compounds. Comparatively, preparing modified proteins has been more challenging. Site-directed mutagenesis provided an important advance but was initially limited to canonical amino acids. New techniques for tRNA activation with noncanonical amino acids subsequently increased the scope of site-directed mutagenesis.Our report in 2012 demonstrated that modification of bacterial ribosomes at key positions enabled the selection of ribosomes capable of introducing β-amino acids into proteins in vitro. The generality of the selection procedure was tested further. Ribosomes capable of incorporating dipeptides, conformationally constrained dipeptides, dipeptidometics with embedded fluorophores, contiguous nucleobase amino acids, and phosphorylated amino acids were successfully identified.In this Account, we focus on the application of the new technology to dramatically alter protein structure in ways that enable new strategies for understanding and altering protein function. To illustrate the robustness of the technology we have provided examples studied in vitro and in cellulo. The first category involves the introduction of nucleobase amino acids into proteins in support of specific interactions with RNA and DNA. The energetic differences between potential protein-nucleic acid complexes formed from two binding partners are often quite small. It seems logical to think that selective binding can be achieved by using a nucleobase moiety in each of the binding partners by utilizing known interactions between nucleic acid bases (located in the protein and nucleic acid) to achieve energetically favorable interactions. We do so both in vitro and in cellulo. A second focus has involved the design of small fluorescent probes not much larger than amino acids that are genetically encodable and which can be incorporated during protein biosynthesis, serving as detectable probes of protein trafficking and interaction with other macromolecules. We provide an in vitro example of strongly fluorescent tryptophan analogues positioned at single sites within dihydrofolate reductase, permitting selective communication with a FRET acceptor at a known position, even in the presence of several tryptophans. An oxazole amino acid, weakly fluorescent in aqueous solution, fluoresced more strongly following incorporation into MreB, a scaffold protein produced in cellulo. Finally, we describe the introduction of a single phosphorylated tyrosine into the p50 subunit of NF-κB. When present at either of two key positions, the resulting NF-κB significantly enhanced binding in vitro to the promoter DNA as well as subsequent mRNA transcription of its client protein CD40 in cellulo. In a separate expression in activated Jurkat cells, an increased production of CD40 protein was observed.

Described herein is the synthesis and photophysical characterization of novel dipeptidomimetic fluorogenic probes. The new dipeptidomimetic cassette is based on the environment-sensitive fluorophore nitrobenzothiadiazole (NBTD) and is designed to be embedded within a polypeptide backbone, not attached as part of a conformationally unrestrained amino acid side chain. Compounds 1-6 were prepared to investigate the effect of introducing a methyl group at positions 5 and 6 of the nitrobenzothiadiazole scaffold on their photophysical properties. Additionally, we investigated the effect of N-methylation of the 4-amino group on their chemical and photophysical properties. The nitrobenzothiadiazole scaffold was functionalized with amino- and carboxy-termini, resulting in a conformationally restricted dipeptidomimetic scaffold (NBTD-Gly) that can potentially be embedded within peptides or proteins of interest and used as a biophysical tool for studying protein structure and function. Thus, we investigated the synthesis and photophysical properties of functionalized nitrobenzothiadiazoles (1-11). The amino-terminus of the dipeptidomimetic scaffold (NBTD-Gly) was introduced at the benzylic position of the benzothiadiazole core using Gabriel synthesis, followed by nitration. The carboxy terminus was introduced via nucleophilic aromatic substitution as part of the glycine moiety. Subsequent steps involved the removal of a phthaloyl protecting group and a condensation reaction to form protected dipeptidomimetic analogues 7-11.

N-Methylated amino acids are constituents of natural bioactive peptides and proteins. Nα-methylated amino acids appear abundantly in natural cyclic peptides, likely due to their constraint of peptide conformation and contribution to peptide stability. Peptides containing Nα-methylated amino acids have long been prepared by chemical synthesis. While such natural peptides are not produced ribosomally, recent ribosomal strategies have afforded Nα-methylated peptides. Presently, we define new strategies for the ribosomal incorporation of Nα-methylated amino acids into peptides and proteins. First, we identify modified ribosomes capable of facilitating the incorporation of six N-methylated amino acids into antibacterial scorpion peptide IsCT. Also synthesized analogously was a protein domain (RRM1) from hnRNP LL; improved yields were observed for nearly all tested N-methylated amino acids. Computational modeling of the ribosomal assembly illustrated how the distortion imposed by N-methylation could be compensated by altering the nucleotides in key 23S rRNA positions. Finally, it is known that incorporation of multiple prolines (an N-alkylated amino acid) ribosomally can be facilitated by bacterial elongation factor P. We report that supplementing endogenous EF-P during IsCT peptide and RRM1 protein synthesis gave improved yields for most of the N-methylated amino acids studied.

Supplementary text, 3 figures, and 1 table.

The introduction of noncanonical amino acids into proteins and peptides has been of great interest for many years and has facilitated the detailed study of peptide/protein structure and mechanism. In addition to numerous nonproteinogenic α-l-amino acids, bacterial ribosome modification has provided the wherewithal to enable the synthesis of peptides and proteins with a much greater range of structural diversity, as has the use of endogenous bacterial proteins in reconstituted protein synthesizing systems. In a recent report, elongation factor P (EF-P), putatively essential for enabling the incorporation of contiguous proline residues into proteins, was shown to facilitate the introduction of an N-methylated amino acid in addition to proline. This finding prompted us to investigate the properties of this protein factor with a broad variety of structurally diverse amino acid analogues using an optimized suppressor tRNAPro that we designed. While these analogues can generally be incorporated into proteins only in systems containing modified ribosomes specifically selected for their incorporation, we found that EF-P could significantly enhance their incorporation into model protein dihydrofolate reductase using wild-type ribosomes. Plausibly, the increased yields observed in the presence of structurally diverse amino acid analogues may result from the formation of a stabilized ribosomal complex in the presence of EF-P that provides more favorable conditions for peptide bond formation. This finding should enable the facile incorporation of a much broader structural variety of amino acid analogues into proteins and peptides using native ribosomes.

Seven d-amino acid derivatives having reactive side chains have been activated to afford their respective 3,5-dinitrobenzyl esters using the Mitsunobu reaction. This esterification was found to be difficult using traditional methods involving 3,5-dinitrobenzyl chloride under alkaline conditions. The conversion of a tRNA to the respective d-glutaminyl-tRNA using d-glutamine 3,5-dinitrobenzyl ester was catalyzed by a flexizyme, followed by purification to remove all the unacylated tRNAs and other byproducts. Both d- and l-glutamine were incorporated from their aminoacyl-tRNAs into a model peptide structurally related to IFN-β.

Significance Base excision repair (BER) is one of the major DNA repair pathways used to fix a myriad of cellular DNA lesions. The enzymes involved in BER, including DNA polymerase β (Polβ), have been identified and characterized, but how they act together to efficiently perform BER has not been fully understood. Through gel electrophoresis, mass spectrometry, and kinetic analysis, we discovered that the two enzymatic activities of Polβ can be interlocked, rather than functioning independently from each other, when processing DNA intermediates formed in BER. The finding prompted us to hypothesize a modified BER pathway. Through conventional and time-resolved X-ray crystallography, we solved 11 high-resolution crystal structures of cross-linked Polβ complexes and proposed a detailed chemical mechanism for Polβ’s 5′-deoxyribose-5-phosphate lyase activity.

The family of NF-κB transcriptional activators controls the expression of many genes, including those involved in cell survival and development. The family consists of homo- and heterodimers constituted by combinations of five subunits. Subunit p50 includes 13 tyrosine residues, but the relationship between specific tyrosine phosphorylations and p50 function is not well understood. Subunits of p50 and p65 prepared in vitro formed a heterodimer, but this NF-κB would not bind to the interleukin-2 (IL-2) promoter DNA. Treatment of p50 with guanosine triphosphate (GTP) and a lysate from activated Jurkat cells, effected rapid p50 phosphorylation, and, in the presence of wild-type subunit p65, was accompanied on the same time scale by IL-2 promoter DNA binding. Modified p50s containing one of seven stoichiometrically phosphorylated tyrosines in NF-κB p50/p65 heterodimers, included three that facilitated binding to the IL-2 DNA promoter region to a greater extent than the wild type. One of these three stoichiometrically phosphorylated p50/p65 heterodimers of NF-κB, containing pTyr60 in the p50 subunit, was treated with a lysate from activated Jurkat cells + GTP and shown to be phosphorylated on the same time scale as wild-type p50. This modified NF-κB also developed IL-2 promoter DNA binding activity on the same time scale as the wild type but exhibited greater binding to the IL-2 DNA promoters than the wild type. The nature of this enhanced binding was studied in greater detail using a metabolically stable pTyr derivative at position 60 of p50 and cellular phosphatases. We suggest that enhanced DNA binding of modified NF-κB containing pTyr60 in the p50 subunit may reflect stoichiometric NF-κB phosphorylation at a site that is not normally fully phosphorylated, or not phosphorylated at all, and is relatively resistant to the effects of Jurkat cell tyrosine phosphatase activity. This conclusion was reinforced by demonstrating that modification of Tyr60 of p50 with a metabolically stable methylenephosphonate moiety further increased the stability of the formed NF-κB p50/p65 heterodimer against the action of activated Jurkat cell phosphatases.

Conformational dynamics contribute importantly to enzyme catalysis, such that targeted conformational constraint may affect catalysis. Firefly luciferases undergo extensive structural change during catalysis; key residues form a hydrophobic pocket, excluding water and enabling maximally energetic light production. Point mutants almost always luminesce at longer wavelengths (lower energy) than the wild type. Conformational constraint, using dipeptide analogue 3 at a position critical for optimized excited state structure, produced luciferase emission at a shorter wavelength by ~10 nm. In comparison, introduction of conformationally constrained analogues 4, 5, or 7 afforded luciferases emitting at longer wavelengths, while a related unconstrained luciferase (analogue 6) exhibited wild-type emission. The constrained luciferases tested were more stable than the wild type. Protein modeling demonstrated that the "inside" or "outside" orientation of the conformationally constrained dipeptide led to the shorter or longer emission wavelength, respectively. More broadly, these results suggest that local conformational constraint can control specific elements of enzyme behavior, both in vitro and in vivo. This represents the first example of studying enzyme function by introducing conformationally constrained dipeptides at a specific protein position. The principles discovered here in luciferase modification will enable studies to control the wavelength emission and photophysical properties of modified luciferases.

Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently accessible by any of several strategies, it is now possible to employ in vitro and in vivo protein biosynthesis to elaborate proteins containing a much larger variety of amino acid building blocks. However, the incorporation of this broader variety of amino acids is limited to those species utilized by the ribosome. As a consequence, virtually all of the substrates utilized over time have been L-α-amino acids. In recent years, a variety of structural and biochemical studies have provided important insights into those regions of the 23S ribosomal RNA that are involved in peptide bond formation. Subsequent experiments, involving the randomization of key regions of 23S rRNA required for peptide bond formation, have afforded libraries of E. coli harboring plasmids with the rrnB gene modified in the key regions. Selections based on the use of modified puromycin derivatives with altered amino acids then identified clones uniquely sensitive to individual puromycin derivatives. These clones often recognized misacylated tRNAs containing altered amino acids similar to those in the modified puromycins, and incorporated the amino acid analogues into proteins. In this fashion, it has been possible to realize the synthesis of proteins containing D-amino acids, β-amino acids, phosphorylated amino acids, as well as long chain and cyclic amino acids in which the nucleophilic amino group is not in the α-position. Of special interest have been dipeptides and dipeptidomimetics of diverse utility.

The elaboration of peptides and proteins containing non-proteinogenic amino acids has been realized using several complementary strategies, including chemical synthesis, ribosome- or non-ribosome-mediated elaboration, intein-mediated polypeptide rearrangements, or some combination of these strategies. All of these have strengths and limitations, and significant efforts have been focused on minimizing the effects of limitations, to improve the overall utility of individual strategies. Our laboratory has studied ribosomally mediated peptide and protein synthesis involving a wide variety of non-proteinogenic amino acids, and in recent years we have described a novel strategy for the selection of modified bacterial ribosomes. These modified ribosomes have enabled the incorporation into peptides and proteins of numerous modified amino acids not accessible using wild-type ribosomes. This has included d-amino acids, β-amino acids, dipeptides and dipeptidomimetic species, as well as phosphorylated amino acids. Presently, we have considered novel strategies for incorporating non-proteinogenic amino acids in improved yields. This has included the incorporation of non-proteinogenic amino acids into contiguous positions, a transformation known to be challenging. We demonstrate the preparation of this type of protein modification by utilizing a suppressor tRNACUA activated with a dipeptide consisting of two identical non-proteinogenic amino acids, in the presence of modified ribosomes selected to recognize such dipeptides. Also, we demonstrate that the use of bis-aminoacylated suppressor tRNAs, shown previously to increase protein yields significantly in vitro, can be extended to the use of non-proteinogenic amino acids.

The NF-κB family of transcriptional activators is responsible for the expression of numerous genes that control key functions such as cell development and survival. Subunit p50 has been studied extensively and is known to include 13 tyrosines, but the extent and pattern of tyrosine phosphorylation that accompanies p50 function has not been defined in the literature, especially at the level of selectivity of gene expression. In this study, phosphorylated tyrosine (pTyr) was site-selectively incorporated into the p50 subunit using an E. coli in vitro expression system containing a modified ribosome. In human T cells, the NF-κBs containing a pTyr at position 60 or 82 of p50 strongly increased the expression of CD40, which is a potential target for cancer or viral immunotherapy. Promoter DNA binding was studied for CD40 promoters, and verified two pTyr residues in NF-κB p50/p65 heterodimers that facilitated this process, and that support the possible importance of phosphorylation stoichiometry. This study defines a new approach for studying tyrosine residues whose phosphorylation alters protein binding to DNA promoters, and contributes to the facility of DNA expression.

The identification of proteins that bind selectively to nucleic acid sequences is an ongoing challenge. We previously synthesized nucleobase amino acids designed to replace proteinogenic amino acids; these were incorporated into proteins to bind specific nucleic acids predictably. An early example involved selective cell free binding of the hnRNP LL RRM1 domain to its i-motif DNA target via Watson-Crick-like H-bonding interactions. In this study, we employ the X-ray crystal structure of transcriptional regulator Rob bound to its micF promoter, which occurred without DNA distortion. Rob proteins modified in vivo with nucleobase amino acids at position 40 exhibited altered DNA promoter binding, as predicted on the basis of their Watson-Crick-like H-bonding interactions with promoter DNA A-box residue Gua-6. Rob protein expression ultimately controls phenotypic changes, including resistance to antibiotics. Although Rob proteins with nucleobase amino acids were expressed in Escherichia coli at levels estimated to be only a fraction of that of the wild-type Rob protein, those modified proteins that bound to the micF promoter more avidly than the wild type in vitro also produced greater resistance to macrolide antibiotics roxithromycin and clarithromycin in vivo, as well as the beta-lactam antibiotic ampicillin. Also demonstrated is the statistical significance of altered DNA binding and antibiotic resistance for key Rob analogues. These preliminary findings suggest the ultimate utility of nucleobase amino acids in altering and controlling preferred nucleic acid target sequences by proteins, for probing molecular interactions critical to protein function, and for enhancing phenotypic changes in vivo by regulatory protein analogues.

There has been little innovation in identifying novel insulin sensitizers. Metformin, developed in the 1920s, is still used first for most Type 2 diabetes patients. Mice with genetic reduction of p52Shc protein have improved insulin sensitivity and glucose tolerance. By high-throughput screening, idebenone was isolated as the first small molecule 'Shc Blocker'. Idebenone blocks p52Shc's access to Insulin Receptor to increase insulin sensitivity. In this work the avidity of 34 novel idebenone analogs and 3 metabolites to bind p52Shc, and to block the interaction of p52Shc with the Insulin receptor was tested. Our hypothesis was that if an idebenone analog bound and blocked p52Shc's access to insulin receptor better than idebenone, it should be a more effective insulin sensitizing agent than idebenone itself. Of 34 analogs tested, only 2 both bound p52Shc more tightly and/or blocked the p52Shc-Insulin Receptor interaction more effectively than idebenone. Of those 2 only idebenone analog #11 was a superior insulin sensitizer to idebenone. Also, the long-lasting insulin-sensitizing potency of idebenone in rodents over many hours had been puzzling, as the parent molecule degrades to metabolites within 1 h. We observed that two of the idebenone's three metabolites are insulin sensitizing almost as potently as idebenone itself, explaining the persistent insulin sensitization of this rapidly metabolized molecule. These results help to identify key SAR = structure-activity relationship requirements for more potent small molecule Shc inhibitors as Shc-targeted insulin sensitizers for type 2 diabetes.

Ferroptosis is an iron-catalyzed, nonapoptotic form of regulated necrosis that has been implicated in the pathological cell death associated with various disorders including neurodegenerative diseases (e.g., Friedreich's ataxia (FRDA), Alzheimer's disease, and Parkinson's disease), stroke, and traumatic brain injury. Recently, we showed that lipophilic methylene blue (MB) and methylene violet (MV) analogues both promoted increased frataxin levels and mitochondrial biogenesis, in addition to their antioxidant activity in cultured FRDA cells. Presently, we report the synthesis of series of lipophilic phenothiazine analogues that potently inhibit ferroptosis. The most promising compounds (1b-5b) exhibited an improved protection compared to the parent phenothiazine against erastin- and RSL3-induced ferroptotic cell death. These analogues have equivalent or better potency than ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1), that are among the most potent inhibitors of this regulated cell death described so far. They represent novel lead compounds with therapeutic potential in relevant ferroptosis-driven disease models such as FRDA.

DNA polymerase β (Pol β) repairs cellular DNA damage. When such damage is inflicted upon the DNA in tumor cells treated with DNA targeted antitumor agents, Pol β thus diminishes their efficacy. Accordingly, this enzyme has long been a target for antitumor therapy. Although numerous inhibitors of the lyase activity of the enzyme have been reported, none has yet proven adequate for development as a therapeutic agent. In the present study, we developed a new strategy to identify lyase inhibitors that critically engage the lyase active site primary nucleophile Lys72 as part of the binding interface. This involves a parallel evaluation of the effect of the inhibitors on the wild-type DNA polymerase β (Pol β) and Pol β modified with a lysine analogue at position 72. A model panel of five structurally diverse lyase inhibitors identified in our previous studies (only one of which has been published) with unknown modes of binding were used for testing, and one compound, cis-9,10-epoxyoctadecanoic acid, was found to have the desired characteristics. This finding was further corroborated by in silico docking, demonstrating that the predominant mode of binding of the inhibitor involves an important electrostatic interaction between the oxygen atom of the epoxy group and Nε of the main catalytic nucleophile, Lys72. The strategy, which is designed to identify compounds that engage certain structural elements of the target enzyme, could find broader application for identification of ligands with predetermined sites of binding.

We have synthesized several conformationally constrained dipeptide analogues as possible substrates for incorporation into proteins. These have included three cyclic dipeptides formed from Boc derivatives of 2,4-diaminobutyric acid, ornithine and lysine, having 5-, 6-, and 7-membered lactam rings, respectively. These dipeptides were used to activate a suppressor tRNA transcript, the latter of which had been prepared by in vitro transcription. Using modified E. coli ribosomes described previously, these activated suppressor tRNAs enabled the incorporation of the three cyclic dipeptides into dihydrofolate reductase (DHFR) at positions 18 and 49. The suppression yields increased with increasing lactam ring size and were found to proceed in suppression yields ranging from 3.4 to 8.9% at two different protein sites for the 5-, 6- and 7-membered lactam dipeptides. The greater facility of incorporation of the 7-membered lactam prompted us to prepare two 7-membered cyclic acylhydrazides (4 and 5) by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)-mediated cyclization of amino acids having selectively protected hydrazine functional groups in their side chains. In common with the lactam dipeptides, acylhydrazide dipeptides 4 and 5 could be used to activate the same suppressor tRNA transcript and to incorporate the cyclic dipeptides into DHFR. They were incorporated into the same two DHFR sites in suppression yields ranging from 8.3 to 11.2%.

Nucleic acid binding proteins have been studied extensively, but the nature of the interactions that control their affinity, selectivity, and DNA and RNA functions is still not well understood. To understand the nature and functional consequences of such interactions, we introduced nucleobase amino acids at specific positions of the transcriptional regulator Rob protein in vivo and succeeded in demonstrating that an alteration of the protein-DNA affinity can affect specific phenotypes associated with Rob protein-DNA interactions. Previously, we inserted different nucleobase amino acids in lieu of Arg40; this residue is known (via X-ray crystallography) to interact with the micF DNA promoter A-box residue Gua6. The interactions predominantly involved Watson-Crick-like H bonding. The present study focused primarily on the micF DNA promoter B-box; the crystallographically determined interaction involves H bonding between the agmatine moiety of Arg90 within an HTH motif of Rob and a phosphate oxygen anion to the 5'-side of Thy14. We had two main goals, the first of which was to demonstrate enhanced Rob-binding to the micF promoter DNA and the functional consequences resulting from the interaction of micF DNA with Rob analogues containing Arg90 nucleobase mimics. The second was to explore the possible functional consequences of enhancing the protein-DNA affinity with nucleobase replacements, which mechanistically mediate interactions differently than those reported to be operative for specific protein-DNA interactions. Nucleobase replacement at position 90 with Arg isosteres enhanced the Rob protein-micF DNA affinity in parallel with increasing antibiotic and Hg2+ resistance, while aromatic amino acid replacements increased the affinity but not the antibiotic or Hg2+ resistance. The demonstration of an increased affinity through strong base stacking interactions was notable.

Genetic code expansion has enabled many noncanonical amino acids to be incorporated into proteins in vitro and in cellulo. These have largely involved alpha-L-amino acids, reflecting the substrate specificity of natural aminoacyl-tRNA synthetases and ribosomes. Recently, modified E. coli ribosomes, selected using a dipeptidylpur-omycin analogue, were employed to incorporate dipeptides and dipeptidomimetics. Presently, we report the in cellulo incorporation of a strongly fluorescent oxazole amino acid (lacking an asymmetric center or alpha-amino group) by using modified ribosomes and pyrrolysyl-tRNA synthetase (Py1RS). Initially, a plasmid encoding the RRM1 domain of putative transcription factor hnRNP LL was cotransformed with plasmid pTECH-Pyl-OP in E. coli cells, having modified ribosomes able to incorporate dipeptides. Cell incubation in a medium containing oxazole 2 resulted in the elaboration of RRM1 containing the oxazole. Green fluorescent protein, previously expressed in vitro with several different oxazole amino acids at position 66, was also expressed in cellulo containing oxazole 2; the incorporation was verified by mass spectrometry. Finally, oxazole 2 was incorporated into position 13 of MreB, a bacterial homologue of eukaryotic cytoskeletal protein actin F. Modified MreB expressed in vitro and in cellulo comigrated with wild type. E. coli cells expressing the modified MreB were strongly fluorescent and retained the E. coli cell rod-like phenotype. For each protein studied, the incorporation of oxazole 2 strongly increased oxazole fluorescence, suggesting its potential utility as a protein tag. These findings also suggest the feasibility of dramatically increasing the repertoire of amino acids that can be genetically encoded for protein incorporation in cellulo.