An enzymatic reaction-based SERS saliva analysis microporous array chip for chiral differentiation and high-throughput detection of D-amino acids.

A Raman-active boronate modified surface-enhanced Raman scattering (SERS) microporous array chip based on the enzymatic reaction was constructed for reliable, sensitive, and quantitative monitoring of D-Proline (D-Pro) and D-Alanine (D-Ala) in saliva. Initially, 3-mercaptophenylboronic acid (3-MPBA) was bonded to Au-coated Si nanocrown arrays (Au/SiNCA) via Au-S bonding. Following this, H2O2 obtained from D-amino acid oxidase (DAAO)-specific catalyzed D-amino acids (D-AAs) further reduced 3-MPBA to 3-hydroxythiophenol (3-HTP) with a new Raman peak at 882 cm-1. Meanwhile, the original characteristic peak at 998 cm-1 remained unchanged. Therefore, the I882/I998 ratio increased with increasing content of D-AAs in the sample to be tested, allowing D-AAs to be quantitatively detected. The Au/SiNCA with large-area periodic crown structure prepared provided numerous, uniform "hot spots," and the microporous array chip with 16 detection units was employed as the platform for SERS analysis, realizing high-throughput, high sensitivity, high specificity and high-reliability quantitative detection of D-AAs (D-Pro and D-Ala). The limits of detection (LOD) were down to 10.1 µM and 13.7 µM throughout the linear range of 20-500 µM. The good results of the saliva detection suggested that this SERS sensor could rapidly differentiate between early-stage gastric cancer patients and healthy individuals.

Insights into the Interaction Landscape of the EVH1 Domain of Mena.

The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed ß7-ß8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 µM). Inhibitor 6c displayed tighter binding (Kd ∼ 0.3 µM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.

Proline, a unique amino acid whose polymer, polyproline II helix, and its analogues are involved in many biological processes: a review.

Proline is a unique amino acid in that its side-chain is cyclised to the backbone, thus giving proline an exceptional rigidity and a considerably restricted conformational space. Polyproline forms two well-characterized helical structures: a left-handed polyproline helix (PPII) and a right-handed polyproline helix (PPI). Usually, sequences made only of prolyl residues are in PPII conformation, but even sequences not rich in proline but which are rich in glycine, lysine, glutamate, or aspartate have also a tendency to form PPII helices. Currently, the only way to study unambiguously PPII structure in solution is to use spectroscopies based on optical activity such as circular dichroism, vibrational circular dichroism and Raman optical activity. The importance of the PPII structure is emphasized by its ubiquitous presence in different organisms from yeast to human beings where proline-rich motifs and their binding domains are believed to be involved in vital biological processes. Some of the domains that are bound by proline-rich motifs include SH3 domains, WW domains, GYF domains and UEV domains, etc. The PPII structure has been demonstrated to be essential to biological activities such as signal transduction, transcription, cell motility, and immune response.

Screening a knowledge-based library of low molecular weight compounds against the proline biosynthetic enzyme 1-pyrroline-5-carboxylate 1 (PYCR1).

Δ1-pyrroline-5-carboxylate reductase isoform 1 (PYCR1) is the last enzyme of proline biosynthesis and catalyzes the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate to L-proline. High PYCR1 gene expression is observed in many cancers and linked to poor patient outcomes and tumor aggressiveness. The knockdown of the PYCR1 gene or the inhibition of PYCR1 enzyme has been shown to inhibit tumorigenesis in cancer cells and animal models of cancer, motivating inhibitor discovery. We screened a library of 71 low molecular weight compounds (average MW of 131 Da) against PYCR1 using an enzyme activity assay. Hit compounds were validated with X-ray crystallography and kinetic assays to determine affinity parameters. The library was counter-screened against human Δ1-pyrroline-5-carboxylate reductase isoform 3 and proline dehydrogenase (PRODH) to assess specificity/promiscuity. Twelve PYCR1 and one PRODH inhibitor crystal structures were determined. Three compounds inhibit PYCR1 with competitive inhibition parameter of 100 µM or lower. Among these, (S)-tetrahydro-2H-pyran-2-carboxylic acid (70 µM) has higher affinity than the current best tool compound N-formyl-l-proline, is 30 times more specific for PYCR1 over human Δ1-pyrroline-5-carboxylate reductase isoform 3, and negligibly inhibits PRODH. Structure-affinity relationships suggest that hydrogen bonding of the heteroatom of this compound is important for binding to PYCR1. The structures of PYCR1 and PRODH complexed with 1-hydroxyethane-1-sulfonate demonstrate that the sulfonate group is a suitable replacement for the carboxylate anchor. This result suggests that the exploration of carboxylic acid isosteres may be a promising strategy for discovering new classes of PYCR1 and PRODH inhibitors. The structure of PYCR1 complexed with l-pipecolate and NADH supports the hypothesis that PYCR1 has an alternative function in lysine metabolism.

Targeting the glutamine-arginine-proline metabolism axis in cancer.

Metabolic abnormalities are an important feature of tumours. The glutamine-arginine-proline axis is an important node of cancer metabolism and plays a major role in amino acid metabolism. This axis also acts as a scaffold for the synthesis of other nonessential amino acids and essential metabolites. In this paper, we briefly review (1) the glutamine addiction exhibited by tumour cells with accelerated glutamine transport and metabolism; (2) the methods regulating extracellular glutamine entry, intracellular glutamine synthesis and the fate of intracellular glutamine; (3) the glutamine, proline and arginine metabolic pathways and their interaction; and (4) the research progress in tumour therapy targeting the glutamine-arginine-proline metabolic system, with a focus on summarising the therapeutic research progress of strategies targeting of one of the key enzymes of this metabolic system, P5CS (ALDH18A1). This review provides a new basis for treatments targeting the metabolic characteristics of tumours.

Incorporation of CF3-pseudoprolines into polyproline type II foldamers confers promising biophysical features.

The development and the use of fluorinated polyproline-type II (PPII) foldamers are still underexplored. Herein, trifluoromethyl pseudoprolines have been incorporated into polyproline backbones without affecting their PPII helicity. The ability of the trifluoromethyl groups to increase hydrophobicity and to act as 19F NMR probes is demonstrated. Moreover, the enzymatic stability and the non-cytotoxicity of these fluorinated foldamers make them valuable templates for use in medicinal chemistry.

Novel selective proline-based peptidomimetics for human cathepsin K inhibition.

Human cathepsin K (CatK) stands out as a promising target for the treatment of osteoporosis, considering its role in degrading the bone matrix. Given the small and shallow S2 subsite of CatK and considering its preference for proline or hydroxyproline, we now propose the rigidification of the leucine fragment found at the P2 position in a dipeptidyl-based inhibitor, generating rigid proline-based analogs. Accordingly, with these new proline-based peptidomimetics inhibitors, we selectively inhibited CatK against other human cathepsins (B, L and S). Among these new ligands, the most active one exhibited a high affinity (pKi = 7.3 - 50.1 nM) for CatK and no inhibition over the other cathepsins. This specific inhibitor harbors two novel substituents never employed in other CatK inhibitors: the trifluoromethylpyrazole and the 4-methylproline at P3 and P2 positions. These results broaden and advance the path toward new potent and selective inhibitors for CatK.

Thioproline-Based Oxidation Strategy for Direct Preparation of N-Terminal Thiazolidine-Containing Peptide Thioesters from Peptide Hydrazides.

We describe a simple and robust oxidation strategy for preparing N-terminal thiazolidine-containing peptide thioesters from peptide hydrazides. We find for the first time that l-thioproline can be used as a protective agent to prevent the nitrosation of N-terminal thiazolidine during peptide hydrazide oxidation. The thioproline-based oxidation strategy has been successfully applied to the chemical synthesis of CC chemokine ligand-2 (69aa) and omniligase-C (113aa), thereby demonstrating its utility in hydrazide-based native chemical ligation.

Selective Binding of Tannic Acid-Conjugated Lipid Nanovesicles to Proline-Rich Proteins Enhances Transdermal Lipophilic-Antioxidant Delivery.

Tannic acid (TA) possesses a notable ability to adhere to proline-rich proteins that make up skin cells and the extracellular matrix (ECM) in the skin tissue. Drug carriers with this specific adhesion ability exhibit improved drug delivery efficiency on the skin. Taking advantage of this, this study presents skin-adhesive TA-conjugated lipid nanovesicles (TANVs) for enhanced transdermal antioxidant delivery. We found that TANVs exhibited selective intermolecular interactions with keratinocyte proline-rich proteins (KPRPs) and collagen that makes up skin cells by hydrogen bonding and van der Waals interactions, further enabling the strong bonding to macroscopic skin itself and ECM. We used vitamin E (α-tocopherol), which is known to effectively reduce oxidative stress but has limited skin penetration, as a drug to verify improved in vitro delivery and therapeutic efficacy. The evaluation revealed that the antioxidant-loaded TANVs exerted excellent scavenging effects against reactive oxygen species induced by ultraviolet light or peroxides in the skin, thereby enabling the development of an active drug delivery system for dermal therapy.

Vesicle-like Nanocapsules Formed by Self-Assembly of Peptides with Oligoproline and -Leucine.

Since drug carriers are envisaged to be used in a wide variety of situations and environments, nanocarriers with diverse properties, such as biocompatibility, biodegradability, nonimmunogenicity, adequate particle size, robustness, and cell permeability, are required. Here, we report the construction of novel nanocapsules with the above-mentioned features by the self-assembly of peptides composed of oligoproline and oligoleucine (i.e., H-Pro10Leu4-NH2 and H-Pro10Leu6-NH2). The peptides self-organized via hydrogen bonds and hydrophobic interactions between oligoleucine moieties to form vesicle-like nanocapsules with cationic oligoproline exposed on the surface. The guest encapsulation experiments revealed that the nanocapsules were capable of uptake of both water-soluble and insoluble compounds. Furthermore, positively charged and/or oligoproline-based peptides are known to improve cell permeability and cellular uptake, suggesting that the peptide nanocapsules are good candidates for nanocarriers to complement liposomes and polymer micelles.

A Simple Generic Model of Elastin-Like Polypeptides with Proline Isomerization.

A generic model of elastin-like polypeptides (ELP) is derived that includes proline isomerization (ProI). As a case study, conformational transition of a -[valine-proline-glycine-valine-glycine]- sequence is investigated in aqueous ethanol mixtures. While the non-bonded interactions are based on the Lennard-Jones (LJ) parameters, the effect of ProI is incorporated by tuning the intramolecular 3- and 4-body interactions known from the underlying all-atom simulations into the generic model. One of the key advantages of such a minimalistic model is that it readily decouples the effects of geometry and the monomer-solvent interactions due to the presence of ProI, thus gives a clearer microscopic picture that is otherwise rather nontrivial within the all-atom setups. These results are consistent with the available all-atom and experimental data. The model derived here may pave the way to investigate large scale self-assembly of ELPs or biomimetic polymers in general.

Green Extraction of Depsidones and Depsides from Hypogymnia physodes (L.) Nyl. Using Natural Deep Eutectic Solvents.

Various studies have shown that Hypogymnia physodes are a source of many biologically active compounds, including lichen acids. These lichen-specific compounds are characterized by antioxidant, antiproliferative, and antimicrobial properties, and they can be used in the cosmetic and pharmaceutical industries. The main aim of this study was to optimize the composition of natural deep eutectic solvents based on proline or betaine and lactic acid for the extraction of metabolites from H. physodes. The design of the experimental method and the response surface approach allowed the optimization of the extraction process of specific lichen metabolites. Based on preliminary research, a multivariate model of the experiment was developed. For optimization, the following parameters were employed in the experiment to confirm the model: a proline/lactic acid/water molar ratio of 1:2:2. Such a mixture allowed the efficient extraction of three depsidones (i.e., physodic acid, physodalic acid, 3-hydroyphysodic acid) and one depside (i.e., atranorin). The developed composition of the solvent mixtures ensured good efficiency when extracting the metabolites from the thallus of H. physodes with high antioxidant properties.

Synthesis of All-Peptide-Based Rotaxane from a Proline-Containing Cyclic Peptide.

Rotaxane cross-linkers enhance the toughness of the resulting rotaxane cross-linked polymers through a stress dispersion effect, which is attributed to the mobility of the interlocked structure. To date, the compositional diversity of rotaxane cross-linkers has been limited, and the poor compatibility of these cross-linkers with peptides and proteins has made their use in such materials challenging. The synthesis of a rotaxane composed of peptides may result in a biodegradable cross-linker that is compatible with peptides and proteins, allowing the fortification of polypeptides and proteins and ultimately leading to the development of innovative materials that possess excellent mechanical properties and biodegradability. However, the chemical synthesis of all-peptide-based rotaxanes has remained elusive because of the absence of strong binding motifs in peptides, which prevents an axial peptide from penetrating a cyclic peptide. Here, we synthesized all-peptide-based rotaxanes using an active template method for proline-containing cyclic peptides. The results of molecular dynamics simulations suggested that cyclic peptides with an expansive inner cavity and carbonyl oxygens oriented toward the center are favorable for rotaxane synthesis. This rotaxane synthesis method is expected to accelerate the synthesis of peptides and proteins with mechanically interlocked structures, potentially leading to the development of peptide- and protein-based materials with unprecedented functionalities.

Discovery and Synthesis of Hydroxy-l-Proline Blockers of the Neutral Amino Acid Transporters SLC1A4 (ASCT1) and SLC1A5 (ASCT2).

As a conformationally restricted amino acid, hydroxy-l-proline is a versatile scaffold for the synthesis of diverse multi-functionalized pyrrolidines for probing the ligand binding sites of biological targets. With the goal to develop new inhibitors of the widely expressed amino acid transporters SLC1A4 and SLC1A5 (also known as ASCT1 and ASCT2), we synthesized and functionally screened synthetic hydroxy-l-proline derivatives using electrophysiological and radiolabeled uptake methods against amino acid transporters from the SLC1, SLC7, and SLC38 solute carrier families. We have discovered a novel class of alkoxy hydroxy-pyrrolidine carboxylic acids (AHPCs) that act as selective high-affinity inhibitors of the SLC1 family neutral amino acid transporters SLC1A4 and SLC1A5. AHPCs were computationally docked into a homology model and assessed with respect to predicted molecular orientation and functional activity. The series of hydroxyproline analogs identified here represent promising new agents to pharmacologically modulate SLC1A4 and SLC1A5 amino acid exchangers which are implicated in numerous pathophysiological processes such as cancer and neurological diseases.

C-Terminal Decarboxylation of Proline-Derived Building Blocks for Protein-Binding Peptides.

Using a set of conformationally restricted Proline-derived Modules (ProMs), our group has recently succeeded in developing inhibitors for the enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain, which is a key mediator of cell migration and plays an important role in tumor metastasis. While these (formally) pentapeptidic compounds show nanomolecular binding affinities towards EVH1, their drug-like properties and cell permeability need to be further optimized before they can be clinically tested as therapeutic agents against metastasis. In this study, we sought to improve these properties by removing the C-terminal carboxylic acid function of our peptoids, either by late-stage decarboxylation or by direct synthesis. For late-stage decarboxylation of ProM-like systems, a method for reductive halo decarboxylation was optimized and applied to several proline-derived substrates. In this way, a series of new decarboxy ProMs suitable as building blocks for decarboxy EVH1 inhibitors were obtained. In addition, we incorporated decarboxy-ProM-1 into the pentapeptide-like compound Ac[2ClF][ProM-2][Decarb-ProM-1], which showed similar affinity towards EVH1 as the methyl ester derivative (Ac[2Cl-F][ProM-2][ProM1]OMe). However, despite better calculated drug-like properties, this compound did not inhibit chemotaxis in a cellular assay.

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

Effects of Proline on Internal Friction in Simulated Folding Dynamics of Several Alanine-Based α-Helical Peptides.

We have studied in silico the effect of proline, a model cosolvent, on local and global friction coefficients in (un)folding of several typical alanine-based α-helical peptides. Local friction is related to dwell times of a single, ensemble-averaged hydrogen bond (HB) within each peptide. Global friction is related to energy dissipated in a series of configurational changes of each peptide experienced by increasing the number of HBs during folding. Both of these approaches are important in relation to future atomic force microscopic-based measurements of internal friction via force-clamp single-molecule force spectroscopy. Molecular dynamics (MD) simulations for six peptides, namely, ALA5, ALA8, ALA15, ALA21, (AAQAA)3, and H2N-GN(AAQAA)2G-COONH2, have been conducted at 2 and 5 M proline solutions in water. Using previously obtained MD data for these peptides in pure water as well as upgraded theoretical models, we obtained variations of local and global internal friction coefficients as a function of solution viscosity. The results showed the substantial role of proline in stabilizing the folded state and slowing the overall folding dynamics. Consequently, larger friction coefficients were obtained at larger viscosities. The local and global internal friction, i.e., respective, friction coefficients approximated to zero viscosity, was also obtained. The evolution of friction coefficients with viscosity was weakly dependent on the number of concurrent folding pathways but was rather dominated by a stabilizing effect of proline on the folded states. Obtained values of local and global internal friction showed qualitatively similar results and a clear dependency on the structure of the studied peptide.

Influence of proline and hydroxyproline as antimicrobial and anticancer peptide components on the silver(I) ion activity: structural and biological evaluation with a new theoretical and experimental SAR approach.

Silver(I) complexes with proline and hydroxyproline were synthesized and structurally characterized and crystal structure analysis shows that the formulas of the compounds are {[Ag2(Pro)2(NO3)]NO3}n (AgPro) (Pro = L-proline) and {[Ag2(Hyp)2(NO3)]NO3}n (AgHyp) (Hyp = trans-4-hydroxy-L-proline). Both complexes crystallize in the monoclinic lattice with space group P21 with a carboxylate bidentate-bridging coordination mode of the organic ligands Pro and Hyp (with NH2+ and COO- groups in zwitterionic form). Both complexes have a distorted seesaw (C2v) geometry around one silver(I) ion with τ4 values of 58% (AgPro) and 51% (AgHyp). Moreover, the results of spectral and thermal analyses correlate with the structural ones. 1H and 13C NMR spectra confirm the complexes species' presence in the DMSO biological testing medium and their stability in the time range of the bioassays. In addition, molar conductivity measurements indicate complexes' behaviour like 1 : 1 electrolytes. Both complexes showed higher or the same antibacterial activity against Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus as AgNO3 (MIC = 0.063 mM) and higher than silver(I) sulfadiazine (AgSD) (MIC > 0.5 mM) against Pseudomonas aeruginosa. In addition, complex AgPro exerted a strong cytotoxic effect against the tested MDA-MB-231 and Jurkat cancer cell lines (IC50 values equal to 3.7 and 3.0 µM, respectively) compared with AgNO3 (IC50 = 6.1 (5.7) µM) and even significantly higher selectivity than cisplatin (cisPt) against MDA-MB-231 cancer cell lines (SI = 3.05 (AgPro); 1.16 (cisPt), SI - selectivity index). The binding constants and the number of binding sites (n) of AgPro and AgHyp complexes with bovine serum albumin (BSA) were determined at four different temperatures, and the zeta potential of BSA in the presence of silver(I) complexes was also measured. The in ovo method shows the safety of the topical and intravenous application of AgPro and AgHyp. Moreover, the complexes' bioavailability was verified by lipophilicity evaluation from the experimental and theoretical points of view.

Proline Peptide Bond Isomerization in Ubiquitin Under Folding and Denaturing Conditions by Pressure-Jump NMR.

Proline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kcal/mole for denatured polypeptides but showed a substantial drop to 12.7 kcal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized 'folding nucleus'. For cis-P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensity revealed a slow component attributed to cis â†’ trans isomerization of P37. This result implies that the NMR-invisible cis-P37 isomer adopts a molten globule state that encompasses the entire length of the ubiquitin chain, suggestive of a structure that mostly resembles the folded state.

Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation.

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami et al. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro.