Tryptophan Residues Research Articles

De Novo Design of Tryptophan Containing Broad-Spectrum Cationic Antimicrobial Octapeptides.

With the advent of antibiotic resistant organisms, development of alternate classes of molecules other than antibiotics to combat microbial infections, have become extremely important. In this context, antimicrobial peptides have taken center stage of antimicrobial therapeutic research. In this work, we have reported two cationic antimicrobial octapeptides WRL and LWRF, with broad spectrum antimicrobial activities against several strains of ESKAPE pathogens. Both the peptides were membrane associative and induced microbial cell death through membranolysis, being selective towards microbial membranes over mammalian membranes. The AMPs were unstructured in water, adopting partial helical conformation in the presence of microbial membrane mimics. Electrostatic interaction formed the primary basis of peptide-membrane interactions. WRL was more potent, salt tolerant and faster acting of the two AMPs, owing to the presence of two tryptophan residues against that of one in LWRF. Increased tryptophan number in WRL enhanced its membrane association ability, resulting in higher antimicrobial potency but lower selectivity. This experimental and computational work, established that an optimum number of tryptophan residues and their position was critical for obtaining high antimicrobial potency and selectivity simultaneously in the designed cationic AMPs. Understanding the peptide membrane interactions in atomistic details can lead to development of better antimicrobial therapeutics in future.

Spectroscopic and Molecular Docking Studies on the Influence of Inulin on the Interaction of Sophoricoside with Whey Protein Concentrate

The influence of inulin on the interaction of sophoricoside (Sop) with whey protein concentrate (WPC) was investigated using various spectroscopic methods, including fluorescence spectroscopy (intrinsic fluorescence, synchronous fluorescence, and three-dimensional fluorescence), ultraviolet-visible (UV–Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and molecular docking. Sop was found to quench the intrinsic fluorescence of WPC by a static mechanism, both with and without the addition of inulin, and to enhance the antioxidant capacity of the protein. The addition of inulin slightly increased the binding distance between WPC and Sop, while reducing the number of binding sites from two to one. Non-covalent interactions, predominantly van der Waals forces and hydrogen bonding, were maintained between Sop and the protein. Synchronous fluorescence spectroscopy revealed that Sop prevents the exposure of hydrophobic groups on tryptophan residues, leading to increased surface hydrophilicity of the WPC complex. This aligns with the decreased protein surface hydrophobicity measured by 8-Anilino-1-naphthalenesulfonic acid (ANS) binding assays. With inulin, the overall hydrophobicity of the protein was lower than in the system without inulin, suggesting that both inulin and Sop improve the solubility of WPC. Three-dimensional fluorescence spectral analysis showed a reduction in fluorescence intensity and a red shift in the presence of both Sop and inulin. FTIR spectroscopy indicated a slight increase in the secondary structure ordering of WPC following the addition of both Sop and inulin, suggesting structural stabilization under heating conditions. Molecular docking highlighted the potential for hydrogen bond formation between Sop and WPC.

Light-Activated Reactivity of Nitrones with Amino Acids and Proteins.

Controlled modifications of amino acids are an indispensable tool for advancing fundamental and translational research based on peptides and proteins. Yet, we still lack methods to chemically modify each naturally occurring amino acid sidechain. To help address this gap, we show that N,α-diaryl oxaziridines expand the scope of bioconjugation methods to chemically modify cysteine, methionine, and tryptophan residues with evidence for additional tyrosine labelling in a proteomic context. Conjugation primarily at tryptophan sites can be accessed by selective cleavage of modifications at other sidechains. The N,α-diaryl oxaziridine reagents are accessed through photoisomerization of nitrones, which serve as photocaged reagents, thus providing an additional level of control over reactivity. Initial guiding principles for the design of nitrone reagents are developed by exploring the impact of structure on UV-vis absorption, photoisomerization, and reactivity. We identify a nitrone structure that maximizes photoisomerization efficiency, the aqueous stability of the oxaziridine, the extent of amino acid modification, and the stability of the resulting amino acid conjugates. We then translate nitrone reagents to modify proteins in aqueous conditions. Finally, we use nitrones to profile reactive residues across the proteome of a mammalian cell line and find that they expand the proteome coverage.

Study on the Structural Characteristics and Foaming Properties of Ovalbumin—Citrus Pectin Conjugates Prepared by the Maillard Reaction

This study explored the structural features and foaming properties of ovalbumin (OVA) and its glycosylated conjugates with citrus pectin (CP) formed through the Maillard reaction. The results demonstrated that OVA and CP were successfully conjugated, with the degree of grafting increasing to 43.83% by day 5 of the reaction. SDS-PAGE analysis confirmed the formation of high-molecular-weight conjugates. Fourier-transform infrared (FT-IR) and fluorescence spectroscopy further revealed alterations in the secondary and tertiary structures of OVA, including an enhanced β-sheet content, a reduced β-turn content, and the depletion of tryptophan residues. Moreover, the surface hydrophobicity of the OVA–CP conjugates significantly increased, enhancing foaming properties. Furthermore, the analysis of foaming properties exhibited that the Maillard reaction improved the foaming capacity of OVA to 66.22% and foaming stability to 81.49%. These findings highlight the potential of glycosylation via the Maillard reaction to significantly improve the foaming properties of OVA, positioning it as a promising novel foaming agent.

Synthesis of Non‐canonical Tryptophan Variants via Rh‐catalyzed C−H Functionalization of Anilines

AbstractTryptophan and its non‐canonical variants play critical roles in pharmaceutical molecules and various enzymes. Facile access to this privileged class of amino acids from readily available building blocks remains a long‐standing challenge. Here, we report a regioselective synthesis of non‐canonical tryptophans bearing C4−C7 substituents via Rh‐catalyzed annulation between structurally diverse tert‐butyloxycarbonyl (Boc)‐protected anilines and alkynyl chlorides readily prepared from amino acid building blocks. This transformation harnesses Boc‐directed C−H metalation and demetalation to afford a wide range of C2‐unsubstituted indole products in a redox‐neutral fashion. This umpolung approach compared to the classic Larock indole synthesis offers a novel mechanism for heteroarene annulation and will be useful for the synthesis of natural products and drug molecules containing non‐canonical tryptophan residues in a highly regioselective manner.

Synthesis and evaluation of cyclic peptide-dasatinib conjugates as anti-melanoma agents

Herein, we synthesized two amphiphilic cyclic peptides, incorporating tryptophan and arginine residues, linked to dasatinib via a Cathepsin B-sensitive tetrapeptide (Gly-Phe-Leu-Gly). To mitigate steric hindrance and optimize drug release, we integrated two different linkers, succinate and glutarate, between the drug and peptide. The synthesis involved solid-phase techniques for the assembly of the peptide, followed by the coupling of the peptide on-resin with the linker, soft cleavage and solution-phase cyclization. Conjugation of the peptide-attached linker with dasatinib was conducted in the presence of N-methylmorpholine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), generating the novel prodrugs. The synthesized compounds were characterized by high-resolution mass spectrometry MALDI, and the purity was confirmed by analytical HPLC. The synthesized cyclic peptide-dasatinib conjugates containing glutarate and succinate linkers were further evaluated against human melanoma A375 cells, which exhibited IC50 values of 4.2 μM and 8.8 μM, respectively. Variations in cytotoxicity could be attributed to linker properties affecting intracellular drug release or prodrug uptake.

Cryptochrome magnetoreception: time course of photoactivation from non-equilibrium coarse-grained molecular dynamics

Magnetoreception, the ability to sense magnetic fields, is widespread in animals but remains poorly understood. The leading model links this ability in migratory birds to the photo-activation of the protein cryptochrome. Magnetic information is thought to induce structural changes in cryptochrome via a transient radical pair intermediate. This signal transduction pathway has been the subject of previous all-atom molecular dynamics (MD) simulations, but insights were limited to short timescales and equilibrium structures. To address this, we developed a non-equilibrium coarse-grained MD simulation approach, exploring cryptochrome’s photo-reduction over 20 replicates of 20 µs each. Our results revealed significant structural changes across the protein, with an overall time constant of 3 µs. The C-terminal (CT) region responded on a timescale of 4.7 µs, followed by the EEE-motif, while the phosphate binding loop (PBL) showed slower dynamics (9 µs). Network analysis highlighted direct pathways connecting the tryptophan tetrad to the CT, and distant pathways involving the EEE and PBL regions. The CT-dynamics are significantly impacted by a rearrangement of tryptophan residues in the central electron transfer chain. Our findings underscore the importance of considering longer timescales when studying cryptochrome magnetoreception and highlight the potential of non-equilibrium coarse-grained MD simulations as a powerful tool to unravel protein photoactivation reactions.

Mechanisms of Copper Selectivity and Release by the Metallochaperone CusF: Insights from CO-Binding, Rapid-Freeze-Quench EXAFS, and Unnatural Amino Acid Substitution.

Metallochaperones are small proteins that shuttle essential metal ions such as Cu selectively to their cellular targets. CusF has unusual Cu(I) coordination, bound by two methionines, one histidine and a capping tryptophan residue, W44. Here we compare the CO binding reactivity of the wild type (WT) protein and its W44A, F, and M variants. Fourier-transform infrared (FTIR) indicates that W44A provides unhindered access for CO, while W44M is unreactive. WT is also largely unreactive to CO suggesting that the tryptophan cap is effective in shielding the Cu(I) center from exogenous adduct formation, while the Phe variant shows partial reactivity suggestive of an equilibrium between cap-on and cap-off conformers. Rates of metal transfer to the partner CusB are consistent with the π-cation cap providing both selectivity and redox protection. Unnatural amino acid substitutions of the W44 ligand with cyano-Phe and Br-Phe underpin the conclusion that the Phe ligand is a less effective capping residue. Finally, density functional theory (DFT) calculations validate the CO-binding strategy. Overall, the study suggests that CusF uses the tryptophan cap to protect against exogenous ligand (O2) attack while the mechanism of protein-protein complex formation allows the cap to swing out of the way, and thus have minimal effect on the rates of metal transfer.

In Vitro Inhibitory Mechanism of Polyphenol Extracts from Multi-Frequency Power Ultrasound-Pretreated Rose Flower Against α-Glucosidase.

This paper explored the in vitro inhibitory mechanism of polyphenol-rich rose extracts (REs) from an edible rose flower against α-glucosidase using multispectral and molecular docking techniques. Results showed that REs had an inhibitory effect on α-Glu activity (IC50 of 1.96 μg/mL); specifically, the samples pretreated by tri-frequency ultrasound (20/40/60 kHz) exhibited a significantly (p < 0.05) stronger inhibitory effect on α-Glu activity with an IC50 of 1.33 μg/mL. The Lineweaver-Burk assay indicated that REs were mixed-type inhibitors and could statically quench the endogenous fluorescence of α-Glu. REs increased the chance of polypeptide chain misfolding by altering the microenvironment around tryptophan and tyrosine residues and disrupting the natural conformation of the enzyme. Molecular docking results showed that polyhydroxy phenolics had a high fit to the active site of α-Glu, so REs with high polymerization and numerous phenolic hydroxyl groups had a stronger inhibitory effect. Therefore, this study provides new insights into polyphenol-rich REs as potential α-glucosidase inhibitors.

Xanthine oxidase inhibitors: Virtual screening and mechanism of inhibition studies

Xanthine oxidase (XO), which plays a key role in purine metabolism, is an important target enzyme for the prevention and treatment of hyperuricemia. Inhibitory activity against XO is a common criterion for the screening of compounds with potential anti-hyperuricemic activity. In this study, 22 XO inhibitors were used to construct a 3D-QSAR pharmacophore model. Subsequently, molecular docking and in vitro activity evaluations were used to identify strong XO inhibitors from a list of 2000 natural compounds. The interaction mechanisms of these compounds with XO were analyzed based on inhibition kinetics and multi-spectral analyses. The pharmacophore model was composed of three hydrogen bond receptors and a hydrophobic center. The screened compounds — Diosmetin, Fisetin, and Genistein — all showed good XO inhibitory activity, with IC50 values of 1.86 ± 0.11 μM, 5.83 ± 0.08 μM, and 7.56 ± 0.10 μM, respectively. Kinetic analysis, fluorescence quenching assays, and molecular docking experiments showed that Diosmetin, Fisetin, and Genistein docked near the same active site of XO, mainly affecting the microenvironment of tryptophan residues. These molecules showed static binding to XO via hydrogen bonds, hydrophobic interactions, and van der Waals forces. Diosmetin and Genistein were competitive inhibitors, whereas Fisetin was a mixed inhibitor. Infrared spectroscopy showed that Diosmetin, Fisetin, and Genistein increased the α-helix content of XO from 7.4 % to 16.6 %, 21.4 %, and 11.2 %, respectively, thereby enhancing its stability. In summary, the pharmacophore model constructed in this study was accurate. The flavonoids Diosmetin, Fisetin, and Genistein effectively inhibited the activity of XO, and the amino acid residues LEU257, ILE353, and VAL259 played a key role in the interaction between the flavonoids and XO. These findings are of great significance for the screening and development of new XO inhibitors.

Temperature-Driven Stopped-Flow Experiments for Investigating the Initial Aggregation of the α-Synuclein Amyloid Protein, Focusing on Active and Inactive Phases.

The primary objective of this research is to further examine the events occurring during the active or burst phase by focusing on the aggregation of the Syn amyloid protein. Regarding this aspect, it was initially conducted rapid temperature variations using stopped-flow spectrometry and tyrosyl group fluorescence emission detection, within the initial 500 milliseconds in buffered Syn solutions at pH 7, exploring various temperature ranges to investigate protein aggregation. The results obtained were contrasted with results obtained for the Nα-acetyl-L-tyrosinamide (NAYA) parent compound in the same conditions. The utilization of the NAYA compound is suitable as it mimics the peptide bonds in proteins and contains a tyrosyl group resembling the four tyrosyl groups found in the Syn protein structure (the protein has no tryptophan residues). Furthermore, the NAYA compound adopts an intramolecularly hydrogen-bonded structure even in an aqueous solution, similar to the interactions seen in the hydrophilic face of β-sheets. Additionally, the Syn protein system can exhibit the presence of β-sheets as a result of the existence of very low abundant Syn amyloid precursor forms or nuclei during the initial stages of the protein aggregation. Thus, a relationship is present between the molecular processes in the NAYA and Syn protein systems, making the NAYA's application crucial in this research. Moreover, to aid in understanding the results, it was also compared the events during the quiescent or inactive phase (30-500 milliseconds) with those in the burst phase (up to 10 milliseconds) using stopped-flow spectrometry conditions. Steady-state measurements were beneficial in comprehending the occurrences in both the quiescent and burst phases examined. Although protein aggregation and disaggregation were observed during the quiescent phase, determining these processes in the burst phase was more challenging. In the latter case, the aggregation of the Syn protein is actually initiated by the interaction of the intrinsically disordered Syn monomers. In the quiescent phase, first-order rate constants were measured and analysis showed that Syn protein aggregation and disaggregation occur simultaneously. At lower temperatures, early protein disaggregation outweighs protein aggregation whereas at higher temperatures protein disaggregation and aggregation are rather similar. It is also need to highlight that the burst phase, while distinct from the quiescent phase, can be considered as a possible structural phase for obtaining details about the aggregation of this specific disordered protein in solution on a very short timescale.

Development of a carbohydrate-binding protein prediction algorithm using structural features of stacking aromatic rings

Carbohydrate-protein interactions play fundamental roles in numerous aspects of biological activities, and the search for new carbohydrate (CHO)-binding proteins (CBPs) has long been a research focus. In this study, through the analysis of CBP structures, we identified significant enrichment of aromatic residues in CHO-binding regions. We further summarized the structural features of these aromatic rings within the CHO-stacking region, namely “exposing” and “proximity” features, and developed a screening algorithm that can identify CHO-stacking Trp (tryptophan) residues based on these two features. Our Trp screening algorithm can achieve high accuracy in both CBP (specificity score 0.93) and CBS (Carbohydrate binding site, precision score 0.77) prediction using experimentally determined protein structures. We also applied our screening algorithm on AlphaGO pan-species predicted models and observed significant enrichment of carbohydrate-related functions in predicted CBP candidates across different species. Moreover, through carbohydrate arrays, we experimentally verified the CHO-binding ability of four candidate proteins, which further confirms the robustness of the algorithm. This study provides another perspective on proteome-wide CBP and CBS prediction. Our results not only help to reveal the structural mechanism of CHO-binding, but also provide a pan-species CBP dataset for future CHO-protein interaction exploration.

Molecular determinants of neuropeptide-mediated activation mechanisms in tachykinin NK1 and NK2 receptors

Substance P and neurokinin A are closely related neuropeptides belonging to the tachykinin family. Their receptors are neurokinin 1 receptor (NK1R) and neurokinin 2 receptor (NK2R), G protein-coupled receptors that transmit Gs and Gq-mediated downstream signaling. We investigate the importance of sequence differences at the bottom of the receptor orthosteric site for activity and selectivity, focusing on residues that closely interact with the C-terminal methionine of the peptide ligands. We identify a conserved serine (NK1R-S2977.45) and the position of the tryptophan residue within the canonical “toggle switch” motif, CWxP of TM6, neighboring a phenylalanine in NK1R (NK1R-F2646.51) and a tyrosine in NK2R (NK2R-Y2666.51), giving rise to distinct micro-environments for the neuropeptide C-terminals. Mutating these residues results in dramatic activity changes in both NK1R and NK2R due to a close interaction between the ligand and toggle switch. Structural analysis of active and inactive NKR structures suggest only a minor change in sidechain rotation of toggle switch residues upon activation. However, extensive molecular dynamics simulations of receptor:neuropeptide:G protein complexes indicate that a major, concerted motion happens in the toggle switch tryptophan indole group and the sidechains of the micro-switch motif PIF. This rotation establishes a tight hydrogen bond interaction from the tryptophan indole to the conserved serine (NK1R-S2977.45) and a mainchain carbonyl (NK1R-A2947.41) in the kink of TM7. This interaction facilitates communication with the NPxxY micro-switch motif of TM7, resulting in stabilization of the G protein binding region. NK1R-S2977.45 is consequently identified as a central hub for the activation of NKRs.

Dynamics and Thermodynamics Study of the Adsorption of Lysozyme to 2D SnSe Nanoflake Surfaces with Associated Unfolding

AbstractIn this work, we explore the interaction as well as corona formation and interfacial phenomena of lysozyme (LYZ) and 2D SnSe nanoflake. The optical results indicate formation of ground state complex with an apparent association constant of 1.5 × 103 (mol)−1. The dynamics of the in situ interaction exhibited surface reorganization (t2 = 73.5 min) and unfolding, which were more prominent than binding (t1 = 3 min) for tryptophan residues of LYZ. The formations of soft and hard corona of the LYZ structure were clearly observed from HRTEM. The emission quenching exhibited tertiary deformation of the LYZ structure with positive cooperative binding (n &gt; 1) and dynamics of quenching followed the sigmoidal Boltzmann nature of the energy transfer process. The ultrafast lifetime of the bioconjugate varied from 1.9–1.35 ns with a maximum 63% energy transfer efficiency within 300 min of the corona evolution. The decrement of α helix (58.87%–21.78%) and increment of β sheet (5.27%–23.98%) were observed after interaction, with the conversion of large amount of α helix into β sheet. The ITC results showed that the injection heat results followed two‐site binding model, and the exothermic binding was dominate over the interaction. The second phase binding is driven by larger positive entropy, where the first binding site has a stronger association.

Performance Study and Application of Antioxidant Peptides CHCIWM and CHCPWM

AbstractWhen the level of reactive oxygen species (ROS) increases abnormally in the body, it damages tissue cells and is closely related to the occurrence of various chronic diseases. Antioxidant peptides (APs) are a class of active short peptides with antioxidant capacity, which have great research value. In this paper, two APs were designed and synthesized based on the structural features of APs: CHIWM (AP1) and CHCPWM (AP2). The reactive sites and activities of APs were analyzed using the quantum chemical density functional theory (DFT) method. The reactive sites are all located in the indolyl heterocycle of tryptophan residues, among which AP1 has a smaller energy gap and single point energy. Multiple free radical scavenging assays showed that AP1 and AP2 had stronger antioxidant activity than the positive control, glutathione (GSH). For scavenging DPPH free radicals, the IC50s values were 0.613 mg/mL and 0.606 mg/mL, respectively. In cellular assays, both AP1 and AP2 were able to scavenge ROS in the cells and attenuate cellular damage. In conclusion, AP1 and AP2 designed in this paper have good antioxidant activity and have the potential to be applied in the treatment of chronic diseases caused by cellular oxidative damage.

Candidate Genes for Brown Fiber in Cotton Revealed Among the R2R3-Myb and bHLH-Myc Gene Families

ABSTRACT Naturally colored cotton varieties attract investigators due to their eco-friendly and hypoallergenic properties. Genetic mechanisms, determining control of pigment biosynthesis pathway in cotton fiber, are being intensively studied nowadays. However, the knowledge, explaining control of proanthocyanidins accumulation in cotton fiber by the MBW complex (R2R3-Myb, bHLH-Myc and WD40), is insufficient. In this connection, we focused on the regulatory genes from a number of the most likely ones involved in proanthocyanidins biosynthesis. We used the Sanger sequencing approach to reveal the allelic diversity of the R2R3-Myb and bHLH-Myc genes among contrast genotype accessions including various fiber shades and hypocotyl color. Having used the number of bioinformatics tools, we concluded that the replacement of conserved tryptophan residue abolished the DNA-binding activity of the GhMYB10-A transcription factor and this, probably, can be associated with the light brown fiber phenotype. Deeper studies are required to definitely establish the GhMYB10-A function. However, our achievements can be practically applied in marker-assisted breeding of cotton varieties with naturally colored fiber.

A comprehensive investigation of the nano-[Cu2-(DIP)2-EA] effects on HSA through spectroscopic procedures and computer simulations

In this research, the toxicity of nano-[Cu2-(DIP)2-EA], a metal nano-complex consisting of ellagic acid and bathophenanthroline ligands, on human serum albumin (HSA) at a protein level was investigated. Molecular docking simulations and spectral analyses were conducted in a simulated physiological environment at pH 7.4 to explore the interaction of nano-[Cu2-(DIP)2-EA] with HSA. The results represented an increase in albumin absorption upon exposure to nano-[Cu2-(DIP)2-EA], demonstrating significant interaction between the two compounds. Steady-state and time-resolved fluorescence measurements pointed out that nano-[Cu2-(DIP)2-EA] induced static quenching of the albumin's intrinsic fluorescence with a high binding affinity of approximately 106 mol/L in a 1:1 interaction ratio. The thermodynamic variables clarified that binding of nano-[Cu2-(DIP)2-EA] to albumin occurs spontaneously and primarily driven by van der Waals interactions and H-bonds. The results of the computer simulations and the binding displacement experiments utilizing the site markers warfarin and ibuprofen revealed that nano-[Cu2-(DIP)2-EA] binds to site I within the subdomain IIA of albumin. Circular dichroism analysis elaborated that nano-[Cu2-(DIP)2-EA] slightly perturbed the microenvironment around of tryptophan residues and diminished the α-helix structure stability to a negligible amount.

Design and Synthesis of Novel Indole-Derived N-Methylcarbamoylguanidinyl Chitinase Inhibitors with Significantly Improved Insecticidal Activity.

Chitinases play an important role in the molting process of insects and are potential targets for the development of green insecticides. Based on the feature that the +1/+2 sites in OfChtI, OfChtII, and OfChi-h have tryptophan residues in mismatch-parallel position, a strategy to introduce indole scaffold into chitinase inhibitors was proposed, and multitarget chitinase inhibitors containing N-methylcarbamoylguanidinyl and indole scaffold were successfully synthesized. The inhibitory activity showed that compound 8u exhibited significant inhibitory activity against OfChtI, OfChtII, and OfChi-h, with IC50 values of 0.7, 0.79, and 0.58 μM, and Ki values of 0.05 ± 0.005, 0.065 ± 0.004, and 0.025 ± 0.006 μM, respectively. In vivo insecticidal activity showed that compounds 8a and 8g exhibited excellent insecticidal activity against Plutella xylostella and Mythimna separata, with LC50 values of 0.79 and 9.17 mg/L against P. xylostella, respectively, and 3.58 and 83.09 mg/L against M. separata, respectively, making them the most potent chitinase inhibitors with in vivo insecticidal activity discovered to date. The inhibition mechanism and binding free energy results suggested that N-methylcarbamoylguanidinyl binds to the -1 catalytic site, while additional interactions acquired by π-π stacking and hydrophobic interactions of the indole scaffold with tryptophan increase the binding affinity of the targets to chitinases. This work provides a new direction for the development of chitinase inhibitors with compounds 8a and 8g potentially serving as promising candidates for pesticide development.

Enhancing solution structural analysis of large molecular proteins through optimal stereo array isotope labeling of aromatic amino acids

The observation of side-chain peaks of aromatic amino acids is the prerequisite for a high-resolution three-dimensional structure determination of proteins by NMR. However, it becomes difficult with increasing molecular size due to an increased transverse relaxation and the control of the relaxation pathway is needed to achieve the observation. We demonstrated that even for the large molecular size of 82 kDa Malate synthase G (MSG), the aromatic 13C-1H (CH) peaks of Tryptophan (Trp) and Phenylalanine (Phe) residues can be observed with high quality using a systematic stable isotope labeling scheme, Stereo-Array Isotope Labeling (SAIL) method. However, the sequence specific assignments of these peaks relied on the use of amino acid substitutions, employing an inefficient method that required many isotopes labeled samples. In this study, we developed novel SAIL amino acids that allow for the observation of the aromatic ring δ,ζ and the aliphatic β position peak of Phe residues. The application of TROSY-based experiment to the isolated CH moieties resulted in the successful observation of discernible and resolved CH peaks in Phe residues in MSG. In MSG, the sequence-specific assignments of the backbone and Cβ positions have already been confirmed. Therefore, using this labeling method, the δ and β position peaks of Phe residues can be clearly assigned in a sequence-specific and stereospecific manner through experiments based on intra-residue NOE. Furthermore, the NOESY experiment also allows for the acquisition of information pertaining to the conformation of Phe residues, such as the χ1 dihedral angle, providing valuable insights for the determination of accurate protein structures and in dynamic analysis. This new SAIL amino acids open an avenue to achieve a variety of NMR analysis of large molecular proteins, including a high-resolution structure determination and dynamics and interaction analysis.

Synthesis of Non-canonical Tryptophan Variants via Rh-catalyzed C-H Functionalization of Anilines.

Tryptophan and its non-canonical variants play critical roles in pharmaceutical molecules and enzymes. Facile access to this privileged class of amino acids from readily available building blocks remains a long-standing challenge. Here, we report a regioselective synthesis of non-canonical tryptophans bearing C4-C7 substituents via Rh-catalyzed annulation between structurally diverse tert-butyloxycarbonyl (Boc)-protected anilines and alkynyl chlorides readily prepared from amino acid building blocks. This transformation harnesses Boc-directed C-H metalation and demetalation to afford a wide range of C2-unsubstituted indole products in a redox-neutral fashion. This umpolung approach compared to the classic Larock indole synthesis offers a novel mechanism for heteroarene annulation and will be useful for the synthesis of natural products and drug molecules containing non-canonical tryptophan residues in a highly regioselective manner.