Hybrid nanostructures of nitrogen-doped carbon dots and aromatic amino acids: Synthesis, interactions at interfaces and optical properties.

Nitrogen-doped carbon dots (NCD) were synthesized using a simple and fast hydrothermal route, employing citric acid and urea as precursors. The resulting NCDs were non-covalently functionalized (conjugated) with aromatic amino acids, namely phenylalanine (Phe) and tryptophan (Trp). Atomic force microscopy revealed that the NCDs exhibit a disk-like morphology with an average diameter of approximately 60 nm and an average height of about 0.5 nm. Following conjugation, the particle height increased to around 3 nm. UV-vis spectroscopy analysis indicated successful conjugation of the amino acids to the NCD nanostructures. Additionally, DFT numerical calculations based on three differently N-doped clusters were performed to elucidate the nature of the non-covalent interactions between NCDs and the corresponding amino acids. Photoluminescent spectra demonstrated a stable and strong fluorescence signal for both hybrids in the UV region. The most significant changes were observed in the case of Trp-conjugation. In contrast to phenylalanine, the non-covalent bonding of tryptophan to NCDs strongly influenced the visible emission (around 500 nm) originating from surface states of the dots.

Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

Chlorination of Aromatic Amino Acids: Elucidating Disinfection Byproducts, Reaction Kinetics, and Influence Factors.

In the face of ongoing water pollution challenges, the intricate interplay between dissolved organic matter and disinfectants like chlorine gives rise to potentially harmful disinfection byproducts (DBPs) during water treatment. The exploration of DBP formation originating from amino acids (AA) is a critical focus of global research. Aromatic DBPs, in particular, have garnered considerable attention due to their markedly higher toxicity compared to their aliphatic counterparts. This work seeks to advance the understanding of DBP formation by investigating chlorination disinfection and kinetics using tyrosine (Tyr), phenylalanine (Phe), and tryptophan (Trp) as precursors. Via rigorous experiments, a total of 15 distinct DBPs with accurate molecular structures were successfully identified. The chlorination of all three AAs yielded highly toxic chlorophenylacetonitriles (CPANs), and the disinfectant dosage and pH value of the reaction system potentially influence chlorination kinetics. Notably, Phe exhibited the highest degradation rate compared to Tyr and Trp, at both the CAA:CHOCl ratio of within 1:2 and a wide pH range (6.0 to 9.0). Additionally, a neutral pH environment triggered the maximal reaction rates of the three AAs, while an acidic condition may reduce their reactivity. Overall, this study aims to augment the DBP database and foster a deeper comprehension of the DBP formation and relevant kinetics underlying the chlorination of aromatic AAs.

Self-Assembled Materials Based on Fully Aromatic Peptides: The Impact of Tryptophan, Tyrosine, and Dopa Residues.

Peptides are able to self-organize in structural elements including cross-ß structures. Taking advantage of this tendency, in the last decades, peptides have been scrutinized as molecular elements for the development of multivalent supramolecular architectures. In this context, different classes of peptides, also with completely aromatic sequences, were proposed. Our previous studies highlighted that the (FY)3 peptide, which alternates hydrophobic phenylalanine and more hydrophilic tyrosine residues, is able to self-assemble, thanks to the formation of both polar and apolar interfaces. It was observed that the replacement of Phe and Tyr residues with other noncoded aromatic amino acids like 2-naphthylalanine (Nal) and Dopa affects the interactions among peptides with consequences on the supramolecular organization. Herein, we have investigated the self-assembling behavior of two novel (FY)3 analogues with Trp and Dopa residues in place of the Phe and Tyr ones, respectively. Additionally, PEGylation of the N-terminus was analyzed too. The supramolecular organization, morphology, and capability to gel were evaluated using complementary techniques, including fluorescence, Fourier transform infrared spectroscopy, and scanning electron microscopy. Structural periodicities along and perpendicular to the fiber axis were detected by grazing incidence wide-angle X-ray scattering. Finally, molecular dynamics studies provided interesting insights into the atomic structure of the cross-ß that constitutes the basic motif of the assemblies formed by these novel peptide systems.

Improved spectral resolution of [13C,1H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive 13C-labelled precursors.

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive 13C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of 13C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [13C,1H]-HSQC cross-peaks free of one-bond 13C-13C couplings. Selective 13C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.

[Advances in separation and analysis of aromatic amino acids in food].

Amino acids are important building blocks of proteins in the human body, which are involved in many metabolic pathways. Patients with metabolic diseases such as phenylketonuria, tyrosinemia, and hepatic encephalopathy are genetically defective and cannot metabolize aromatic amino acids (AAA) in food; hence, a regular diet may lead to permanent physiological damage. For this reason, it is necessary to restrict the intake of AAA in their daily diet by limiting natural protein intake, while ensuring normal intake of low protein foods and supplementation with low-AAA protein equivalents. Sources of low-AAA protein equivalents currently rely on free amino acid complex mixtures and low-AAA peptides (also known as high-Fischer-ratio peptides), which have better absorption availability and palatability. AAA separation and analysis techniques are essential for the preparation and detection of low-AAA peptides. Researchers in this field have explored a variety of efficient adsorption materials to selectively remove AAA from complex protein hydrolysates and thus prepare low-AAA peptide foods, or to establish analysis strategies for AAA. Covering more than 70 publications on AAA removal and separation in the last decade from Web of Science Core Collection and China National Knowledge Infrastructure, this review analyzes the structural characteristics and physicochemical properties of AAA, and summarizes the technological progress of AAA removal based on adsorbents such as activated carbon and resin. The applications of two-dimensional nanomaterials, molecular imprinting, cyclodextrins, and metal-organic frameworks in AAA adsorption and analysis from three dimensions, i. e., sample pretreatment, chiral separation and adsorption sensing, are also reviewed. The mainstream adsorbents for AAA removal, such as activated carbon, still suffer from poor specificity and cause environmental pollution during post-use treatment. Existing AAA separating materials show impressive selective adsorption capability in food samples and chiral mixtures as well as high sensitivity in adsorption sensing. The development of an efficient detection technology for AAA may help in detecting trace AAA in food and in evaluating chiral AAA adulteration in food samples. By exploring the advantages and disadvantages of each type of technology, we provide support for the advancement of the removal and analysis techniques for AAA.

Clustering of Aromatic Residues in Prion-like Domains Can Tune the Formation, State, and Organization of Biomolecular Condensates.

In immature oocytes, Balbiani bodies are conserved membraneless condensates implicated in oocyte polarization, the organization of mitochondria, and long-term organelle and RNA storage. In Xenopus laevis, Balbiani body assembly is mediated by the protein Velo1. Velo1 contains an N-terminal prion-like domain (PLD) that is essential for Balbiani body formation. PLDs have emerged as a class of intrinsically disordered regions that can undergo various different types of intracellular phase transitions and are often associated with dynamic, liquid-like condensates. Intriguingly, the Velo1 PLD forms solid-like assemblies. Here we sought to understand why Velo1 phase behavior appears to be biophysically distinct from that of other PLD-containing proteins. Through bioinformatic analysis and coarse-grained simulations, we predict that the clustering of aromatic residues and the amino acid composition of residues between aromatics can influence condensate material properties, organization, and the driving forces for assembly. To test our predictions, we redesigned the Velo1 PLD to test the impact of targeted sequence changes in vivo. We found that the Velo1 design with evenly spaced aromatic residues shows rapid internal dynamics, as probed by fluorescent recovery after photobleaching, even when recruited into Balbiani bodies. Our results suggest that Velo1 might have been selected in evolution for distinctly clustered aromatic residues to maintain the structure of Balbiani bodies in long-lived oocytes. In general, our work identifies several tunable parameters that can be used to augment the condensate material state, offering a road map for the design of synthetic condensates.

Altered Peptide Self-Assembly and Co-Assembly with DNA by Modification of Aromatic Residues.

Aromatic residues are widely used as building blocks for driving self-assemblies in natural and designer biomaterials. The noncovalent interactions involving aromatic rings determine proteins' structure and biofunction. Here, we studied the effects of changes in the proximity of the aromatic rings in a self-assembling peptide for modulating interactions involving the aromatic residues. By changing the distance between the aromatic ring and peptide backbone and replacing the side chain with a sulfur atom, we altered the nanostructures and gene transfection efficiency of peptide-DNA co-assemblies. This study demonstrates the significance of subtle alterations in aromatic interactions and facilitates deeper understanding of the aromatic-involving interactions.

Aromatic and aliphatic residues of the disordered region of TDP-43 are on a fast track for self-assembly.

The C-terminal, intrinsically disordered, prion-like domain (PrLD) of TDP-43 promotes liquid condensate and solid amyloid formation. These phase changes are crucial to the normal biological functions of the protein but also for its abnormal aggregation, which is implicated in amyotrophic lateral sclerosis (ALS) and certain dementias. We and other previously found that certain amyloid forms emerge from an intermediate condensed state that acts as a nucleus for fibrillization. To quantitatively ascertain the role of individual residues within TDP-43's PrLD in its early self-assembly we have followed the kinetics of NMR 1H-15N HSQC signal loss to obtain values for the lag time, elongation rate and extent of condensate formation at equilibrium. The results of this analysis represent a robust corroboration that aliphatic and aromatic residues are key drivers of condensate formation.

Mapping the role of aromatic amino acids within a blue-light sensing LOV domain.

Photosensing LOV (Light, Oxygen, Voltage) domains detect and respond to UVA/Blue (BL) light by forming a covalent adduct between the flavin chromophore and a nearby cysteine, via the decay of the flavin triplet excited state. LOV domains where the reactive cysteine has been mutated are valuable fluorescent tools for microscopy and as genetically encoded photosensitisers for reactive oxygen species. Besides being convenient tools for applications, LOV domains without the reactive cysteine (naturally occurring or engineered) can still be functionally photoactivated via formation of a neutral flavin radical. Tryptophans and tyrosines are held as the main partners as potential electron donors to the flavin excited states. In this work, we explore the relevance of aromatic amino acids in determining the photophysical features of the LOV protein Mr4511 from Methylobacterium radiotolerans by introducing point mutations into the C71S variant that does not form the covalent adduct. By using an array of spectroscopic techniques we measured the fluorescence quantum yields and lifetimes, the triplet yields and lifetimes, and the efficiency of singlet oxygen (SO) formation for eleven Mr4511 variants. Insertion of Trp residues at distances between 0.6 and 1.5 nm from the flavin chromophore results in strong quenching of the flavin excited triplet state and, at the shorter distances even of the singlet excited state. The mutation F130W (ca. 0.6 nm) completely quenches the singlet excited state, preventing triplet formation: in this case, even if the cysteine is present, the photo-adduct is not formed. Tyrosines are also quenchers for the flavin excited states, although not as efficient as Trp residues, as demonstrated with their substitution with the inert phenylalanine. For one of these variants, C71S/Y116F, we found that the quantum yield of formation for singlet oxygen is 0.44 in aqueous aerobic solution, vs 0.17 for C71S. Based on our study with Mr4511 and on literature data for other LOV domains we suggest that Trp and Tyr residues too close to the flavin chromophore (at distances less than 0.9 nm) reduce the yield of photoproduct formation and that introduction of inert Phe residues in key positions can help in developing efficient, LOV-based photosensitisers.

Genesis of Neurotoxic Hybrid Nanofibers from the Coassembly of Aromatic Amino Acids.

Considering the relevance of accumulation and self-assembly of metabolites and aftermath of biological consequences, it is important to know whether they undergo coassembly and what properties the resultant hybrid higher-order structures would exhibit. This work reveals the inherent tendency of aromatic amino acids to undergo a spontaneous coassembly process under physiologically mimicked conditions, which yields neurotoxic hybrid nanofibers. Resultant hybrid nanostructures resembled the ß-structured conformers stabilized by H-bonds and π-π stacking interactions, which were highly toxic to human neuroblastoma cells. The hybrid nanofibers also showed strong cross-seeding potential that triggered in vitro aggregation of diverse globular proteins and brain extract components, converting the native structures into cross-ß-rich amyloid aggregates. The heterogenic nature of the hybrid nanofibers seems crucial for their higher toxicity and faster cross-seeding potential as compared to the homogeneous amino acid nanofibers. Our findings reveal the importance of aromaticity-driven optimized intermolecular arrangements for the coassembly of aromatic amino acids, and the results may provide important clues to the fundamental understanding of metabolite accumulation-related complications.

Quantitative helix handedness bias through a single H vs. CH3 stereochemical differentiation.

A novel chiral aromatic δ-amino acid building block was shown to fully induce handedness in quinoline oligoamide foldamers with the possibility of further increasing the bias by combining multiples of these units in the same sequence. Through its incorporation within the helix, both N- and C-termini are still accessible for further functionalisation.

Enhancing the binding of the ß-sheet breaker peptide LPFFD to the amyloid-ß fibrils by aromatic modifications: A molecular dynamics simulation study.

Alzheimer's is a fatal neurodegenerative disease for which there is no cure at present. The disease is characterized by the presence of plaques in the brains of a patient, which are composed mainly of aggregates of the amyloid-ß peptide in the form of ß-sheet fibrils. Here, we investigated the possibility of exploiting the superior binding ability of aromatic amino acids to a particular model of the amyloid-ß fibrils. which is a difficult target for drug design. The ß-sheet breaker peptide LPFFD was modified with aromatic amino acids and its binding to these fibrils was studied. We found that the orientation and the electrostatic complementarity of the modified peptide with respect to the fibrils played a crucial role in determining whether its binding was improved by the aromatic amino acids. The modified LPFFD peptides were able to bind to those fibril residues. which are important in the aggregation of amyloid-ß peptides and thus can potentially inhibit the further aggregation of the amyloid-beta peptides by blocking their interactions. We found that the tryptophan modified LPFFD peptides had the best binding affinities. In most cases, the aromatic amino acids in the N-terminus of the modified peptides made more contacts with the fibrils than those in the C-terminus. We also found that increasing the aromatic content did not significantly improve the binding of the LPFFD peptide to the fibrils. Our study can serve as a basis for the design of novel peptide-based drugs for Alzheimer's disease in which aromatic interactions play an important role.

Halogenation Regulates Supramolecular Chirality at Hierarchical Levels of Self-Assembled N-Terminal Aromatic Amino Acids.

Halogenation brings about dramatic variations to the performance of self-assembled organic species, such as luminescence and crystallinity, but it has seldom been utilized for chirality control. Here we show the halogenation effect of self-assembling organic building units on supramolecular chirality and chiroptical responses. N-terminal aromatic amino acids with different substituted halogen atoms at p-phenylalanine residues self-assembled into one-dimensional fibrous structures. Halogenation induced the emergence of macroscopic chirality regardless of halogen properties like electronegativity, generating exclusive homochiral helical structures. Solid-state X-ray structures and time-dependent density functional theory were utilized for calculated electronic circular dichroism spectra, which evidenced the diverse driving forces to enable chiral molecular arrangements, including H-bonds and halogen bonds. Red-shifted luminescence was observed in brominated building units, giving rise to active circularly polarized luminescence. This work elucidates the multiple roles of halogen in chiral self-assembly systems, which provides insight into the rational control over supramolecular chirality and their chiroptical applications.

Novel quaternary ammonium compounds derived from aromatic and cyclic amino acids: Synthesis, physicochemical studies and biological evaluation.

Novel quaternary ammonium surfactants (QUATs) derived from phenylalaninyl-proline dipeptide with chain length C12 and C14 were synthesised as potential active ingredients to be used in body cleansing formulations. The physicochemical properties and biological activities of the QUATs were determined in both single and in mixed surfactant system with either the conventional anionic surfactant sodium dodecyl sulphate (SDS) or sodium N-dodecyl prolinate. The C12 QUAT derivative showed antagonistic behaviour in both SDS and sodium N-dodecyl prolinate mixed surfactant system. Comparing the mixed system of the C12 QUAT with SDS and sodium N-dodecyl prolinate, it was found that the latter displayed better antibacterial activity together with the lower ocular irritation. The C12 QUAT-sodium N-dodecyl prolinate mixture were non cytotoxic at a concentration corresponding to its MIC value, showing that the mixture was selective towards bacterial cells rather than mammalian cell lines. Diffusion measurements showed that the sodium N-dodecyl prolinate surfactant consisted of 26 molecules per micelle in water but only 3 molecules per micelle in DMSO/water (1:1). On the other hand, C12 QUAT did not form a micelle in DMSO/Water. Membrane permeability studies of the C12 QUAT and sodium N-dodecyl prolinate showed that these surfactants are capable to penetrate into deeper skin layers to exert their antibacterial and cleansing action and hence can be used as a promising candidate as active ingredients in body wash formulations.

Clustering of Aromatic Amino Acid Residues around Methionine in Proteins.

Short-range, non-covalent interactions between amino acid residues determine protein structures and contribute to protein functions in diverse ways. The interactions of the thioether of methionine with the aromatic rings of tyrosine, tryptophan, and/or phenylalanine has long been discussed and such interactions are favorable on the order of 1-3 kcal mol-1. Here, we carry out a new bioinformatics survey of known protein structures where we assay the propensity of three aromatic residues to localize around the [-CH2-S-CH3] of methionine. We term these groups "3-bridge clusters". A dataset consisting of 33,819 proteins with less than 90% sequence identity was analyzed and such clusters were found in 4093 structures (or 12% of the non-redundant dataset). All sub-classes of enzymes were represented. A 3D coordinate analysis shows that most aromatic groups localize near the CH2 and CH3 of methionine. Quantum chemical calculations support that the 3-bridge clusters involve a network of interactions that involve the Met-S, Met-CH2, Met-CH3, and the π systems of nearby aromatic amino acid residues. Selected examples of proposed functions of 3-bridge clusters are discussed.

Aromatic Interactions Drive the Coupled Folding and Binding of the Intrinsically Disordered Sesbania mosaic Virus VPg Protein.

The plant Sesbania mosaic virus [a (+)-ssRNA sobemovirus] VPg protein is intrinsically disordered in solution. For the virus life cycle, the VPg protein is essential for replication and for polyprotein processing that is carried out by a virus-encoded protease. The nuclear magnetic resonance (NMR)-derived tertiary structure of the protease-bound VPg shows it to have a novel tertiary structure with an α-ß-ß-ß topology. The quaternary structure of the high-affinity protease-VPg complex (≈27 kDa) has been determined using HADDOCK protocols with NMR (residual dipolar coupling, dihedral angle, and nuclear Overhauser enhancement) restraints and mutagenesis data as inputs. The geometry of the complex is in excellent agreement with long-range orientational restraints such as residual dipolar couplings and ring-current shifts. A "vein" of aromatic residues on the protease surface is pivotal for the folding of VPg via intermolecular edge-to-face π···π stacking between Trp271 and Trp368 of the protease and VPg, respectively, and for the CH···π interactions between Leu361 of VPg and Trp271 of the protease. The structure of the protease-VPg complex provides a molecular framework for predicting sites of important posttranslational modifications such as RNA linkage and phosphorylation and a better understanding of the coupled folding upon binding of intrinsically disordered proteins. The structural data presented here augment the limited structural data available on viral proteins, given their propensity for structural disorder.

Amino acid-based hydrophobic affinity cryogel for protein purification from ora-pro-nobis (Pereskia aculeata Miller) leaves.

The surfaces of the polyacrylamide cryogels were coated with L-tryptophan (cryogel-Trp) or L-phenylalanine (cryogel-Phe) to enhance crude leaf extract-derived ora-pro-nobis (OPN) protein binding via pseudo-specific hydrophobic interactions. Cryogels functionalized with amino acids were prepared and characterized through morphological, hydrodynamic, and thermal analyses. The adsorption capacities of cryogel-Phe and cryogel-Trp were evaluated in terms of type (sodium sulfate or sodium phosphate) and concentration (0.02 or 0.10 mol∙L-1) of saline solution, pH (4.0, 5.5, or 7.0), and NaCl concentration (0.0 or 0.5 mol∙L-1). The cryogel-Phe presented a higher adsorptive capacity, achieving its maximum value (q = 92.53 mg∙g-1) when the crude OPN crude leaf extract was diluted in sodium sulfate 0.02 mol∙L-1 + NaCl 0.50 mol∙L-1, at pH = 7.0. The dilution rate significantly (p < 0.05) affected the recovered protein amount after the adsorption and elution processes, reaching 94.45% when the feedstock solution was prepared with a crude extract 5 times. The zeta potential for the eluted OPN proteins was 5.76 mV (pH = 3.23) for both dilution rates. The secondary structure composition mainly included ß-sheets (46.50%) and α-helices (13.93%). The cryogel-Phe exhibited interconnected pores ranging 20-300 µm in size, with a Young modulus of 1.51 MPa, and thermal degradation started at 230 °C. These results indicate that the cryogel-Phe exhibited satisfactory properties as promising chromatography support for use in high-throughput purification of crude leaf extract-derived OPN proteins.

Nonenzymatic post-translational modifications in peptides by cold plasma-derived reactive oxygen and nitrogen species.

Cold physical plasmas are emerging tools for wound care and cancer control that deliver reactive oxygen species (ROS) and nitrogen species (RNS). Alongside direct effects on cellular signaling processes, covalent modification of biomolecules may contribute to the observed physiological consequences. The potential of ROS/RNS generated by two different plasma sources (kINPen and COST-Jet) to introduce post-translational modifications (PTMs) in the peptides angiotensin and bradykinin was explored. While the peptide backbone was kept intact, a significant introduction of oxidative PTMs was observed. The modifications cluster at aromatic (tyrosine, histidine, and phenylalanine) and neutral amino acids (isoleucine and proline) with the introduction of one, two, or three oxygen atoms, ring cleavages of histidine and tryptophan, and nitration/nitrosylation predominantly observed. Alkaline and acidic amino acid (arginine and aspartic acid) residues showed a high resilience, indicating that local charges and the chemical environment at large modulate the attack of the electron-rich ROS/RNS. Previously published simulations, which include only OH radicals as ROS, do not match the experimental results in full, suggesting the contribution of other short-lived species, i.e., atomic oxygen, singlet oxygen, and peroxynitrite. The observed PTMs are relevant for the biological activity of peptides and proteins, changing polarity, folding, and function. In conclusion, it can be assumed that an introduction of covalent oxidative modifications at the amino acid chain level occurs during a plasma treatment. The introduced changes, in part, mimic naturally occurring patterns that can be interpreted by the cell, and subsequently, these PTMs allow for prolonged secondary effects on cell physiology.

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids.

In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as ß-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation.