Amino Acid Side Chains Research Articles

Hierarchical Self-Assembly of Short Peptides: Nanostructure Formation, Function Tailoring, and Applications.

This article explores the hierarchical self-assembly of short peptides, which refers to the structured spatial arrangements of these molecules over long distances. This phenomenon is commonly found in nature and has important implications for biological structure and function. Short peptides are preferred for self-assembly because they have the ability to spontaneously create various nanostructures. This process, known as bottom-up assembly, allows for the addition of functional groups at the carboxyl or amine ends of the peptides. These functional groups enable specific functions that are extremely valuable in the fields of biotechnology and biomedicine. This text discusses the basic processes involved in the self-assembly of short peptides, such as the characteristics of amino acid side chains, the categorization of short peptides according to their chemical structure, the influence of intermolecular forces, and the dynamic nature of the self-assembly process. In addition, the paper discusses the various uses of short peptides in the disciplines of biomedicine and optoelectronics, including stimulus-responsive hydrogels, tissue engineering, and drug delivery. The article also suggests rational design principles for controlling the hierarchical self-assembly of short peptides, creating new commercial applications, particularly with functional hydrogels, and offers insights into the future of the discipline.

Impact of Phosphorylation and O-GlcNAcylation on the Binding Affinity of R4 Tau Peptide to Microtubule and Its Conformational Preference upon Dissociation.

Tau is a microtubule (MT)-associated protein that binds to and stabilizes the MTs of neurons. Due to its intrinsically disordered nature, it undergoes several post-translational modifications (PTMs) that are intricately linked to both the physiological and pathophysiological roles of Tau. Prior research has shown phosphorylation and O-GlcNAcylation to have contrasting effects on Tau aggregation; however, the precise molecular mechanisms and potential synergistic effects of these modifications remain elusive. In this article, we study the impact of phosphorylation at S352, and S356, as well as the phosphorylation of O-GlcNAcylation at S356, individually and in combination, on the binding of the R4 (336-367) peptide with MTs by performing classical molecular dynamics (MD) simulations. By analyzing the binding free energies of the Tau-MT complex, we found that both individual and combined phosphorylation at S352 and S356 sites decreased the affinity of the R4 peptide toward MT. Surprisingly, O-GlcNAcylation, a likely neuroprotective modification, at S356 also decreased the binding affinity of Tau to MT similar to the single phosphorylation systems (pS352 or pS356) but was observed to maintain major interactions with MT comparable to unmodified R4. Additionally, we investigated the impact of phosphorylation at both sites and the interplay between phosphorylation at S352 and O-GlcNAcylation at S356, which showed that the latter preserved the interactions and affinity of the Tau with MT better than dual phosphorylation, though still not as effectively as single phosphorylation. These findings suggest that O-GlcNAcylation at residue S356 has a moderate destabilizing effect. We also performed replica-exchange MD simulations of the R4 peptide to understand the changes in conformational preferences upon phosphorylation, O-GlcNAcylation, and a combination of both modifications. Both individual and combined phosphorylation of R4 peptide at S352, and S356, sites induced salt-bridge interactions with positively charged side chains of lysine and arginine amino acids. However, O-GlcNAcylation at S356 induced secondary structural changes on the R4 peptide, leading to the formation of a β-sheet structure, consistent with previous experimental observations. Interestingly, simultaneous phosphorylation at S352 and the phosphorylation of O-GlcNAcylation at S356 resulted in conformations promoting salt-bridges and β-sheets. Thus, our study provides atomistic insights into the impact of PTMs on the binding of Tau peptide to MT and its conformational preferences upon dissociation.

Local structural dynamics of Rad51 protomers revealed by cryo-electron microscopy of Rad51-ssDNA filaments.

Homologous recombination (HR) is a high-fidelity repair mechanism for double-strand breaks. Rad51 is the key enzyme that forms filaments on single-stranded DNA (ssDNA) to catalyze homology search and DNA strand exchange in recombinational DNA repair. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) to ascertain the density map of the wild-type budding yeast Rad51-ssDNA filament bound to ADP-AlF3, achieving a resolution of 2.35 Å without imposing helical symmetry. The model assigned 6 Rad51 protomers, 24 nt of DNA, and 6 bound ADP-AlF3. It shows 6-fold symmetry implying monomeric building blocks, unlike the structure of the Rad51-I345T mutant filament with three-fold symmetry implying dimeric building blocks, for which the structural comparisons provide a satisfying mechanistic explanation. This image analysis enables comprehensive comparisons of individual Rad51 protomers within the filament and reveals local conformational movements of amino acid side chains. Notably, R293 in Loop 1 adopts multiple conformations to facilitate L296 and V331 in separating and twisting the DNA triplets. We also analyzed the crystal structure of Rad51-I345T and the predicted structure of yeast Rad51-K342E using the Rad51-ssDNA structure from this study as a reference.

Dual Inhibitors of SARS-CoV-2 3CL Protease and Human Cathepsin L Containing Glutamine Isosteres Are Anti-CoV-2 Agents.

SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P2 position of a peptidomimetic inhibitor. At the P1 position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir, and PF-835321. Our inhibitors contain glutamine isosteres at the P1 position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from Ki = 0.6-18 nM (cathepsin L) and Ki = 2.6-124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC50 = 0.47-15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys145, and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P3 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.

Cell-Active, Arginine-Targeting Irreversible Covalent Inhibitors for Non-Kinases and Kinases.

Targeted covalent inhibitors (TCIs) play an essential role in the fields of kinase research and drug discovery. TCI strategies to target more common amino acid side-chains have yet to be demonstrated. Targeting other amino acids would also expand the pharmaceutical industry's toolbox for targeting other tough-to-drug proteins. We report herein a glyoxal-based, arginine-reactive strategy to generate potent and selective small-molecule TCIs of Mcl-1 (an important anti-apoptotic protein) by selectively targeting the conserved arginine (R263) in the protein. We further validated the generality of this strategy by developing glyoxal-based, irreversible covalent inhibitors of AURKA (a cancer-related kinase) that showed exclusive reactivity with a solvent-exposed arginine (R220) of this enzyme. We showed the resulting compounds were potent, selective and cell-active, capable of covalently engaging endogenous AURKA in MV-4-11 cells with long residence time. Finally, we showed the potential application of glyoxal-based TCIs in targeting an acquired drug-resistance mutant of ALK kinase (G1202R).

Advances in bacterial glycoprotein engineering: A critical review of current technologies, emerging challenges, and future directions.

Protein glycosylation, which involves the addition of carbohydrate chains to amino acid side chains, imparts essential properties to proteins, offering immense potential in synthetic biology applications. Despite its importance, natural glycosylation pathways present several limitations, highlighting the need for new tools to better understand glycan structures, recognition, metabolism, and biosynthesis, and to facilitate the production of biologically relevant glycoproteins. The field of bacterial glycoengineering has gained significant attention due to the ongoing discovery and study of bacterial glycosylation systems. By utilizing protein glycan coupling technology, a wide range of valuable glycoproteins for clinical and diagnostic purposes have been successfully engineered. This review outlines the recent advances in bacterial protein glycosylation from the perspective of synthetic biology and metabolic engineering, focusing on the development of new glycoprotein therapeutics and vaccines. We provide an overview of the production of high-value, customized glycoproteins using prokaryotic glycosylation platforms, with particular emphasis on four key elements: (i) glycosyltransferases, (ii) carrier proteins, (iii) glycosyl donors, and (iv) host bacteria. Optimization of these elements enables precise control over glycosylation patterns, thus enhancing the potential of the resulting products. Finally, we discuss the challenges and future prospects of leveraging synthetic biology technologies to develop microbial glyco-factories and cell-free systems for efficient glycoprotein production.

Non-enzymatic posttranslational protein modifications in protein aggregation and neurodegenerative diseases.

Highly reactive metabolic intermediates and other small molecules frequently react with amino acid side chains, leading to non-enzymatic posttranslational modifications (nPTMs) of proteins. The abundance of these modifications increases under high metabolic activity or stress conditions and can dramatically impact protein structure and function. Although protein quality control mechanisms typically mitigate the effects of these impaired proteins, in long-lived and degradation-resistant proteins, nPTMs accumulate. In some cases, such as cataract development and diabetes, clear links between nPTMs, aging, and disease progression have been established. In neurodegenerative diseases such as Alzheimer's and Parkinson's disease, a key question is whether accumulation of nPTMs is a cause or consequence of protein aggregation. This review focuses on major nPTMs found on proteins with central roles in neurodegenerative diseases such as α-synuclein, β-amyloid, and tau. We summarize current knowledge on the formation of these modifications and discuss their potential impact on disease onset and progression. Additionally, we examine what is known to date about how nPTMs impair cellular detoxification, repair, and degradation systems. Finally, we critically discuss the available methodologies to systematically investigate nPTMs at the molecular level and outline suitable approaches to study their effects on protein aggregation. We aim to foster more research into the role of nPTMs in neurodegeneration by adapting methodologies that have proven successful in studying enzymatic posttranslational modifications. Specifically, we advocate for site-specific incorporation of these modifications into target proteins using advanced chemical and molecular biology techniques.

A modular and extensible CHARMM-compatible model for all-atom simulation of polypeptoids.

Peptoids (N-substituted glycines) are a class of sequence-defined synthetic peptidomimetic polymers with applications including drug delivery, catalysis, and biomimicry. Classical molecular simulations have been used to predict and understand the conformational dynamics of single chains and their self-assembly into morphologies including sheets, tubes, spheres, and fibrils. The CGenFF-NTOID model based on the CHARMM General Force Field has demonstrated success in accurate all-atom molecular modeling of peptoid structure and thermodynamics. Extension of this force field to new peptoid side chains has historically required reparameterization of side chain bonded interactions against abinitio data. This fitting protocol improves the accuracy of the force field but is also burdensome and precludes modular extensibility of the model to arbitrary peptoid sequences. In this work, we develop and demonstrate a Modular Side Chain CGenFF-NTOID (MoSiC-CGenFF-NTOID) as an extension of CGenFF-NTOID employing a modular decomposition of the peptoid backbone and side chain parameterizations, wherein arbitrary side chains within the large family of substituted methyl groups (i.e., -CH3, -CH2R, -CHRR', and -CRR'R″) are directly ported from CGenFF. We validate this approach against abinitio calculations and experimental data to develop a MoSiC-CGenFF-NTOID model for all 20 natural amino acid side chains along with 13 commonly used synthetic side chains and present an extensible paradigm to efficiently determine whether a novel side chain can be directly incorporated into the model or whether refitting of the CGenFF parameters is warranted. We make the model freely available to the community along with a tool to perform automated initial structure generation.

Multiplex Trifluoromethyl and Hydroxyl Radical Chemistry Enables High-Resolution Protein Footprinting.

Hydroxyl radical-based protein footprinting (HRPF) coupled with mass spectrometry is a valuable medium-resolution technique in structural biology, facilitating the assessment of protein structure and molecular-level interactions in solution conditions. In HRPF with X-rays (XFP), hydroxyl radicals generated by water radiolysis covalently label multiple amino acid (AA) side chains. However, HRPF technologies face challenges in achieving their full potential due to the broad (>103) dynamic range of AA reactivity with •OH and difficulty in detecting slightly modified residues, most notably in peptides with highly reactive residues like methionine, or where all residues have low •OH reactivities. To overcome this limitation, we developed a multiplex labeling chemistry that utilizes both CF3 radicals (•CF3) produced from a trifluoromethylation (TFM) reagent and OH radicals (•OH), under controlled and optimized radiolysis doses generated by X-rays. We optimized the dual •CF3/•OH chemistry using model peptides and proteins, thereby extending the existing •OH labeling platform to incorporate simultaneous •CF3 labeling. We labeled >50% of the protein sequence and >80% of protein solvent-accessible AAs via multiplex TFM labeling resulting in high-resolution footprinting, primarily by enhancing the labeling of AAs with low •OH reactivity via the •CF3 channel, while labeling moderate and highly •OH-reactive AAs in both •CF3 and •OH channels. Moreover, the low reactivity of methionine with •CF3 enabled the detection and quantification of additional AAs labeled by •CF3 within methionine-containing peptides. Finally, we found that the solvent accessibility of protein AAs directly correlated with •CF3 labeling, demonstrating that multiplex TFM labeling enables a high-resolution assessment of molecular interactions for enhanced HRPF.

Positive regulation of a LuxR family protein, MilO, in mildiomycin biosynthesis.

Mildiomycin is a representative peptidyl nucleoside antibiotic and was first isolated from Streptoverticillium rimofaciens, which has been used as an important biological agent to control powdery mildew in plants. Despite its importance, the biosynthetic pathways and regulatory mechanisms remain to be fully elucidated. In this study, we identified MilO as a positive pathway-specific regulator of mildiomycin biosynthesis in the heterologous host Streptomyces avermitilis. Gene disruption of milO resulted in almost loss of mildiomycin production, and it was restored to the level comparable to that in the wild-type strain in complemented strain. Overexpression of milO using host native promoter rpsJp, engineered promotor SP44, and kasOp* led to a 50%, 6.5-fold, and 9.2-fold increase in mildiomycin production compared with the wild-type strain, respectively. Quantitative real-time PCR and electrophoretic mobility shift assay (EMSA) experiments revealed that MilO directly enhances the transcription of the milA gene by 20 folds after 48 h fermentation and indirectly regulates the transcription levels of other genes from milB to milM. Using DNase I footprinting assays, milO was revealed to bind to a 44 bp DNA sequence of the milA promoter region. The binding region consists of three imperfect direct repeats of TGTC(N)3CGGT separated by two-nucleotide spacers and each repeat is important to efficient binding to MilO. In addition, we identified two related compounds by overexpressing milO in a structural gene milN-deficient mutant. Taken together, this study indicates that pathway-specific regulator MilO is essential for mildiomycin biosynthesis and provides an effective strategy to improve the production of mildiomycin and its intermediates.IMPORTANCEAs an important biological agent to control powdery mildew on plants, mildiomycin has been commercialized and used in various plants. However, its regulatory mechanisms and biosynthetic pathways remain unknown. This study provides new insights into the regulation of mildiomycin biosynthesis through MilO, a LuxR family protein that modulates mildiomycin production by directly enhancing the transcription of milA. The yield of mildiomycin was significantly improved by overexpressing milO in a heterologous host. In addition, the positive regulatory effect of milO helped to discover two related compounds, which provide important clues for the timing of uploading of two amino acid side chains during mildiomycin biosynthesis for the first time. In brief, our findings on transcriptional regulation of mildiomycin biosynthesis by milO will be valuable to further increase the yield of mildiomycin and explore its biosynthetic pathways.

Synthesis, Antibacterial Properties and Molecular Docking of Nitrobenzoylthiourea Compounds and their Copper(II) Complex

The rise of multidrug-resistant microbial pathogens has increased the demand for highly effective antibiotics. Five nitrobenzoylthiourea ligands (1–5) with amino acid side chains and their corresponding Cu(II) complexes (6–10) were synthesised with yields ranging from 43% to 90%. The successful synthesis of ligands 1-5 were confirmed by the absence of the ν(NCS) stretching band and the presence of the ν(NH) band, indicating the complete reaction of all (NCS) with a series of amino acids as well as the appearance of two N-H signals in the 1H NMR spectra of all the synthesised ligands. On the other hand, the shift of the (C=O) carboxylic peaks in the Cu(II) complexes suggested successful coordination of ligands to the metal ion via the carboxylate group. The antibacterial activities of these compounds were tested against six bacteria: Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa using the disc diffusion method. The Cu(II) complexes (6-10) exhibited enhanced antibacterial activity compared to the ligands (1-5), especially against gram-negative bacteria (E. coli, K. pneumoniae, and P. aeruginosa). For example, compound 4 showed moderate activity against K. pneumoniae with a 14 mm inhibition zone while its Cu(II) complexes, 8 recorded better inhibition against K. pneumoniae with a 16 mm inhibition zone. Molecular docking studies on all complexes (6-10) also revealed higher binding affinity with targeted proteins with binding energy between -10.4 kcal/mol to -9.0 kcal/mol, in comparison with ligand 2 and 4 with the binding energy of only -7.7 kcal/mol (against S. aureus) and -6.9 kcal/mol (against K. pneumoniae). The enhanced antibacterial activity of all complexes correlates with the higher binding affinity obtained for all complexes. Hence, this study concludes that the nitrobenzoylthiourea derivatives, and particularly their Cu(II) complexes can show potential as antibacterial agent although more thorough investigation are required to develop these compounds into useful drugs.

Pyridoxal 5'-Phosphate (PLP)-Dependent β- and γ-Substitution Reactions Forming Nonproteinogenic Amino Acids in Natural Product Biosynthesis.

Living organisms synthesize various nonproteinogenic amino acids (NPAAs) as the building blocks of natural products. These NPAAs are often biosynthesized by pyridoxal 5'-phosphate (PLP)-dependent enzymes, which catalyze β- or γ- substitutions. These enzymes contribute to the structural diversification of NPAAs by installing new functional groups to amino acid side chains. Recent developments in genome mining have led to the identification of various PLP-dependent enzymes catalyzing β- or γ- substitutions, which form NPAAs in secondary metabolism. This short review summarizes recently investigated PLP-enzymes catalyzing β- or γ-substitutions in the biosynthesis of NPAAs by covering the literature published from 2015 through 2024.

Divergent Metabolism of Cyclo-l-Trp-l-Leu in Streptomyces albofaciens by Hydroxylation and Nucleobase Transfer with Two Cytochrome P450 Enzymes.

A three-gene salb cluster from Streptomyces albofaciens was proven to be responsible for the formation of cyclo-l-Trp-l-Leu (cWL) derivatives. An Escherichia coli strain harboring the cyclodipeptide synthase (CDPS) gene salbA produced cWL. Expression of the whole cluster or genes of various combinations in Streptomyces coelicolor revealed different metabolites of cWL by two cytochrome P450 enzymes. Isolation and structure elucidation proved the conversion of cWL to guatrypleumycine A by nucleobase transfer with SalbB and to cyclo(trans-10-hydroxy-l-Trp-l-Leu) by hydroxylation with SalbC. Incubation with 15NH4Cl supported the incorporation of guanine in guatrypleumycine A and an X-ray crystallographic study confirmed the stereospecific hydroxylation at C-10 of the tryptophanyl residue. Cultivation of the salbB or salbC expression strains with different substrates further proved the divergent metabolisms of cWL. To the best of our knowledge, SalbC is the first report of the P450 enzyme from CDPS-associated pathways to catalyze β-hydroxylation at the amino acid side chain.

Insights through molecular modeling into the structural requirement of Phenyl(6-phenylpyrazin-2-yl)methanone derivatives as aldose reductase inhibitors

Aldose reductase, play an important role in the pathogenesis of diabetic complications such as neuropathy, nephropathy and retinopathy. Therefore design of aldose reductase inhibitors has received considerable attention. In the present study molecular insights were explored through docking and QSAR. The quantification of the structural features of Phenyl(6-phenylpyrazin-2-yl)methanone analogs inferred that atomic Senderson electronegativity, van der Waals volume and charge distribution on the molecule are decisive for the aldose reductase inhibitory activity. Moreover, docking study depicted that benzonoid system of scaffold is crucial for - interaction with the side chain of key aromatic amino acids i.e. TRP111 and TRP20. These structural insights might provide significant roadmap for continuing search of potential ARIs prior to synthesis.

Molecular Structure of Paraoxonase-1 and Its Modifications in Relation to Enzyme Activity and Biological Functions-A Comprehensive Review.

PON1 is a Ca2+-dependent enzyme that indicates a hydrolytic activity towards a broad spectrum of substrates. The mechanism of hydrolysis catalyzed by this enzyme is poorly understood. It was shown that the active site of PON1 is highly dynamic. The catalytic center of this enzyme consists of side chains of amino acids binding two calcium ions, from which the first one performs a structural function and the other one is responsible for the catalytic properties of PON1. This review summarizes available information on the structure of PONs, the role of amino acids located in the active site in specificity, and multiple substrate affinity of enzymes for understanding and explaining the basis of the physiological function of PONs. Moreover, in this paper, we described the changes in the structure of PONs induced by environmental and genetic factors and their association with diseases. The detoxification efficiency depends on the polymorphism of the PON1 gene, especially Q192R. However, data on the association between single-nucleotide polymorphisms (SNPs) in the PON1 gene and cardiovascular or neurodegenerative diseases are insufficient. The reviewed papers may confirm that PON1 is a very promising tool for diagnostics, but further studies are required.

Amino Acid Transfer Free Energies Reveal Thermodynamic Driving Forces in Biomolecular Condensate Formation.

The self-assembly of intrinsically disordered proteins into biomolecular condensates shows a dependence on the primary sequence of the protein, leading to sequence-dependent phase separation. Methods to investigate this sequence-dependent phase separation rely on effective residue-level interaction potentials that quantify the propensity for the residues to remain in the dilute phase versus the dense phase. The most direct measure of these effective potentials are the distribution coefficients of the different amino acids between the two phases, but due to the lack of availability of these coefficients, proxies, most notably hydropathy, have been used. However, recent work has demonstrated the limitations of the assumption of hydropathy-driven phase separation. In this work, we address this fundamental gap by calculating the transfer free energies associated with transferring each amino acid side chain analog from the dilute phase to the dense phase of a model biomolecular condensate. We uncover an interplay between favorable protein-mediated and unfavorable water-mediated contributions to the overall free energies of transfer. We further uncover an asymmetry between the contributions of positive and negative charges in the driving forces for condensate formation. The results presented in this work provide an explanation for several non-trivial trends observed in the literature and will aid in the interpretation of experiments aimed at elucidating the sequence-dependent driving forces underlying the formation of biomolecular condensates.

Structural Convergence and Water-Mediated Substrate Mimicry Enable Broad Neuraminidase Inhibition by Human Antibodies.

Influenza has been responsible for multiple global pandemics and seasonal epidemics and claimed millions of lives. The imminent threat of a panzootic outbreak of avian influenza H5N1 virus underscores the urgent need for pandemic preparedness and effective countermeasures, including monoclonal antibodies (mAbs). Here, we characterize human mAbs that target the highly conserved catalytic site of viral neuraminidase (NA), termed NCS mAbs, and the molecular basis of their broad specificity. Cross-reactive NA-specific B cells were isolated by using stabilized NA probes of non-circulating subtypes. We found that NCS mAbs recognized multiple NAs of influenza A as well as influenza B NAs and conferred prophylactic protections in mice against H1N1, H5N1, and influenza B viruses. Cryo-electron microscopy structures of two NCS mAbs revealed that they rely on structural mimicry of sialic acid, the substrate of NA, by coordinating not only amino acid side chains but also water molecules, enabling inhibition of NA activity across multiple influenza A and B viruses, including avian influenza H5N1 clade 2.3.4.4b viruses. Our results provide a molecular basis for the broad reactivity and inhibitory activity of NCS mAbs targeting the catalytic site of NA through substrate mimicry.

Substitution of the Axial Type 1 Cu Ligand Affords Binding of a Water Molecule in Axial Position Affecting Kinetics, Spectral, and Structural Properties of the Small Laccase Ssl1.

Multicopper oxidases use Cu ions as cofactors to oxidize various substrates. High reduction potential at Type 1 Cu is considered as crucial for effective catalysis. Previous studies have shown that replacing the axial methionine ligand of the Type 1 Cu with leucine or phenylalanine leads to an increased reduction potential, but not always to higher enzyme activity. Here we present a study on six variants of the small laccase Ssl1 from Streptomyces sviceus, where the axial methionine ligand was substituted, and the effect of the axial ligand on reduction potential, activity, spectral properties and structure was investigated. Absorption, electronic circular dichroism and EPR spectra revealed the presence of a stronger coordinating axial ligand like oxygen, which influences the electronic and catalytic properties more than the nature of the amino acid side chain. The crystal structures of the Ssl1 variants were solved, which show that none of the amino acid side chains coordinate to the Cu. Instead, a water molecule is found in the axial coordination site, which support the spectroscopic data. Our findings highlight the importance of combining structural and spectroscopic methods to investigate the effect of amino acid exchange on multicopper oxidases.

Controlled Helical Organization in Supramolecular Polymers of Pseudo-Macrocyclic Tetrakisporphyrins.

Tetrakisporphyrin monomers with amino acid side chains at each end form intramolecular antiparallel hydrogen-bonds to adopt chirally twisted pseudo-macrocyclic structures that result in right-handed and left-handed (P)- and (M)-conformations. The pseudo-macrocyclic tetrakisporphyrin monomers self-assembled to form supramolecular helical pseudo-polycatenane polymers via head-to-head complementary dimerization of the bisporphyrin cleft units in an isodesmic manner. The formation of one-handed supramolecular helical pseudo-polycatenane polymers was confirmed by circular dichroism (CD) spectroscopy. The methyl and iso-propyl groups at the stereogenic center greatly enhanced the induced circular dichroism in the Soret bands of the supramolecular helical pseudo-polycatenane polymers. The induced CDs were reduced upon the introduction of large iso-butyl and tert-butyl groups. Atomic force microscopy revealed well-grown and long supramolecular helical pseudo-polycatenane polymer chains with chain lengths in the range of 361 to 13.6 nm. The right-handed helical chains were established by the self-assembly of the right-handed (P)-conformation of the pseudo-macrocyclic monomer.

Controlled Helical Organization in Supramolecular Polymers of Pseudo‐Macrocyclic Tetrakisporphyrins

AbstractTetrakisporphyrin monomers with amino acid side chains at each end form intramolecular antiparallel hydrogen‐bonds to adopt chirally twisted pseudo‐macrocyclic structures that result in right‐handed and left‐handed (P)‐ and (M)‐conformations. The pseudo‐macrocyclic tetrakisporphyrin monomers self‐assembled to form supramolecular helical pseudo‐polycatenane polymers via head‐to‐head complementary dimerization of the bisporphyrin cleft units in an isodesmic manner. The formation of one‐handed supramolecular helical pseudo‐polycatenane polymers was confirmed by circular dichroism (CD) spectroscopy. The methyl and iso‐propyl groups at the stereogenic center greatly enhanced the induced circular dichroism in the Soret bands of the supramolecular helical pseudo‐polycatenane polymers. The induced CDs were reduced upon the introduction of large iso‐butyl and tert‐butyl groups. Atomic force microscopy revealed well‐grown and long supramolecular helical pseudo‐polycatenane polymer chains with chain lengths in the range of 361 to 13.6 nm. The right‐handed helical chains were established by the self‐assembly of the right‐handed (P)‐conformation of the pseudo‐macrocyclic monomer.