Amino Acid Side Chains Research Articles

PH-Induced structural changes in xylanase GH11 from Thermoanaerobacterium saccharolyticum

Background Glycosyl hydrolase 11 (GH11) xylanase is utilized in various in industrial applications such as baking, fruit juice production, pulp processing, and animal feed. Thermophilic GH11 from Thermoanaerobacterium saccharolyticum (TsaGH11) exhibits maximum activity at acid pH with high catalytic efficiency toward beechwood xylan. TsaGH11 activity is pH dependent, exhibiting relative low hydrolase activity at basic pH. However, the effect of a basic pH environment on the structure of TsaGH11 correlated with enzyme activity remains unknown. To understand pH-dependent activity changes, the crystal structure of TsaGH11 at basic pH was determined and compared with that of TsaGH11 at acid pH. Methods TsaGH11 was crystallized at basic pH of 8.5, and the crystal structure was determined at 1.95 Å resolution. The structure, flexibility, and water molecules of TsaGH11 at pH 8.5 and pH 4.3 were compared. Results The open and closed conformations of TsaGH11 at pH 8.5 are reported. Subtle movements of the side chains of amino acids involved in the substrate-binding cleft and catalytic residues were observed. The overall temperature factor of TsaGH11 at pH 8.5 was higher than that at pH 4.6. The position of water molecules near the catalytic residues in TsaGH11 exhibited variations in different pH environments. Conclusions The structural comparison of TsaGH11 at basic and acidic pH offers valuable insights into the pH-dependent functionality of TsaGH11, enhancing our understanding of these structural alterations.

Effects of moderate dielectric barrier discharge (DBD) plasma treatment on the structure, antigenicity, and digestibility of casein

Casein is the primary allergen in the dairy products. This study investigated the effects of moderate dielectric barrier discharge (DBD) plasma treatment on the structure, antigenicity, and digestibility of casein. The results showed that exposure to moderate DBD plasma for 4 min decreased the antigenicity, digestibility, total amino acid content, α-helix content, surface hydrophobicity, and total free sulfhydryl (SH) groups of casein by 18.2%, 9.5%, 42.2%, 4.5%, 51.0%, and 50.9%, respectively. The amino acid side chains of casein were oxidized, but cleavage was not observed on the peptide bone. Moderate DBD plasma treatment induced structural modification and oxidation of the amino acid side chains, which disrupted both the conformational and linear epitopes of casein, resulting in a reduction of its antigenicity. The modification of amino acid may have also disrupted the enzymatic cleavage site of casein, leading to a decrease in its digestibility. This study provides evidence that moderate DBD plasma treatment can decrease the antigenicity of casein, potentially broadening the application of dairy products in the food industry.

Revving up a Designed Copper Nitrite Reductase Using Non-Coded Active Site Ligands.

Herein, we report a three stranded coiled-coil (3SCC) de novo protein containing a type II copper center (CuT2) composed of 6-membered ring N-heterocycles. This design yields the most active homogenous copper nitrite reductase (CuNiR) mimic in water. We achieved this result by controlling three factors. First, previous studies with Nδ and Nε -Methyl Histidine had indicated that a ligand providing pyridine-like electronic character to the copper site was superior to the more donating Nδ for nitrite reduction. By substitution of the parent histidine with the non-coded amino acids pyridyl alanine (3'-Pyridine [3'Py] vs 4'-Pyridine [4'Py]), an authentic pyridine donor was employed without the complications of the coupling of both electronic and tautomeric effects of histidine or methylated histidine. Second, by changing the position of the nitrogen atom within the active site (4'-Pyridine vs. 3'Pyridine) a doubling of the enzyme's catalytic efficiency resulted. This effect was driven exclusivity by substrate binding to the copper site. Third, we replaced the leucine layer adjacent to the active site with an alanine, and the disparity between the 3'Py and 4'Py became more apparent. The decreased steric bulk minimally impacted the 3'Py derivative; however, the 4'Py K m decreased by an order of magnitude (600 mM to 50 mM), resulting in a 40-fold enhancement in the k cat/K m compared to the analogues histidine site and a 1500-fold improvement compared with the initially reported CuNiR catalyst of this family, TRIW-H. When combined with XANES/EXAFS data, the relaxing of the Cu(I) site to a more 2-coordinate Cu(I) like structure in the resting state increases the overall catalytic efficiency of nitrite reduction via the lowering of K m. This study illustrates how by combining advanced spectroscopic methods, detailed kinetic analysis, and a broad toolbox of amino acid side chain functionality, one can rationally design systems that optimize biomimetic catalysis.

Unveiling the affinity-stability relationship in anti-measles virus antibodies: a computational approach for hotspots prediction.

Recent years have seen an uptick in the use of computational applications in antibody engineering. These tools have enhanced our ability to predict interactions with antigens and immunogenicity, facilitate humanization, and serve other critical functions. However, several studies highlight the concern of potential trade-offs between antibody affinity and stability in antibody engineering. In this study, we analyzed anti-measles virus antibodies as a case study, to examine the relationship between binding affinity and stability, upon identifying the binding hotspots. We leverage in silico tools like Rosetta and FoldX, along with molecular dynamics (MD) simulations, offering a cost-effective alternative to traditional in vitro mutagenesis. We introduced a pattern in identifying key residues in pairs, shedding light on hotspots identification. Experimental physicochemical analysis validated the predicted key residues by confirming significant decrease in binding affinity for the high-affinity antibodies to measles virus hemagglutinin. Through the nature of the identified pairs, which represented the relative hydropathy of amino acid side chain, a connection was proposed between affinity and stability. The findings of the study enhance our understanding of the interactions between antibody and measles virus hemagglutinin. Moreover, the implications of the observed correlation between binding affinity and stability extend beyond the field of anti-measles virus antibodies, thereby opening doors for advancements in antibody research.

Switchable Coacervate Formation via Amino Acid Functionalization of Poly(dehydroalanine).

Our group recently developed a family of side-chain amino acid-functionalized poly(S-alkyl-l-homocysteines), Xaa-CH (Xaa = generic amino acid), which possess the ability to form environmentally responsive coacervates in water. In an effort to further study how the molecular structure affects polypeptide coacervate formation, we prepared side-chain amino acid-functionalized poly(S-alkyl-rac-cysteines), Xaa-rac-C, via post-polymerization modification of poly(dehydroalanine), ADH. The use of the ADH platform allowed straightforward synthesis of a diverse range of side-chain amino acid-functionalized polypeptides via direct reaction of unprotected l-amino acid 2-mercaptoethylamides with ADH. Despite their differences in the main-chain structure, we found that Xaa-rac-C can form coacervates with properties similar to those seen with Xaa-CH. These results suggest that the incorporation of side-chain amino acids onto polypeptides may be a way to generally favor coacervation. The incorporation of l-methionine in Met-rac-C allowed the preparation of coacervates with improved stability against high ionic strength media. Further, the presence of additional thioether groups in Met-rac-C resulted in an increased solubility change upon oxidation allowing facile reversible redox switching of coacervate formation in aqueous media.

A Comprehensive Review of Patented Antimicrobial Peptides from Amphibian Anurans.

Since the 1980s, studies of antimicrobial peptides (AMPs) derived from anuran skin secretions have unveiled remarkable structural diversity and a wide range of activities. This study explores the potential of these peptides for drug development by examining granted patents, amino acid modifications related to patented peptides, and recent amphibians' taxonomic updates influencing AMP names. A total of 188 granted patents related to different anuran peptides were found, with Asia and North America being the predominant regions, contributing 65.4% and 15.4%, respectively. Conversely, although the Neotropical region is the world's most diversified region for amphibians, it holds only 3.7% of the identified patents. The antimicrobial activities of the peptides are claimed in 118 of these 188 patents. Additionally, for 160 of these peptides, 66 patents were registered for the natural sequence, 69 for both natural and derivative sequences, and 20 exclusively for sequence derivatives. Notably, common modifications include alterations in the side chains of amino acids and modifications to the peptides' N- and C-termini. This review underscores the biomedical potential of anuran-derived AMPs, emphasizing the need to bridge the gap between AMP description and practical drug development while highlighting the urgency of biodiversity conservation to facilitate biomedical discoveries.

Cysteine Is the Only Universally Affected and Disfavored Proteomic Amino Acid under Oxidative Conditions in Animals.

Oxidative modifications of amino acid side chains in proteins are a hallmark of oxidative stress, and they are usually regarded as structural damage. However, amino acid oxidation may also have a protective effect and may serve regulatory or structural purposes. Here, we have attempted to characterize the global redox role of the 20 proteinogenic amino acids in animals by analyzing their usage frequency in 5 plausible evolutionary paradigms of increased oxidative burden: (i) peroxisomal proteins versus all proteins, (ii) mitochondrial proteins versus all proteins, (iii) mitochondrially encoded respiratory chain proteins versus all mitochondrial proteins, (iv) proteins from long-lived animals versus those from short-lived animals, and (v) proteins from aerobic, free-living animals versus those from facultatively anaerobic animals. We have found that avoidance of cysteine in the oxidative condition was the most pronounced and significant variation in the majority of comparisons. Beyond this preeminent pattern, only local signals were observed, primarily increases in methionine and glutamine as well as decreases in serine and proline. Hence, certain types of cysteine oxidation appear to enforce its proteome-wide evolutionary avoidance despite its essential role in disulfide bond formation and metal ligation. The susceptibility to oxidation of all other amino acids appears to be generally unproblematic, and sometimes advantageous.

DNA three-way junction structures with amino acid side chains prepared via post-synthetic amide condensation

DNA three-way junction (3WJ) structures with three amino acid side chains in the core have been synthesized via post-synthetic DNA modification. Amide condensation reactions of oligonucleotides containing 2′-aminouridine with activated esters yielded DNA strands modified with His, Cys and Asp side chains to form modified 3WJs. Even a 3WJ with three negatively charged Asp side chains formed stably at room temperature. Furthermore, DNA hybridization alone placed two (His and Asp) and three (His, Cys, and Asp) side chains within the 3WJs, indicating that the DNA 3WJs are a useful platform for spatial arrangement of amino acid side chains.

Egg White Protein-Proanthocyanin Complexes Stabilized Emulsions: Investigation of Physical Stability, Digestion Kinetics, and Free Fatty Acid Release Dynamics.

Egg white proteins pose notable limitations in emulsion applications due to their inadequate wettability and interfacial instability. Polyphenol-driven alterations in proteins serve as an effective strategy for optimizing their properties. Herein, covalent and non-covalent complexes of egg white proteins-proanthocyanins were synthesized. The analysis of structural alterations, amino acid side chains and wettability was performed. The superior wettability (80.00° ± 2.23°) and rigid structure (2.95 GPa) of covalent complexes established favorable conditions for their utilization in emulsions. Furthermore, stability evaluation, digestion kinetics, free fatty acid (FFA) release kinetics, and correlation analysis were explored to unravel the impact of covalent and non-covalent modification on emulsion stability, dynamic digestion process, and interlinkages. Emulsion stabilized by covalent complex exhibited exceptional stabilization properties, and FFA release kinetics followed both first-order and Korsmeyer-Peppas models. This study offers valuable insights into the application of complexes of proteins-polyphenols in emulsion systems and introduces an innovative approach for analyzing the dynamics of the emulsion digestion process.

The logic of protein post-translational modifications (PTMs): Chemistry, mechanisms and evolution of protein regulation through covalent attachments.

Protein post-translational modifications (PTMs) play a crucial role in all cellular functions by regulating protein activity, interactions and half-life. Despite the enormous diversity of modifications, various PTM systems show parallels in their chemical and catalytic underpinnings. Here, focussing on modifications that involve the addition of new elements to amino-acid sidechains, I describe historical milestones and fundamental concepts that support the current understanding of PTMs. The historical survey covers selected key research programmes, including the study of protein phosphorylation as a regulatory switch, protein ubiquitylation as a degradation signal and histone modifications as a functional code. The contribution of crucial techniques for studying PTMs is also discussed. The central part of the essay explores shared chemical principles and catalytic strategies observed across diverse PTM systems, together with mechanisms of substrate selection, the reversibility of PTMs by erasers and the recognition of PTMs by reader domains. Similarities in the basic chemical mechanism are highlighted and their implications are discussed. The final part is dedicated to the evolutionary trajectories of PTM systems, beginning with their possible emergence in the context of rivalry in the prokaryotic world. Together, the essay provides a unified perspective on the diverse world of major protein modifications.

Using Redox Proteomics to Gain New Insights into Neurodegenerative Disease and Protein Modification.

Antioxidant defenses in biological systems ensure redox homeostasis, regulating baseline levels of reactive oxygen and nitrogen species (ROS and RNS). Oxidative stress (OS), characterized by a lack of antioxidant defenses or an elevation in ROS and RNS, may cause a modification of biomolecules, ROS being primarily absorbed by proteins. As a result of both genome and environment interactions, proteomics provides complete information about a cell's proteome, which changes continuously. Besides measuring protein expression levels, proteomics can also be used to identify protein modifications, localizations, the effects of added agents, and the interactions between proteins. Several oxidative processes are frequently used to modify proteins post-translationally, including carbonylation, oxidation of amino acid side chains, glycation, or lipid peroxidation, which produces highly reactive alkenals. Reactive alkenals, such as 4-hydroxy-2-nonenal, are added to cysteine (Cys), lysine (Lys), or histidine (His) residues by a Michael addition, and tyrosine (Tyr) residues are nitrated and Cys residues are nitrosylated by a Michael addition. Oxidative and nitrosative stress have been implicated in many neurodegenerative diseases as a result of oxidative damage to the brain, which may be especially vulnerable due to the large consumption of dioxygen. Therefore, the current methods applied for the detection, identification, and quantification in redox proteomics are of great interest. This review describes the main protein modifications classified as chemical reactions. Finally, we discuss the importance of redox proteomics to health and describe the analytical methods used in redox proteomics.

Amino‐Acid Side‐Chain Nanoarchitectonics for Tuning the Chiroptical Properties and Supramolecular Structure of Pentameric Oligothiophenes

AbstractOligothiophenes with specific photophysical properties and molecular organization are of great interest, since this class of materials are used in organic electronics and bioelectronics, as well as biosensing. Herein, 8 different pentameric oligothiophenes, denoted proteophenes, with different amino acid substitution patterns at distinct positions along the thiophene backbone were investigated. Spectroscopic and microscopic studies of the ligands revealed the formation of optically active self‐assembled materials under acidic or basic conditions. The distinct photophysical characteristics, including induced circular dichroism, as well as the supramolecular structures of the assemblies deduced from light scattering and transmission electron microscopy, were highly influenced by the positioning of distinct amino acid moieties along the thiophene backbone. Proteophenes functionalized with only glutamate residues or these functionalities in combination with hydrophobic valine moieties formed fibrillar structures with excellent chiroptical properties under acidic conditions. In addition, the amino acid functionality at the β‐position of distinct thiophene moieties influenced the induced circular dichroism pattern observed from the proteophenes. Overall, the obtained results demonstrate how changes in the position of various amino acid functionalities, as well as the chemical nature of the amino acid side chain functionality greatly affect the optical properties as well as the architecture of the self‐assembled materials.

Thermodynamics of Metal-Acetate Interactions.

Metal ions play crucial roles in protein- and ligand-mediated interactions. They not only act as catalysts to facilitate biological processes but are also important as protein structural elements. Accurately predicting metal ion interactions in computational studies has always been a challenge, and various methods have been suggested to improve these interactions. One such method is the 12-6-4 Lennard-Jones (LJ)-type nonbonded model. Using this model, it has been possible to successfully reproduce the experimental properties of metal ions in aqueous solution. The model includes induced dipole interactions typically ignored in the standard 12-6 LJ nonbonded model. In this we expand the applicability of this model to metal ion-carboxylate interactions. Using 12-6-4 parameters that reproduce the solvation free energies of the metal ions leads to an overestimation of metal ion-acetate interactions, thus, prompting us to fine-tune the model to specifically handle the latter. We also show that the standard 12-6 LJ model significantly falls short in reproducing the experimental binding free energy between acetate and 11 metal ions (Ni(II), Mg(II), Cu(II), Zn(II), Co(II), Cu(I), Fe(II), Mn(II), Cd(II), Ca(II), and Ag(I)). In this study, we describe optimized C4 parameters for the 12-6-4 LJ nonbonded model to be used with three widely employed water models (Transferable Intermolecular Potential with 3 Points (TIP3P), Simple Point Charge Extended (SPC/E), and Optimal Point Charge (OPC) water models). These parameters can accurately match the experimental binding free energy between 11 metal ions and acetate. These parameters can be applied to the study of metalloproteins and transition metal ion channels and transporters, as acetate serves as a representative of the negatively charged amino acid side chains from aspartate and glutamate.

TRNA: The operational RNA code and protein folding

tRNA: The operational RNA code and protein folding Charles W. Carter, Jr., from the Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, relates molecular recognition used in genetic coding to structures of aminoacyl-tRNA synthetases and their cognate tRNAs. Genetic coding is simultaneously widely understood and little appreciated. Widely understood because Marshall Nierenberg worked out the codon assignments within a decade or so of the double helical structure of DNA by Watson and Crick (1). Little appreciated because a code, as far as we know, is something Nature had never produced – a symbolic mapping of the physical chemistry of the 20 amino acid side chains. The Morse code is a widely appreciated example. Like inheritance, the genetic code is a property of biology.

Design of C2-symmetric pseudopeptides for in vivo detection of Cu(II) through controlled supramolecular nano-assembly.

Pseudopeptides are emerging next-generation soft bioinspired materials for biological applications. Therefore, a new class of C2-symmetric L-valine-derived pseudopeptides has been designed and developed. The newly developed pseudopeptides exhibit intracellular Cu(II) ion detection in live-cell fluorescence studies on RAW264.7 cells. We find that the changes in the amino acid side chain in desired pseudopeptidic moieties lead to a drastic change in their selectivity towards different metal ions. The L-valine-derived pseudopeptides exhibit selectivity towards Cu(II) ions through turn-off fluorescence, and the L-phenylalanine-derived pseudopeptides exhibit selectivity towards Zn(II) ions through turn-on fluorescence. In addition, the L-valine-derived pseudopeptides show an increase in spherical-shaped structures upon incubation with Cu(II) ions during supramolecular nano-assembly formation. In contrast, the L-phenylalanine-derived pseudopeptides show a decrease in spherical-shaped structures upon adding Zn(II) ions. The judiciously designed L-valine-derived and L-phenylalanine-derived bioinspired pseudopeptides are promising for exploring similar effects in various peptidomimetics in advanced biological applications.

Perspective: intrinsic interactions of metal ions with biological molecules as studied by threshold collision-induced dissociation and infrared multiple photon dissociation.

In this perspective, gas-phase studies of group 1 monocations and group 12 dications with amino acids and small peptides are highlighted. Although the focus is on two experimental techniques, threshold collision-induced dissociation and infrared multiple photon dissociation action spectroscopy, these methods as well as complementary approaches are summarized. The synergistic interplay with theory, made particularly powerful by the small sizes of the systems explored and the absence of solvent and support, is also elucidated. Importantly, these gas-phase methods permit quantitative insight into the structures and thermodynamics of metal cations interacting with biological molecules. Periodic trends in how these interactions vary as the metal cations get heavier are discussed as are quantitative trends with changes in the amino acid side chain and effects of hydration. Such trends allow these results to transcend the limitations associated with the biomimetic model systems.

Plant peptides - redefining an area of ribosomally synthesized and post-translationally modified peptides.

Covering 1965 to February 2024Plants are prolific peptide chemists and are known to make thousands of different peptidic molecules. These peptides vary dramatically in their size, chemistry, and bioactivity. Despite their differences, all plant peptides to date are biosynthesized as ribosomally synthesized and post-translationally modified peptides (RiPPs). Decades of research in plant RiPP biosynthesis have extended the definition and scope of RiPPs from microbial sources, establishing paradigms and discovering new families of biosynthetic enzymes. The discovery and elucidation of plant peptide pathways is challenging due to repurposing and evolution of housekeeping genes as both precursor peptides and biosynthetic enzymes and due to the low rates of gene clustering in plants. In this review, we highlight the chemistry, biosynthesis, and function of the known RiPP classes from plants and recommend a nomenclature for the recent addition of BURP-domain-derived RiPPs termed burpitides. Burpitides are an emerging family of cyclic plant RiPPs characterized by macrocyclic crosslinks between tyrosine or tryptophan side chains and other amino acid side chains or their peptide backbone that are formed by copper-dependent BURP-domain-containing proteins termed burpitide cyclases. Finally, we review the discovery of plant RiPPs through bioactivity-guided, structure-guided, and gene-guided approaches.

Interaction of Au(III) with amino acids: a vade mecum for medicinal chemistry and nanotechnology.

Au(III) is highly reactive. At odds with its reduced counterpart, Au(I), it is hardly present in structural databases. And yet, it is the starting reactant to form gold nanoclusters (AuNCs) and the constitutive component of a new class of drugs. Its reactivity is a world apart from that of the iso-electronic Pt(II) species. Rather than DNA, it targets proteins. Its interaction with amino acid residues is manifold. It can strongly interact with the residue backbones, amino acid side chains and protein ends, it can form appropriate complexes whose stabilization energy reaches up to more than 40 kcal mol-1, it can affect the pKa of amino acid residues, and it can promote charge transfer from the residues to the amount that it is reduced. Here, quantum chemical calculations provide quantitative information on all the processes where Au(III) can be involved. A myriad of structural arrangements are examined in order to determine the strongest interactions and quantify the amount of charge transfer between protonated and deprotonated residues and Au(III). The calculated interaction energies of the amino acid side chains with Au(III) quantitatively reproduce the experimental tendency of Au(III) to interact with selenocysteine, cysteine and histidine and negatively charged amino acids such as Glu and Asp. Also, aromatic residues such as tyrosine and tryptophan strongly interact with Au(III). In proteins, basic pH plays a role in the deprotonation of cysteine, lysine and tyrosine and strongly increases the binding affinity of Au(III) toward these amino acids. The amino acid residues in the protein can also trigger the reduction of Au(III) ions. Sulfur-containing amino acids (cysteine and methionine) and selenocysteine provide almost one electron to Au(III) upon binding. Tyrosine also shows a considerable tendency to act as a reductant. Other amino acids, commonly identified in Au-protein adducts, such as Ser, Trp, Thr, Gln, Glu, Asn, Asp, Lys, Arg and His, possess a notable reducing power toward Au(III). These results and their discussion form a vade mecum that can find application in medicinal chemistry and nanotech applications of Au(III).

Quasi-spherical silver nanoparticles for human prolactin detection by surface-enhanced Raman spectroscopy.

Prolactin is a polypeptide hormone made of 199 amino acids; 50% of the amino acid chain forms helices, and the rest forms loops. This hormone is typically related to initiating and maintaining lactation, although it is also elevated in various pathological conditions. Serum prolactin levels of 2 to 18 ng ml-1 in men, up to 30 ng ml-1 in women, and 10 to 210 ng ml-1 in pregnant women are considered normal. Immunoassay techniques used for detection are susceptible to error in different clinical conditions. Surface-enhanced Raman spectroscopy (SERS) is a technique that allows for obtaining the protein spectrum in a simple, fast, and reproducible manner. Nonetheless, proper characterization of human prolactin's Raman/SERS spectrum at different concentrations has so far not been deeply discussed. This study aims to characterize the Raman spectrum of human prolactin at physiological concentrations using silver nanoparticles (AgNPs) as the SERS substrate. The Raman spectrum of prolactin at 20 ng ul-1 was acquired. Quasi-spherical AgNPs were obtained using chemical synthesis. For SERS characterization, decreasing dilutions of the protein were made by adding deionized water and then a 1 : 1 volume of the AgNPs colloid. For each mixture, the Raman spectrum was determined. The spectrum of prolactin by SERS was obtained with a concentration of up to 0.1 ng ml-1. It showed characteristic bands corresponding to the side chains of aromatic amino acids in the protein's primary structure and the alpha helices of the secondary structure of prolactin. In conclusion, using quasi-spherical silver nanoparticles as the SERS substrate, the Raman spectrum of human prolactin at physiological concentration was determined.

Exploiting the full potential of cryo-EM maps

The Coulomb potential maps generated by electron microscopy (EM) experiments contain not only information about the position but also about the charge state of the atom. This feature of EM maps allows the identification of specific ions and the protonation state of amino acid side chains in the sample. Here, we summarize qualitative observations of charges in EM maps, discuss the difficulties in interpreting the charge in Coulomb potential maps with respect to distinguishing it from radiation damage, and outline considerations to implement the correct charge in fitting algorithms.