Changes In Tertiary Structure Research Articles

Manothermosonication, high-pressure homogenization, and their combinations with pH-shifting improve the techno-functionality and digestibility of hemp protein

The growing interest in plant-based proteins, particularly hemp protein, has driven the exploration of new methods to address the limitations of its industrial utilization, such as its poor functional properties. This study examined how manothermosonication (MTS) and high-pressure homogenization (HPH) techniques, along with pH-shifting, could enhance the techno-functionality of hemp protein isolate (HPI) and produce functionalized hemp protein nanoaggregates. Optimal processing conditions of MTS were 50 °C, 200 kPa, and 90 s, as determined by response surface methodology. The pH-shifting + MTS and pH-shifting + HPH treatments reduced the size of hemp protein nanoaggregates by 3.2- and 1.9-fold, respectively, compared to the untreated HPI. The changes in secondary (FTIR, CD), tertiary (SH group, fluorescence intensity), and 3D structures (XRD) confirmed modifications of treated hemp proteins. The XRD patterns showed an increase in the %crystallinity values from 21% to 29% upon the pH-shifting + MTS treatment. The combination of pH-shifting + MTS resulted in the highest solubility and digestibility, 2.1- and 1.3-fold higher than the untreated HPI, respectively, followed by the MTS-alone and HPH treatments. In addition, the emulsion properties (emulsion activity, stability, creaming index, and droplet sizes) exhibited the highest values in HPI treated by pH-shifting + MTS and + HPH. The MTS and HPH technologies provide a promising viewpoint for producing protein nanoaggregates with enhanced functional properties for microencapsulation, edible film, and other applications.

Biotransformation approaches using solid-state fermentation and germination with high-intensity ultrasound to produce added-value hemp protein nanoaggregates

Hemp protein, with superior nutritional content and high digestibility, has attracted significant interest in recent years. However, like other plant protein sources, hemp protein has still suffered from underutilization in industrial applications due to its poor solubility attributes. To overcome this limitation, this study aimed to use biotechnological approaches such as germination (1, 3, 5 days) and solid-state fermentation using Lactobacillusplantarum and Bacillus subtilis to enhance the physicochemical, functional, and bioactive properties of hemp protein. The high-intensity ultrasound combined with these treatments for further modifications. The protein recovery in the defatted flour increased by 42% in germinated isolates compared to the untreated isolate. The primary structure changes were proven by degrading high molecular weight proteins to lower weights in SDS-PAGE. The secondary and tertiary structure changes of isolates were observed in FTIR, CD spectra, DSC, and free SH groups. While the particle sizes of isolates were reduced by 83%, solubility increased by 133% in fermented and ultrasonicated isolates. Water, oil absorption capacities and emulsion activities were increased by 103%, 194%, and 300%, respectively, in modified isolates compared to the untreated isolate. Total phenolic and antioxidant activities of isolates improved in germinated and fermented isolates. Fermentation, germination, and ultrasonication are promising, green, safe, alternative biomodification techniques that may effectively improve protein functionality. These clean-labeled proteins can also be used as bioactive agents in the food industry.

Sulfonamides stimulate ROS formation upon glycation of human carbonic anhydrase II

Advanced glycation end products are the most important species of glycation pathway, and cause disorders such as oxidative stress and diabetes. Sulfonamide compounds, which are generally known as widespread inhibitors, are potential agents used in different drug products, which can readily enter biological matrices. In the present work, the structure and activity of human carbonic anhydrase II studied in the presence of glucose as well as four sulfonamide agents from different views. These included enzyme kinetics, free lysine content, fluorescence spectroscopy, circular dichroism, and ROS measurement. Our results indicated that upon glycation, the structure of HCA II collapses and 8 to 13 lysine residues will be more available based on ligand incubation. Secondary and tertiary structural changes were also observed in the presence and absence of sulfonamide agents using fluorescence and circular dichroism methods, respectively. These spectroscopic data also showed a remarkable increase in hydrophobicity and decrease in α-helix contents during glycation, especially after 35 days of incubation. ROS assay was studied in the presence of glucose and sulfonamide compounds, that demonstrated the role of sulfonamide compounds in ROS formation in the presence of glucose in a synergistic manner. Overall, our data indicated that sulfonamides act as a stimulant factor upon prolonged interaction with HCA II and may intensify the complications of some disorders, such as diabetes and other conformational diseases.

Effect of non-covalently bound polyphenols on the structural and functional properties of wheat germ protein

This study investigated the effects of epigallocatechin-3-gallate (EGCG), chlorogenic acid (CA), piceatannol (PIC) and ferulic acid (FA) on the conformational and functional properties of wheat germ protein (WGP). The WGP-polyphenol interaction was confirmed by native polyacrylamide gel electrophoresis and detection of polyphenol content in the complex. Among the four polyphenols, EGCG showed the highest binding affinity to WGP. Moreover, FT-IR analysis indicated that the polyphenols changed the secondary structure of WGP, inducing an enhancement in alpha-helix content and a reduction in β-sheet structure. Fluorescence spectrum analysis demonstrated the presence of polyphenols induced tertiary structure change of WGP, leading to the deconstruction and unfolding of protein. In addition, the main interaction force for WGP-EGCG was hydrogen bonding and van der Waals forces, and for WGP-CA complexes was electrostatic interaction, while interaction force for WGP-PIC and WGP-FA complexes were hydrophobic interaction. Moreover, the addition of polyphenols significantly increased the absolute value of zeta potential while decreased surface hydrophobicity and free sulfhydryl content of WGP. The solubility and functionality of WGP were mainly improved by CA and EGCG. The findings in this study suggest that WGP-polyphenol complexes have the potential to be applied as novel functional food ingredients.

Hexametaphosphate, a Common Food Additive, Aggregated the Hen Egg White Lysozyme.

Polyphosphate polymers are chains of phosphate monomers chemically bonded together via phosphoanhydride bonds. They are found in all prokaryotic and eukaryotic organisms and are among the earliest, most anionic, and most mysterious molecules known. They are everywhere, from small cellular components to additives in our food. There is a strong association between hyperphosphatemia and mortality. That is why it is crucial to assess how polyphosphates, as food additives, affect the quality of edible proteins. This study investigated the effect of inexpensive and widely used food additives (hexametaphosphate labeled as E452) on bakery items, meat products, fish, and soft drinks. Using various spectroscopic and microscopic techniques, we examined how hexametaphosphate affected the aggregation propensity, structure, and stability of a commonly used food protein: hen egg white lysozyme (HEWL). The solubility of HEWL is affected in a bimodal fashion by the concentration of hexametaphosphate. The bimodal concentration-dependent effect was also observed in the tertiary and secondary structural changes. Hexametaphosphate-induced HEWL aggregates were amorphous, as evidenced by ThT fluorescence, far-UV CD, and TEM imaging. This study showed that the food additive (hexametaphosphate) may denature and aggregate proteins and may lead to undesirable health issues.

Effect of ultrasonic assisted grafting on the structural and functional properties of mung bean protein isolate conjugated with maltodextrin through maillard reaction

Four different methods of maillard reaction including ultrasound (150 W, 10 min) assisted, classical wet heating (80 °C, 60min), moderate water bath heating (60°C, 12 to 30 h) and dry state method (60 °C, 79 % relative humidity and 48 h) were used to Mung bean protein isolate – Maltodexrtin conjugates (MPI-MD) preparation. The samples prepared under ultrasound and wet heating were chosen for further analysis according to degree of graft and UV-absorbance at 420 nm. Higher glycosylation at short time and lower browning were obtained under ultrasound treatment. Covalent attachment in conjugates confirmed by SDS-polyacrylamide gel electrophoresis. The structural analysis revealed prominent unfolding effect of ultrasound waves on the protein's molecules. The decrease of α-helix content was related to the exposure of buried amino group residues during reaction. Glycation of MPI under ultrasound caused changes in tertiary structure of protein and leads to decrease in the fluorescence intensity compared with native and wet heating treatments. FTIR spectra confirmed the conjugation of the MPI and MD and suggested that protein structure was changed and ultrasound promoted the graft reaction more than wet heating treatment. Conjugated MPI showed higher emulsification and solubility index than MPI, moreover the effect of ultrasonic waves on ameliorated functional properties was impressive than those for wet heating treatment. Overall, this study showed use of ultrasonication in maillard reaction was a suitable method for producing MPI- MD conjugates and improved the efficiency of graft reaction and functional properties of grafts.

Mitigation of the Deleterious Effect of Heavy Metals on the Conformational Stability of Ubiquitin through Osmoprotectants.

The Ubiquitin-Proteasome System (UPS) is important in protein homeostasis and is involved in many cell processes. UPS's wide range of regulatory activities is based on the unique and diverse signals transmitted through all-encompassing processes. Cells need a fully functional UPP to cope with oxidative stress, so cellular redox status modulates ubiquitin activity. However, these protein quality control systems are compromised under adverse conditions such as heavy metal stress, resulting in pathological conditions. Heavy metals disrupt the physiological action of sensitive proteins by forming complexes with side-chain functional groups or by dislocating critical metal ions in metalloproteins. In addition, perturbation in the structure of Ubiquitin may affect the ubiquitin-proteasome pathway. In this study, it has been investigated the effects of heavy metals likewise chromium (Cr), cadmium (Cd), and mercury chloride (HgCl2) on the conformational stability of Ubiquitin as well as overcome their hazardous effect, the interaction of osmo-protectants such as Sesamol, gallic acid, Glycine, and ascorbic acid have also been explored in the study. The near and far UV-circular dichroism measurements deduced the secondary and tertiary structural changes. The size of the Ubiquitin before and after exposure to heavy metals was measured by DLS (dynamic light scattering). Docking research was also used to investigate the interaction of Ubiquitin with various heavy metals. Near and far UV-circular dichroism (CD) measurements revealed that mercury, chromium, and cadmium disrupt Ubiquitin's secondary and tertiary structure. The effect of chromium, even at low concentrations, was significantly deleterious compared to cadmium and mercury chloride. Ubiquitin's far-UV circular dichroism spectra subjected to heavy metals were recorded in several osmo-protectants, such as ascorbic acid, Glycine, gallic acid, and Sesamol, which offset the adverse effects of heavy metals. DLS studies revealed a noteworthy change in the hydrodynamic radius of Ubiquitin in the presence of heavy metals. Docking analysis revealed a significant binding affinity of mercury and cadmium ions with Ubiquitin. This study can infer the heavy metals' disruption of Ubiquitin's secondary and tertiary structure. Osmo-protectants produced by animal cells are more effective against heavy metals than plant antioxidants.

Effect of Palmitic Acid on Tertiary Structure of Glycated Human Serum Albumin

Non-enzymatic glycation is a process, which can be best described as a significant posttranslational modification of various proteins. It emerges in hyperglycemic conditions and may have an impact on albumin stability as well as its activity and physical and chemical properties, essentially affecting all its physiological functions. The goal of this research was to answer the following questions: (i) how does the glycation of defatted human serum albumin by glucose–fructose syrup (GFS) alter its tertiary structure; (ii) does palmitic acid (PA), a component of palm oil, affect the in vitro glycation process and cause conformational changes of glycated albumin; and (iii) does PA inhibit the formation of Advanced Glycation End-Products (AGEs)? Therefore, in order to point out differences in the tertiary structure of macromolecules, the absorption and emission of fluorescence spectra and their second derivatives, excitation fluorescence and synchronous spectra, Red-Edge Excitation Shift (REES effect), and the degree of modification of sulfhydryl groups of defatted, non-glycated (HSA) and glycated human serum albumin (gHSA) with GFS and glycated with GFS and PA were investigated. In the present study, it has been confirmed that the glycation of albumin in the presence of GFS and PA causes changes in both HSA and gHSA tertiary structures, respectively. Moreover, palmitic acid, at ratios of 1.5:1 and 3:1 with glycated albumin, does not exhibit inhibition of AGEs formation. This study indicates the fact that the structural changes, especially those of glycated albumin, are important for treatment planning because the type of the interaction between the components and their primary transporter may be altered as the disease progresses or in the elderly.

Improving AlphaFold2-based protein tertiary structure prediction with MULTICOM in CASP15

Since the 14th Critical Assessment of Techniques for Protein Structure Prediction (CASP14), AlphaFold2 has become the standard method for protein tertiary structure prediction. One remaining challenge is to further improve its prediction. We developed a new version of the MULTICOM system to sample diverse multiple sequence alignments (MSAs) and structural templates to improve the input for AlphaFold2 to generate structural models. The models are then ranked by both the pairwise model similarity and AlphaFold2 self-reported model quality score. The top ranked models are refined by a novel structure alignment-based refinement method powered by Foldseek. Moreover, for a monomer target that is a subunit of a protein assembly (complex), MULTICOM integrates tertiary and quaternary structure predictions to account for tertiary structural changes induced by protein-protein interaction. The system participated in the tertiary structure prediction in 2022 CASP15 experiment. Our server predictor MULTICOM_refine ranked 3rd among 47 CASP15 server predictors and our human predictor MULTICOM ranked 7th among all 132 human and server predictors. The average GDT-TS score and TM-score of the first structural models that MULTICOM_refine predicted for 94 CASP15 domains are ~0.80 and ~0.92, 9.6% and 8.2% higher than ~0.73 and 0.85 of the standard AlphaFold2 predictor respectively.

Structure of the Hepatitis B virus capsid quasi-6-fold with a trapped C-terminal domain reveals capsid movements associated with domain exit

Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/β complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impβ as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T= 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impβ-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.

Effect of Soft Preheating of Bovine Serum Albumin on the Complexation with Oligochitosan: Structure and Conformation of BSA in the Complex.

Phase analysis, spectroscopic and light scattering methods are applied to investigate the peculiarities of the interaction of oligochitosan (OCHI) with native and preheated BSA as well as the conformational and structural changes of BSA in BSA/OCHI complex. As shown, untreated BSA binds with OCHI mainly forming soluble electrostatic nanocomplexes, with the binding causing an increase in BSA helicity without a change in the local tertiary structure and thermal stability of BSA. In contrast, soft preheating at 56°C enhances the complexation of BSA with OCHI and slightly destabilizes the secondary and local tertiary structures of BSA within the complex particles. Preheating at 64°C (below the irreversible stage of BSA thermodenaturation) leads to further enhancement in the complexation and formation of insoluble complexes stabilized by both Coulomb forces and hydrophobic interactions. The finding can be promising for the preparation of biodegradable BSA/chitosan-based drug delivery systems. This article is protected by copyright. All rights reserved.

Deciphering the enigma of RNA editing in the ATP1_alpha subunit of ATP synthase in Triticum aestivum

There is evidence that RNA editing is related to plant cellular stress as well as electron transport organelles, such as mitochondria. The mitochondrial atp1 gene encodes the alpha-subunit of Atp synthase. Control as well as two periods of drought stress treatments were analyzed in the cDNAs generated from the mitochondrial atp1 gene of two cultivars of Triticum aestivum [Giza 168 (G168) and Gemmiza 10 (GM10)]. Following RNA-seq data assembly, atp1 cDNAs from the control (acc. no. OQ129415), 2-hour (acc. no. OQ129416), and 12-hour (acc. no. OQ129417) time points of the T. aestivum cultivar G168 were obtained. Control (acc. no. OQ129419), 2-hour (acc. no. OQ129420), and 12-hour (acc. no. OQ129421) samples all included reconstructed atp1 transcripts from Gemmiza 10. Atp1 transcripts were assembled using the wheat atp1 gene (acc. no. NC_036024). RNA-seq raw data was utilized to identify 11 RNA editing sites in atp1 in the tolerant cultivar Giza168 and 6 in the sensitive cultivar Gemmiza10. The significant difference in RNA editing observed between control and drought stress conditions in sites led to synonymous amino acids. This led to no change in tertiary structure between tolerant and sensitive cultivars. But the change was focused between produced protein and its correspondence sequence on DNA.

Effect of ultrahigh-pressure treatment on the structure and allergenicity of peach allergenic proteins

Peach is a common plant-derived allergenic food and ultrahigh-pressure treatment is often used in peach products. In our study, an in-depth analysis of the structural and allergenicity changes of peach allergenic proteins after UHP treatment was performed by spectroscopy, mass spectrometry combined with serology and cytology. The results indicated that UHP treatment could reduce the content of peach soluble proteins and cause changes in secondary and tertiary structures. In addition, more hydrophobic residues were exposed and proteins tended to polymerize after UHP-treatment. The results of immunological assays showed that UHP treatment could reduce the IgE binding capacity of peach proteins and affect the ability of basophil degranulation, the upregulation of some cytokines may contribute to the reduction of peach protein allergenicity. Notably, UHP treatment may lead to the masking of some digestion sites in Pru p 3 epitopes, thus impeding human digestion and increasing the potential risk of allergenicity.

Extended physicochemical stability of cetuximab in opened vials and infusion bags when stored at 4°C and 25°C.

This study aimed to determine the stability of cetuximab: (1) under "in-use" conditions after dilution to 1 mg/mL in 0.9% sodium chloride in polyolefin bags and (2) as an undiluted solution (5 mg/mL) repackaged in polypropylene bags or kept in the vial after opening. Ready-to-use 500 mg/100 mL vials of cetuximab solution were diluted to 1 mg/mL in 100 mL bags of 0.9% sodium chloride or repackaged as a 5 mg/mL solution into empty 100 mL bags. Bags and vials were stored at 4°C for 90 days and 25°C for 3 days. A syringe sample of 7 mL was taken from each bag for the initial determinations. The sampled bags were weighed to determine their initial weight and placed under the planned storage conditions. The physicochemical stability of cetuximab was estimated using validated methods. No changes in turbidity, no protein loss, and no changes in cetuximab tertiary structure were observed after 30 days of storage or when subjected to a temperature excursion of 3 days at 25°C and when stored at 4°C for up to 90 days, regardless of the concentrations and batches. The colligative parameters did not change under any of the tested conditions. No evidence of microbial growth was found in bags after 90 days of storage at 4°C. These results support the extended in-use shelf-life of cetuximab vials and bags, which can be cost-effective for healthcare providers.

Structural basis for the cold activation and adaptation of an α-agarase from marine bacterium Catenovulum agarivorans STB13

Enzymes have high activity at low temperatures are defined as cold-adapted enzymes, but few enzymes show an activation effect after low-temperature incubation. In this study, we cloned an agarase from the marine bacterium Catenovulum agarivorans STB13 (Ca-AGA) and expressed the protein in Bacillus subtilis. The enzyme exhibited a peculiar cold activation, which was enhanced by 5-fold after incubation at −20 °C or 4 °C for 6 h. Fluorescence and CD spectra studies confirmed the secondary and tertiary structural changes in Ca-AGA at low temperature, which formed compact protein structures after low-temperature treatment. The enzyme also showed outstanding cold adaptation, retaining approximately 60% of its maximum activity at 10 °C. Based on a bioinformatics analysis, the carbohydrate-binding modules (CBMs) and long linker region were presumed to play a crucial role in cold adaptation due to a relatively high percentage of acidic residues and a negative charge density compared to the catalytic domain. A mutation that truncated the linker-CBM region resulted in a 59.97% reduction of the activity at 10 °C. This study describes an α-agarase of cold activation and adaptation in detail, which may have great potential for producing functional agaro-oligosaccharides in food and pharmaceutical industrial applications at ambient temperature.

Comparison of different ultrafiltration-recovered soy protein hydrolysate fractions and their effects on the stability of mulberry anthocyanin extract

The effects of three ultrafiltration-recovered soy protein hydrolysate fractions (USHFs) with different molecular weights (>10 kDa for USHF-1, 1–10 kDa for USHF-2, and ＜1 kDa for USHF-3) on mulberry anthocyanin extract (MAE) was investigated under the condition of neutral pH for the first time in this study. Generally, all USHFs showed pronounced color- and anthocyanin-stabilized effects. The addition of 1 mg/mL of USHF-3 showed best protection for anthocyanin stability in this study. Based on the spectroscopic results, the electrostatic interactions were the main forces in the binding system between USHF-3 and cyanidin-3-O-glucoside (C3G) indicated by the negative ΔH and positive ΔS values. USHF-1 and USHF-2 and their complexes with C3G were mainly driven by hydrophobic interactions determined with positive ΔH and positive ΔS values. C3G addition resulted in tertiary structural changes of the USHFs. Overall, the complexation of USHFs and C3G participates in maintaining anthocyanin stability.

Improvement of Structural, Rheological, and physicochemical properties of type I collagen by calcium lactate combined with ultrasound

Type I collagen has a relatively stable quality while quite resistant to digestion because of the complex triple helix structure. This study was conducted to explore the acoustic conditions of ultrasound (UD)-assisted calcium lactate processing of collagen and control the processing process through its sono-physico-chemical effects. The findings demonstrated that UD might lower the average particle size of collagen and increase its zeta potential. In contrast, the rise in calcium lactate concentration could dramatically limit the impact of UD processing. This may be because of its low acoustic cavitation effect, as demonstrated by the phthalic acid method (the fluorescence value decreased from 81245.67 to 18243.67). Poor changes in tertiary and secondary structures confirmed the detrimental effect of calcium lactate concentration on UD-assisted processing. Although UD-assisted calcium lactate processing can significantly alter the structure of collagen, the integrity of the collagen is basically preserved. Furthermore, the addition of UD and a trace amount of calcium lactate (0.1%) increased the roughness of the fiber structure. At this relatively low calcium lactate concentration, ultrasound improved the gastric digestibility of collagen by nearly 20%.

Secondary structure characterization of mixed food protein complexes using microfluidic modulation spectroscopy (MMS)

Microfluidic modulation spectroscopy (MMS) has been recognised as a novel technology for producing high quality and reproducible data at a wide concentration range to characterise protein structure. In this study, the use of MMS for food applications was explored through the structural analysis of pea protein, whey protein, and pea/whey protein complexes prepared by transglutaminase cross-linking. MMS generated reproducible data with >99.5% repeatability in all protein samples. The cross-linking seemed to alter the structure of whey protein with isopeptide and hydrogen bond formation, more significantly than that of pea protein. The larger secondary and tertiary structural changes were observed with higher whey protein content in cross-linked pea/whey protein complex, which eventually increased the surface hydrophobicity. According to the second derivatives analysis by MMS, β-sheet and random coil increased and β-turn decreased in the pea protein, whereas α-helix, β-sheet, and β-turn decreased and random coil increased in the whey protein after cross-linking. In the pea/whey protein complex, β-turn shifted to β-sheet due to cross-linking. There are some discrepancies between the relative secondary structure contents obtained from MMS data compared to the data acquired from circular dichroism. However, MMS has the considerable advantage of being able to directly measure original samples without significant dilution as required for circular dichroism measurements. This study provides evidence that MMS technology could be a robust technique to quantify the secondary structures of typical proteins in food with high reliability.

Molecular structure, IgE binding capacity and gut microbiota of ovalbumin conjugated to fructose and galactose:A comparative study

Ovalbumin (OVA) was modified by fructose (Fru) and galactose (Gal) to study the structure, IgG/IgE binding capacity and effects on human intestinal microbiota of the conjugated products. Compared with OVA-Fru, OVA-Gal has a lower IgG/IgE binding capacity. The reduction of OVA is not only associated with the glycation of R84, K92, K206, K263, K322 and R381 in the linear epitopes, but also with conformational epitope changes, manifested as secondary and tertiary structural changes caused by Gal glycation. In addition, OVA-Gal could alter the structure and abundance of gut microbiota at phylum, family, and genus levels and restore the abundance of bacteria associated with allergenicity, such as Barnesiella, Christensenellaceae_R-7_group, and Collinsela, thereby reducing allergic reactions. These results indicate that OVA-Gal glycation can reduce the IgE binding capacity of OVA and change the structure of human intestinal microbiota. Therefore, Gal glycation may be a potential method to reduce protein allergenicity.

Pseudouridimycin-A Potent Nucleoside Inhibitor of the RNA Polymerase Beta Prime Subunit of Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococcus, GAS), a Gram-positive bacterium, is a major cause of mild to severe life-threatening infections. Antibacterial resistance to penicillin and macrolides poses a major threat in the treatment of GAS and necessitates alternate drugs and newer antibiotics. In this direction, nucleotide-analog inhibitors (NIAs) have emerged as important antiviral, antibacterial, and antifungal agents. Pseudouridimycin (PUM), a nucleoside analogue inhibitor discovered from the soil bacterium Streptomyces sp., has proven to be effective against multidrug-resistant S. pyogenes. However, the mechanism of its activity remains elusive. In this study, subunits of the RNA polymerase of GAS have been identified as targets for PUM inhibition and the binding regions have been mapped to the N-terminal domain of the β' subunit, using computational methods. The antibacterial activity of PUM against macrolide-resistant GAS was evaluated. PUM showed effective inhibition at 0.1-1 μg/mL concentration, which was higher when compared to earlier reports. The molecular interaction between PUM and the RNA polymerase β'-N terminal subunit was investigated using isothermal titration calorimetry (ITC), circular dichorism (CD), and intrinsic fluorescence spectroscopy. The thermodynamic characterization by ITC showed an affinity constant of 6.175 × 105 M-1 denoting a moderate affinity. Fluorescence studies revealed that the interaction of protein-PUM was spontaneous in nature and follows a static quenching of tyrosine signals from the protein. The near- and far-UV CD spectral analysis concluded that PUM induced local tertiary structural changes in the protein, predominantly contributed by aromatic amino acids rather than notable changes in the secondary structure. Hence PUM could be a promising lead drug target for macrolide-resistant strains of S. pyogenes and enable eradication of pathogen in the host system.