Synthesis and characterization of novel histidine functionalized chitosan nanoformulations and its bioactivity in tomato plant.

The use of novel active ingredients for the functional modification of chitosan nanoformulations has attracted global attention. In this study, chitosan has been functionalized via histidine to craft novel chitosan-histidine nanoformulation (C-H NF) using ionic gelation method. C-H NF exhibited elite physico-biochemical properties, influencing physiological and biochemical dynamics in Tomato. These elite properties include homogenous-sized nanoparticles (314.4 nm), lower PDI (0.218), viscosity (1.43 Cps), higher zeta potential (11.2 mV), nanoparticle concentration/ml (3.53 × 108), conductivity (0.046 mS/cm), encapsulation efficiency (53%), loading capacity (24%) and yield (32.17%). FTIR spectroscopy revealed histidine interaction with C-H NF, while SEM and TEM exposed its porous structure. Application of C-H NF to Tomato seedling and potted plants through seed treatment and foliar spray positively impacts growth parameters, antioxidant-defense enzyme activities, reactive oxygen species (ROS) content, and chlorophyll and nitrogen content. We claim that the histidine-functionalized chitosan nanoformulation enhances physico-biochemical properties, highlighting its potential to elevate biochemical and physiological processes of Tomato plant.

Exploring How System Dimensions and Periodic Boundary Conditions Influence the Molecular Dynamics Simulation of A6H Peptide Self-Assembly Nanostructures.

This work presents a study on the effects of periodic boundary conditions (PBC) on the energetic/structural properties and hydrogen bond dynamics (HB) using molecular dynamics (MD) simulations of peptide membranes composed of alanine and histidine. Our results highlight that simulations using small surface areas for the peptide membrane may result in nonconvergent values for membrane properties, which are only observed in regions simulated at a certain distance from the PBCs. Specifically, regarding hydrogen bonds, a property pervasive in peptide membranes, our findings indicate a significant increase in the lifetime of these interactions, reaching values ∼19% higher when observed in structures free from PBCs. For peptide mobility in these nanomembranes, our results compare regions simulated directly under the influence of PBCs with regions free from these conditions, emphasizing greater mobility of amino acid psi/phi angles in the latter model.

Genetically encoded Nδ-vinyl histidine for the evolution of enzyme catalytic center.

Genetic code expansion has emerged as a powerful tool for precisely introducing unnatural chemical structures into proteins to improve their catalytic functions. Given the high catalytic propensity of histidine in the enzyme pocket, increasing the chemical diversity of catalytic histidine could result in new characteristics of biocatalysts. Herein, we report the genetically encoded Nδ-Vinyl Histidine (δVin-H) and achieve the wild-type-like incorporation efficiency by the evolution of pyrrolysyl tRNA synthetase. As histidine usually acts as the nucleophile or the metal ligand in the catalytic center, we replace these two types of catalytic histidine to δVin-H to improve the performance of the histidine-involved catalytic center. Additionally, we further demonstrate the improvements of the hydrolysis activity of a previously reported organocatalytic esterase (the OE1.3 variant) in the acidic condition and myoglobin (Mb) catalyzed carbene transfer reactions under the aerobic condition. As histidine is one of the most frequently used residues in the enzyme catalytic center, the derivatization of the catalytic histidine by δVin-H holds a great potential to promote the performance of biocatalysts.

Oral histidine affects gut microbiota and MAIT cells improving glycemic control in type 2 diabetes patients.

Amino acids, metabolized by host cells as well as commensal gut bacteria, have signaling effects on host metabolism. Oral supplementation of the essential amino acid histidine has been shown to exert metabolic benefits. To investigate whether dietary histidine aids glycemic control, we performed a case-controlled parallel clinical intervention study in participants with type 2 diabetes (T2D) and healthy controls. Participants received oral histidine for seven weeks. After 2 weeks of histidine supplementation, the microbiome was depleted by antibiotics to determine the microbial contribution to histidine metabolism. We assessed glycemic control, immunophenotyping of peripheral blood mononucelar cells (PBMC), DNA methylation of PBMCs and fecal gut microbiota composition. Histidine improves several markers of glycemic control, including postprandial glucose levels with a concordant increase in the proportion of MAIT cells after two weeks of histidine supplementation. The increase in MAIT cells was associated with changes in gut microbial pathways such as riboflavin biosynthesis and epigenetic changes in the amino acid transporter SLC7A5. Associations between the microbiome and MAIT cells were replicated in the MetaCardis cohort. We propose a conceptual framework for how oral histidine may affect MAIT cells via altered gut microbiota composition and SLC7A5 expression in MAIT cells directly and thereby influencing glycemic control. Future studies should focus on the role of flavin biosynthesis intermediates and SLC7A5 modulation in MAIT cells to modulate glycemic control.

Heterologous Expression and Purification of Eukaryotic ALA Synthase from E. coli.

This chapter presents a method for the heterologous expression and purification of human ALA synthase from Escherichia coli. Mature ALAS is produced with an N-terminal hexahistidine affinity tag followed by a SUMO fusion tag for solubility and ease of purification. The plasmid is introduced into competent E. coli cells, and robust protein expression is induced with IPTG. The ALAS cofactor, pyridoxal 5'-phosphate, is inserted during protein production to yield an active enzyme upon purification. After cell lysis, the tagged ALAS protein is isolated via a multistep purification that involves an initial nickel-affinity step, affinity tag cleavage and removal, and a final size exclusion chromatography polishing step. Importantly, this protocol is amenable to various ALAS truncations and mutations, opening the door to understanding ALAS biology and its intersections with iron utilization across several organisms.

Highly selective chemical modification of poly-His tagged peptides and proteins.

Chemical modifications to proteins have wide applications. They may be used in, for example, the production of biopharmaceuticals and fluorescent probes. Despite their importance, highly regioselective chemical protein modifications are often challenging to achieve. We have developed two highly selective methods for protein acylation using poly-His tags inserted either at the N-terminus or in combination with a specific Lys residue. For this, we used an N-terminal Gly-His6 (Gly-His tag) or the sequence Hism-Lys-Hisn (Lys-His tag), respectively. The Gly-His tag directed the acylation to the N-terminal Nα-amine when reacted with 4-methoxyphenyl esters to yield stable conjugates. Next, the Lys-His tag was developed to allow modifications at the C-terminus or in loop regions of proteins. This gave a high selectivity of acylation of the designated Lys Nε-amine in the tag over native Lys residues in the protein under mild conditions. Here, we describe the synthesis of aromatic esters carrying different functionalities and reactivity tuning substituents on the phenol. The expression of poly-His tagged proteins, and the procedure for the highly selective peptide and protein acylations are detailed in this contribution. The versatility of these methods has been demonstrated by the attachment of different functionalities such as fluorophores, biotin, and azides to different proteins and an antibody.

Development of Robust Freeze-Drying Process for Long-Term Stability of rVSV-SARS-CoV-2 Vaccine.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

The Angiotensin Metabolite His-Leu Is a Strong Copper Chelator Forming Highly Redox Active Species.

His-Leu is a hydrolytic byproduct of angiotensin metabolism, whose concentration in the bloodstream could be at least micromolar. This encouraged us to investigate its Cu(II) binding properties and the concomitant redox reactivity. The Cu(II) binding constants were derived from isothermal titration calorimetry and potentiometry, while identities and structures of complexes were obtained from ultraviolet-visible, circular dichroism, and room-temperature electronic paramagnetic resonance spectroscopies. Four types of Cu(II)/His-Leu complexes were detected. The histamine-like complexes prevail at low pH. At neutral and mildly alkaline pH and low Cu(II):His-Leu ratios, they are superseded by diglycine-like complexes involving the deprotonated peptide nitrogen. At His-Leu:Cu(II) ratios of ≥2, bis-complexes are formed instead. Above pH 10.5, a diglycine-like complex containing the equatorially coordinated hydroxyl group predominates at all ratios tested. Cu(II)/His-Leu complexes are also strongly redox active, as demonstrated by voltammetric studies and the ascorbate oxidation assay. Finally, numeric competition simulations with human serum albumin, glycyl-histydyl-lysine, and histidine revealed that His-Leu might be a part of the low-molecular weight Cu(II) pool in blood if its abundance is >10 µM. These results yield further questions, such as the biological relevance of ternary complexes containing His-Leu.

Magnetic Titanium Dioxide Nanocomposites as a Recyclable SERRS Substrate for the Ultrasensitive Detection of Histidine.

A highly sensitive, selective and recyclable histidine detection method based on magnetic Fe3O4@mTiO2 (M-TiO2) nanocomposites with SERRS was developed. Mesoporous M-TiO2 nanoparticles were functionalized with 4-aminothiophenol and then coupled with histidine through an azo coupling reaction in 5 min, producing the corresponding azo compound. The strong and specific SERRS response of the azo product allowed for ultrasensitive and selective detection for histidine with an M-TiO2 device loaded with Ag NPs due to the molecular resonance effect and plasmonic effect of Ag NPs under a 532 nm excitation laser. The sensitivity was further enhanced with the magnetic enrichment of M-TiO2. The limit of detection (LOD) was as low as 8.00 × 10-12 mol/L. The M-TiO2 demonstrated applicability towards histidine determination in human urine without any sample pretreatment. Additionally, the M-TiO2 device can be recycled for 3 cycles with the photodegradation of the azo product under UV irradiation due to TiO2-assisted and plasmon-enhanced photocatalysis. In summary, a multifunctional and recyclable M-TiO2 device was synthesized based on azo coupling and SERRS spectroscopy for ultra-sensitive and specific histidine sensing. In addition, the proposed system demonstrated the potential for the multiplex determination of toxic compounds in the fields of food safety, industrial production and environmental protection, which benefit from the fingerprint property and universality of SERRS.

Delivery of mRNA with Histidine-Lysine Peptides.

Transfection with mRNA has been considered superior to that with plasmids since the mRNA can be translated to a protein in the cytosol without entering the nucleus. One disadvantage of using mRNA is its susceptibility to enzymatic biodegradability, and consequently, significant research has occurred to determine nonviral carriers that will sufficiently stabilize this nucleic acid for cellular transport. Histidine-lysine peptides (HK) are one such class of mRNA carriers, which we think serves as a model for other peptides and polymeric carrier systems. When the HK peptide and mRNA are mixed and interact through ionic and nonionic bonds, mRNA polyplexes are formed, which can transfect cells. In contrast to linear HK peptides, branched HK peptides protected and efficiently transfected mRNA into cells. After describing the preparation and biophysical characterization of these polyplexes, we will provide protocols for in vitro and in vivo transfection for these mRNA polyplexes.

Purification of Recombinant N-terminal Histidine-Tagged Arabidopsis thaliana Phosphoglycolate Phosphatase 1, Glycolate Oxidase 1 and 2, and Hydroxypyruvate Reductase 1.

To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni2+-NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase, and hydroxypyruvate reductase) from Escherichia coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD.

Construction and application of H1R ligand screening materials based on SMA stabilization and his-tag covalent immobilization of membrane proteins.

The histamine H1 receptor (H1R) plays a pivotal role in allergy initiation and undergoes the necessity of devising a high-throughput screening approach centered on H1R to screen novel ligands effectively. This study suggests a method employing styrene maleic acid (SMA) extraction and His-tag covalent bonding to immobilize H1R membrane proteins, minimizing the interference of nonspecific proteins interference while preserving native protein structure and maximizing target exposure. This approach was utilized to develop a novel material for high-throughput ligand screening and implemented in cell membrane chromatography (CMC). An H1R-His-SMALPs/CMC model was established and its chromatographic performance (selectivity, specificity and lifespan) validated, demonstrating a significant enhancement in lifespan compared to previous CMC models. Subsequently, this model facilitated high-throughput screening of H1R ligands in the compound library and preliminary activity verification of potential H1R antagonists. Identification of a novel H1R antagonist laid the foundation for further development in this area.

Ultrasound-assisted modification of soybean protein isolate with L-histidine: Relationship between structure and function.

Herein, the effects of ultrasound-assisted L-histidine (L-His) on the physicochemical properties and conformation of soybean protein isolate (SPI) were investigated. Particle size, zeta potential, turbidity, and solubility were used to evaluate protein aggregation, and the relationship between structural and functional changes of the proteins was characterized using spectral analysis, surface hydrophobicity, emulsification, and antioxidant properties. After ultrasound-assisted L-His treatment, SPI exhibited a smaller particle size, higher solubility, and more homogeneous micromorphology owing to the decrease in alpha-helix content and subsequent increases in zeta potential and active sulfhydryl content. In addition, spectral analysis showed that L-His and SPI could form a complex, which changed the microenvironment of the amino acid residues in SPI, thus improving its emulsification and antioxidant properties. At the concentration of L-His was 0.3 % w/w, the nanocomplex had a smaller particle size (140.03 nm), higher ζ-potential (-23.63 mV), and higher emulsification stability (22.48 min).

Trivalent Copper Ion-Mediated Dual Oxidation in the Copper-Catalyzed Fenton-Like System in the Presence of Histidine.

The Cu(II)/H2O2 system is recognized for its potential to degrade recalcitrant organic contaminants and inactivate microorganisms in wastewater. We investigated its unique dual oxidation strategy involving the selective oxidation of copper-complexing ligands and enhanced oxidation of nonchelated organic compounds. L-Histidine (His) and benzoic acid (BA) served as model compounds for basic biomolecular ligands and recalcitrant organic contaminants, respectively. In the presence of both His and BA, the Cu(II)/H2O2 system rapidly degraded His complexed with copper ions within 30 s; however, BA degraded gradually with a 2.3-fold efficiency compared with that in the absence of His. The primary oxidant responsible was the trivalent copper ion [Cu(III)], not hydroxyl radical (•OH), as evidenced by •OH scavenging, hydroxylated BA isomer comparison with UV/H2O2 (a •OH generating system), electron paramagnetic resonance, and colorimetric Cu(III) detection via periodate complexation. Cu(III) selectively oxidized His owing to its strong chelation with copper ions, even in the presence of excess tert-butyl alcohol. This selectivity extended to other copper-complexing ligands, including L-asparagine and L-aspartic acid. The presence of His facilitated H2O2-mediated Cu(II) reduction and increased Cu(III) production, thereby enhancing the degradation of BA and pharmaceuticals. Thus, the Cu(II)/H2O2 system is a promising option for dual-target oxidation in diverse applications.

High-efficient depletion and separation of histidine-rich proteins via Cu2+-chelated porous polymer microspheres.

Depletion and separation of histidine-rich proteins from complicated biosamples are crucial for various downstream applications in proteome research and clinical diagnosis. Herein, porous polymer microspheres coated with polyacrylic acid (SPSDVB-PAA) were fabricated through double emulsion interfacial polymerization technique and followed by immobilization of Cu2+ ions on the surface of SPSDVB-PAA. The as-prepared SPSDVB-PAA-Cu with uniform size and nanoscale pore structure enabled coordination interaction of Cu2+ with histidine residues in his-rich proteins, resulting in high-performance adsorption. As metal affinity adsorbent, the SPSDVB-PAA-Cu exhibited favorable selectivity for adsorbing hemoglobin (Hb) and human serum albumin (HSA) with the maximum adsorption capacities of 152.2 and 100.7 mg g-1. Furthermore, the polymer microspheres were used to isolate histidine-rich proteins from human whole blood and plasma, underscoring their effectiveness. The liquid chromatography tandem mass spectrometry (LC-MS/MS) results indicated that the content of 14 most abundant proteins in human plasma was depleted from 81.6 % to 30.7 % and low-abundance proteins were enriched from 18.4 % to 69.3 % after treatment with SPSDVB-PAA-Cu, illustrating potential application of SPSDVB-PAA-Cu in proteomic research.

Histidine modified Fe3O4 nanoparticles improving the ethanol yield and tolerance of Saccharomyces cerevisiae.

Saccharomyces cerevisiae, the primary microorganism involved in ethanol production, is hindered by the accumulation of ethanol, leading to reduced ethanol production. In this study, we employed histidine-modified Fe3O4 nanoparticles (His-Fe3O4) for the first time, to the best of our knowledge, as a method to enhance ethanol yield during the S. cerevisiae fermentation process. The results demonstrated that exposing S. cerevisiae cells to Fe3O4 nanoparticles (Fe3O4 NPs) led to increased cell proliferation and glucose consumption. Moreover, the introduction of His-Fe3O4 significantly boosted ethanol content by 17.3% (p < 0.05) during fermentation. Subsequent findings indicated that the increase in ethanol content was associated with enhanced ethanol tolerance and improved electron transport efficiency. This study provided evidence for the positive effects of His-Fe3O4 on S. cerevisiae cells and proposed a straightforward approach to enhance ethanol production in S. cerevisiae fermentation. The mediation of improved ethanol tolerance offers significant potential in the fermentation and bioenergy sectors.

Ultrasmall CuMn-His Nanozymes with Multienzyme Activity at Neutral pH: Construction of a Colorimetric Sensing Array for Biothiol Detection and Disease Identification.

Biothiol assays offer vital insights into health assessment and facilitate the early detection of potential health issues, thereby enabling timely and effective interventions. In this study, we developed ultrasmall CuMn-Histidine (His) nanozymes with multiple enzymatic activities. CuMn-His enhanced peroxidase (POD)-like activity at neutral pH was achieved through hydrogen bonding and electrostatic effects. In addition, CuMn-His possesses laccase (LAC)-like and superoxide dismutase (SOD)-like activities at neutral pH. Based on three different enzyme mimetic activities of CuMn-His at neutral pH, the colorimetric sensing array without changing the buffer solution was successfully constructed. The array was successfully used for the identification of three biothiols, glutathione (GSH), cysteine (Cys), and homocysteine (Hcy). Subsequently, excellent application results were shown in complex serum and cellular level analyses. This study provides an innovative strategy for the development of ultrasmall bimetallic nanozymes with multiple enzymatic activities and the construction of colorimetric sensing arrays.

Trading in catalytic power for sensing.

In this issue of Structure, Yin et al.1 present the CryoEM structure of the HisRS-like domain of human GCN2 and demonstrate that it is a pseudoenzyme, which binds uncharged tRNA in a different manner than HisRS and does not bind histidine and ATP.

Kinetic isotope effect reveals rate-limiting step in green-to-red photoconvertible fluorescent proteins.

Photoconvertible fluorescent proteins (pcFPs) undergo a slow photochemical transformation when irradiated with blue light. Since their emission is shifted from green to red, pcFPs serve as convenient fusion tags in several cutting-edge biological imaging technologies. Here, a pcFP termed the Least Evolved Ancestor (LEA) was used as a model system to determine the rate-limiting step of photoconversion. Perdeuterated histidine residues were introduced by isotopic enrichment and chromophore content was monitored by absorbance. pH-dependent photoconversion experiments were carried out by exposure to 405-nm light followed by dark equilibration. The loss of green chromophore correlated well with the rise of red, and maximum photoconversion rates were observed at pH 6.5 (0.059 ± 0.001 min-1 for red color acquisition). The loss of green and the rise of red provided deuterium kinetic isotope effects (DKIEs) that were identical within error, 2.9 ± 0.9 and 3.8 ± 0.6, respectively. These data indicate that there is one rate-determining step in the light reactions of photoconversion, and that CH bond cleavage occurs in the transition state of this step. We propose that these reactions are rate-limited on the min time scale by the abstraction of a proton at the His62 beta-carbon. A conformational intermediate such as a twisted or isomerized chromophore is proposed to slowly equilibrate in the dark to generate the red form. Additionally, His62 may shuttle protons to activate Glu211 to serve as a general base, while also facilitating beta-elimination. This idea is supported by a recent X-ray structure of methylated His62.

Structure and Stability of Ago2 MID-Nucleotide Complexes: All-in-One (Drop) His6-SUMO Tag Removal, Nucleotide Binding, and Crystal Growth.

The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5'-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His6-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2'-3' linked α-L-threofuranosyl thymidine-3'-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (Kd) of MID-nucleotide interactions for nucleoside 5'-monophosphates and nucleoside 3',5'-bisphosphates. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Crystallization of Ago2 MID-nucleotide complexes Basic Protocol 2: Measurement of dissociation constant Kd between Ago2 MID and nucleotides.