Synthesis of histidine, serine and folic acid-functionalized and boron and iron-doped graphene quantum dot with excellent optical behavior and peroxidase-like activity for colorimetric and fluorescence detection of H2O2 in food.

Low fluorescence under visible light excitation and catalytic activity limit many applications of graphene quantum dots in optical detection, biosensing, catalysis and biomedical. The paper reports design and synthesis of histidine, serine and folic acid-functionalized and boron and iron-doped graphene quantum dot (Fe/B-GQD-HSF). The Fe/B-GQD-HSF shows excellent fluorescence behavior and peroxidase-like activity. Excitation of 330 nm ultraviolet light produces the strongest blue fluorescence and excitation of 480 nm visible light produces the strongest yellow fluorescence. The specific activity reaches 92.67 U g-1, which is higher than that of other graphene quantum dots. The Fe/B-GQD-HSF can catalyze oxidation of 3,3',5,5'-tetramethylbenzidine with H2O2 to form blue compound. Based on this, it was used for colorimetric and fluorescence detection of H2O2. The absorbance at 652 nm linearly increases with the increase of H2O2 concentration between 0.5 and 100 µM with detection limit of 0.43 µM. The fluorescence signal linearly decreases with the increase of H2O2 concentration between 0.05 and 100 µM with detection limit of 0.035 µM. The analytical method has been satisfactorily applied in detection of H2O2 in food. The study also paves one way for design and synthesis of functional graphene quantum dots with ideal fluorescence behavior and catalytic activity.

Facile Synthesis of Aptamer-Functionalized Polydopamine-Coated Magnetic Graphene Oxide Nanocomposites for Highly Efficient Purification of His-Tagged Proteins.

Recombinant proteins hold significant importance in numerous disciplines. As the demand for expressing and purifying these proteins grows, the scientific community is in dire need of a simple yet versatile methodology that can efficiently purify these proteins. Aptamers as synthetic nucleic acid-based ligands with high affinity have shown promise in this regard, as they can capture targets through molecular recognition. In this study, novel aptamer-functionalized polydopamine-coated magnetic graphene oxide nanocomposites were facilely prepared, achieving an impressive average aptamer coverage density (45 nmol/mg). These nanocomposites exhibited a uniform structure and robust magnetic responsiveness. The findings indicated that they possess several advantages, such as rapid adsorption, substantial capacity (171.4 mg/g), and excellent reusability. Notably, due to the inherent properties of nucleic acids, the immobilized aptamer-magnetic beads can be utilized repeatedly with high purification efficiency. Finally, the nanocomposites were further employed to purify His-tagged proteins from actual samples. Remarkably, they were able to selectively and efficiently isolate His-tagged retinoid X receptor alpha protein from complex Escherichia coli lysate. The purified His-tagged retinoid X receptor alpha protein was analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This confirmed the efficacy of developed nanocomposites, reinforcing their vast potential for purification of His-tagged recombinant proteins.

A pH-Dependent Coarse-Grained Model for Disordered Proteins: Histidine Interactions Modulate Conformational Ensembles.

Histidine (His) presents a unique challenge for modeling disordered protein conformations, as it is versatile and occurs in both the neutral (His0) and positively charged (His+) states. These His charge states, which are enabled by its imidazole side chain, influence the electrostatic and short-range interactions of His residues, which potentially engage in cation-π, π-π, and charge-charge interactions. Existing coarse-grained (CG) models often simplify His representation by assigning it an average charge, thereby neglecting these potential short-range interactions. To address this gap, we developed a model for intrinsically disordered proteins (IDPs) that accounts for the properties of histidine (H). The resulting IDPH model is a 21-amino acid CG model incorporating both His charge states. We show that interactions involving previously neglected His0 are critical for accurate modeling at high pH, where they significantly influence the compaction of His-rich IDPs such as Histatin-5 and CPEB4. These interactions contribute to structural stabilizations primarily via His0-His0 and His0-Arg interactions, which are overlooked in models focusing solely on the charged His+ state.

Effect of histidine and L-Tyrosine supplementation in maturation medium on in-vitro developmental outcomes of buffalo oocytes.

The present study was designed to investigate the effects of amino acid (histidine and L-Tyrosine) on in vitro maturation (IVM), in vitro fertilization (IVF), cleavage (CR) rates, and in vitro embryonic cultivation (IVC; Morula and Blastocyst stage) in buffaloes. Within two hours of buffalo slaughter, the ovaries were collected and transported to the laboratory. Follicles with a diameter of 2 to 8 mm were aspirated to recover the cumulus oocyte complexes (COCs). Histidine (0.5, 1, and 3 mg/ml) or L-Tyrosine (1, 5, and 10 mg/ml) were added to the synthetic oviductal fluid (SOF) and Ferticult media. The IVM, IVF, CR, and IVC (Morula and Blastocyst) rates were evaluated. The results showed that SOF maturation media containing histidine at 0.5 mg/ml significantly (P ≤ 0.01) improved the oocyte maturation when compared to control and other concentrations. The addition of histidine to FertiCult media at 0.5, 1, and 3 mg/ml did not improve the IVM, IVF, CR, or IVC percentages. However, the embryos in the control group were unable to grow into a morula or blastocyst in the SOF or Ferticult, while addition of L-Tyrosine to the SOF or Ferticult at various concentrations improved IVC (morula and blastocyst rates). There was a significant (P ≤ 0.01) increase in IVM when histidine was added to SOF medium at a concentration of 0.5 mg/ml compared with L-Tyrosine. Also, there were significant (P ≤ 0.01) increases in IVC when L-Tyrosine was added to SOF medium at concentrations of 1 and 10 mg/ml compared with histidine. In conclusion, the supplementation of the SOF and FertiCult with the amino acids histidine and L-Tyrosine improve the maturation rate of oocytes and development of in vitro-produced buffalo embryos.

Structural transition of GP64 triggered by a pH-sensitive multi-histidine switch.

The fusion of viruses with cellular membranes is a critical step in the life cycle of enveloped viruses. This process is facilitated by viral fusion proteins, many of which are conformationally pH-sensitive. The specifics of how changes in pH initiate this fusion have remained largely elusive. This study presents the cryo-electron microscopy (cryo-EM) structures of a prototype class III fusion protein, GP64, in its prefusion and early intermediate states, revealing the structural intermediates accompanying the membrane fusion process. The structures identify the involvement of a pH-sensitive switch, comprising H23, H245, and H304, in sensing the low pH that triggers the initial step of membrane fusion. The pH sensing role of this switch is corroborated by assays of cell-cell syncytium formation and dual dye-labeling. The findings demonstrate that coordination between multiple histidine residues acts as a pH sensor and activator. The involvement of a multi-histidine switch in viral fusion is applicable to fusogens of human-infecting thogotoviruses and other viruses, which could lead to strategies for developing anti-viral therapies and vaccines.

The effect of histidine, histamine, and imidazole on electrochemical properties of Cu(II) complexes of Aß peptides containing His-2 and His-3 motifs.

The N-truncation of amyloid beta (Aß) peptides could lead to peptide sequences with the histidine residue at the second and third positions, creating His-2 and His-3 motifs, known as high-affinity Cu(II) binding sites. In such complexes, the Cu(II) ion is arrested in a rigid structure of a square-planar arrangement of nitrogen donors, which highly limits its susceptibility to Cu(II) reduction. Cu(II) reduction fuels the Cu(II)/Cu(I) redox cycle, which is engaged in the production of reactive oxygen species (ROS). Employing electrochemical techniques, cyclic voltammetry (CV) and differential pulse voltammetry (DPV), together with UV-vis spectroscopy, we showed that low-molecular-weight (LMW) substances, such as imidazole, histamine, and histidine, could enhance the redox activity of Cu(II) complexes of three models of N-truncated Aß peptides, Aß4-9, Aß5-9, and Aß12-16, identifying three main mechanisms. LMW compounds could effectively compete with Aß peptides for Cu(II) ions, forming Cu(II)/LMW species, which are more prone to Cu(II) reduction. LMW substances could also shift the equilibrium between the Cu(II)/Aß species towards the species with higher susceptibility to Cu(II) reduction. Finally, the presence of LMW molecules could promote Cu(I) reoxidation in ternary Cu(II)/Aß/LMW systems. The obtained results raise further questions regarding the Cu(II) redox activity in Alzheimer's disease.

Alternative buffer systems in biopharmaceutical formulations and their effect on protein stability.

The formulation of biopharmaceutical drugs is designed to eliminate chemical instabilities, increase conformational and colloidal stability of proteins, and optimize interfacial stability. Among the various excipients involved, buffer composition plays a pivotal role. However, conventional buffers like histidine and phosphate buffers may not always be the optimal choice for all monoclonal antibodies (mAbs). In this study, we investigated the effects of several alternative buffer systems on seven different mAbs, exploring various combinations of ionic strengths, concentrations of the main buffer component, mAb concentrations, and stress conditions. Protein stability was assessed by analyzing soluble aggregate formation through size exclusion chromatography. At low protein concentrations, protein instability after temperature stress was exclusively observed in the bis-TRIS/ glucuronate buffer. Conversely, freeze-thaw stress led to a significant increase in aggregate formation in tested formulations, highlighting the efficacy of several alternative buffers, particularly arginine/ citrate, in preserving protein stability. Under temperature stress, the introduction of arginine to histidine buffer systems provided additional stabilization, while the addition of lysine resulted in protein destabilization. Similarly, the incorporation of arginine into histi-dine/HCl buffer further enhanced protein stability during freeze--thaw cycles. At high protein concentrations, the histidine/citrate buffer emerged as one of the most optimal choices for addressing temperature and light-induced stress. The efficacy of histidine buffers in combating light stress might be attributed to the light-absorbing properties of histidine molecules. Our findings demonstrate that the development of biopharmaceutical formulations should not be confined to conventional buffer systems, as numerous alternative options exhibit comparable or even superior performance.

Ultrafast His-Tagged Protein Purification.

This article details how to use a vortex fluidic device (VFD) to accelerate protein purification via immobilized metal affinity chromatography (IMAC). Building upon a previous report of VFD-based purification, we introduce a membrane insert to simplify the purification protocol and the resin recovery step. This new platform can be adapted to different types of IMAC resins and purification membranes. Proteins can be purified directly from clarified lysate, non-clarified lysate, and even non-lysed cultures without concerns of system clogging. Strong binding between the Ni2+ and the target protein's His6-tag effectively captures the target protein on IMAC resins or membranes placed in the VFD. Continuous flow of different solutions through the VFD allows dynamic binding, washing, and elution of the target protein. Furthermore, the system dramatically accelerates protein purification; a typical purification from cell lysate requires approximately 4 min. Herein, we demonstrate the single-step purification of two His6-tagged proteins from both clarified and non-clarified cell lysates without requiring batch binding. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of the resin-loaded membrane insert and the vortex fluidic device (VFD) setup prior to purification Basic Protocol 2: Purification of His6-tagged proteins using the VFD Alternate Protocol: VFD-mediated His6-tagged protein purification from non-clarified lysate Support Protocol: Preparation of chemically modified glass fiber membrane for VFD-mediated immobilized metal affinity chromatography purification.

Metabolic labeling based methylome profiling enables functional dissection of histidine methylation in C3H1 zinc fingers.

Protein methylation is a functionally important post-translational modification that occurs on diverse amino acid residues. The current proteomics approaches are inefficient to discover the methylation on residues other than Arg and Lys, which hinders the deep understanding of the functional role of rare protein methylation. Herein, we present a methyl-specific metabolic labeling approach for global methylome mapping, which enable the acquisition of methylome dataset covering diverse methylation types. Interestingly, of the identified methylation events, His methylation is found to be preferably occurred in C3H1 zinc fingers (ZFs). These His methylation events are determined to be Nπ specific and catalyzed by CARNMT1. The His methylation is found to stabilize the structure of ZFs. U2AF1 is used as a proof-of-concept to highlight the functional importance of His methylation in ZFs in RNA binding and RNA metabolism. The results of this study enable novel understanding of how protein methylation regulates cellular processes.

Discovery, Characterization and Engineering of the Free l-Histidine C4-Prenyltransferase.

Prenylation of amino acids is a critical step for synthesizing building blocks of prenylated alkaloid family natural products, where the corresponding prenyltransferase that catalyzes prenylation on free l-histidine (l-His) has not yet been identified. Here, we first discovered and characterized a prenyltransferase FunA from the antifungal agent fungerin pathway that efficiently performs C4-dimethylallylation on l-His. Crystal structure-guided engineering of the prenyl-binding pocket of FunA, a single M181A mutation, successfully converted it into a C4-geranyltransferase. Furthermore, FunA and its variant FunA-M181A show broad substrate promiscuity toward substrates that vary in substituents of the imidazole ring. Our work furthers our knowledge of free amino acid prenyltransferase and expands the arsenal of alkylation biocatalysts for imidazole-containing small molecules.

Broad-spectrum degradation of fluoroquinolone antibiotics by Hemin-His-Fe nanozymes with peroxidase-like activity.

Fluoroquinolones are a widely used class of antibiotics, with a large variety, which are frequently monitored in the aqueous environment, threatening ecological and human health. To date, effective degradation of fluoroquinolone antibiotics remains a major challenge. Focused on the broad-spectrum degradation of fluoroquinolone antibiotics, a novel biomimetic peroxidase nanozyme named Hemin-His-Fe (HHF)-peroxidase nanozyme was synthesized through a green and rapid "one-pot" method involving hemin, Fmoc-L-His and Fe2+ as precursors. After systematic optimization of the reaction conditions, fluoroquinolone antibiotics can be degraded by the HHF-peroxidase nanozyme when supplemented with H2O2 in acidic environments. Through validation and analysis, it was proved that the generated strong oxidative hydroxyl radicals are the main active species in the degradation process. In addition, it was verified that this method shows great universal applicability in real water samples.

Purification of his-tagged proteins using printed monolith adsorption columns.

Bio-separation is a crucial process in biotechnology and biochemical engineering for separating biological macromolecules, and the field has long relied on bead-based and expanded bed chromatography. Printed monolith adsorption (PMA) is a new alternative to which uses a 3D-printed monolithic structure containing self-supporting, ordered flow channels. PMA allows for direct purification of biological molecules from crude cell lysates and cell cultures, and like the other technologies, can functionalized to specifically target a molecule and enable affinity chromatography. Here we have combined PMA technology with an immobilized metal affinity ligand (iminodiacetic acid) to provide selectivity of binding to polyhistidine-tagged proteins during PMA chromatography. Two different PMA structures were created and tested for both static and dynamic protein-binding capacity. At comparative linear flow rates, the dynamic binding capacity of both columns was ≈3 mg/mL, while static capacity was shown to differentiate based on column voidage. We show that a polyhistidine-tagged protein can be directly purified from crude lysate with comparable results to the available commercial providers of IMAC, and with a substantially reduced purification time.

A cocktail of histidine, carnosine, cysteine and serine reduces adiposity and improves metabolic health and adipose tissue immunometabolic function in ovariectomized rats.

Many women have sought alternative therapies to address menopause. Recently, a multi-ingredient supplement (MIS) containing L-histidine, L-carnosine, L-serine, and L-cysteine has been shown to be effective at ameliorating hepatic steatosis (HS) in ovariectomized (OVX) rats, a postmenopausal oestrogen deficiency model. Considering that HS frequently accompanies obesity, which often occurs during menopause, we aimed to investigate the effects of this MIS for 8 weeks in OVX rats. Twenty OVX rats were orally supplemented with either MIS (OVX-MIS) or vehicle (OVX). Ten OVX rats received vehicle orally along with subcutaneous injections of 17ß-oestradiol (OVX-E2), whereas 10 rats underwent a sham operation and received oral and injected vehicles (control group). MIS consumption partly counteracted the fat mass accretion observed in OVX animals, leading to decreased total fat mass, adiposity index and retroperitoneal white adipose tissue (RWAT) adipocyte hypertrophy. OVX-MIS rats also displayed increased lean mass and lean/fat ratio, suggesting a healthier body composition, similar to the results reported for OVX-E2 animals. MIS consumption decreased the circulating levels of the proinflammatory marker CRP, the total cholesterol-to-HDL-cholesterol ratio and the leptin-to-adiponectin ratio, a biomarker of diabetes risk and metabolic syndrome. RWAT transcriptomics indicated that MIS favourably regulated genes involved in adipocyte structure and morphology, cell fate determination and differentiation, glucose/insulin homeostasis, inflammation, response to stress and oxidative phosphorylation, which may be mechanisms underlying the beneficial effects described for OVX-MIS rats. Our results pave the way for using this MIS formulation to improve the body composition and immunometabolic health of menopausal women.

Bioinspired Dual Hemin-Bonded G-Quadruplex and Histidine-Functionalized Metal-Organic Framework for Sensitive Biosensing.

Biomimetic enzymes have emerged as ideal alternatives to natural enzymes, and there is considerable interest in designing biomimetic enzymes with enhanced catalytic performance to address the low activity of the current biomimetic enzymes. In this study, we proposed a meaningful strategy for constructing an efficient peroxidase-mimicking catalyst, called HhG-MOF, by anchoring histidine (H) and dual hemin-G-quadruplex DNAzyme (double hemin covalently linked to 3' and 5' terminals of G-quadruplex DNA, short as hG) to a mesoporous metal-organic framework (MOF). This design aims to mimic the microenvironment of natural peroxidase. Remarkably, taking a terbium MOF as a typical model, the initial rate of the resulting catalyst was found to be 21.1 and 4.3 times higher than that of Hh-MOF and hG-MOF, respectively. The exceptional catalytic properties of HhG-MOF can be attributed to its strong affinity for substrates. Based on the inhibitory effect of thiocholine (TCh) produced by the reaction between acetylcholinesterase (AChE) and acetylthiocholine, a facile, cost-effective, and sensitive colorimetric method was designed based on HhG-MOF for the measurement of AChE, a marker of several neurological diseases, and its inhibitor. This allowed a linear response in the 0.002 to 1 U L-1 range, with a detection limit of 0.001 U L-1. Furthermore, the prepared sensor demonstrated great selectivity and performed well in real blood samples, suggesting that it holds promise for applications in the clinical field.

A pH-sensitive motif in an outer membrane protein activates bacterial membrane vesicle production.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella Typhimurium increases OMV production inside the acidic vacuoles of host cells by changing expression of its outer membrane proteins and modifying the composition of lipid A. However, the molecular mechanisms that translate pH changes into OMV production are not completely understood. Here, we show that the outer membrane protein PagC promotes OMV production through pH-dependent interactions between its extracellular loops and surrounding lipopolysaccharide (LPS). Structural comparisons and mutational studies indicate that a pH-responsive amino acid motif in PagC extracellular loops, containing PagC-specific histidine residues, is crucial for OMV formation. Molecular dynamics simulations suggest that protonation of histidine residues leads to changes in the structure and flexibility of PagC extracellular loops and their interactions with the surrounding LPS, altering membrane curvature. Consistent with that hypothesis, mimicking acidic pH by mutating those histidine residues to lysine increases OMV production. Thus, our findings reveal a mechanism for sensing and responding to environmental pH and for control of membrane dynamics by outer membrane proteins.

Effect of amino acids on the formation and distribution of volatile aldehydes in high oleic sunflower oil during frying.

This research aims to assess the effect of amino acids as lipid antioxidants in reducing the formation of volatile aldehydes in frying oil. Methionine, histidine, and glycine at concentrations of 2.5, 5, and 10 mM were added to high oleic sunflower oil (HOSO) to investigate their effects on the distribution and formation of saturated, monounsaturated, and polyunsaturated volatile aldehydes. The results showed that the proportion of saturated volatile aldehydes was greater than that of unsaturated ones; Methionine exhibited the best inhibitory effect, after 12 h of frying, 10 mM methionine reduced the content of saturated volatile aldehydes by 24.21 %, monounsaturated by 52.4 %, and polyunsaturated by 54.73 % compared to the control. Methionine's sulfur-containing side chain was also proven to have strong antioxidant activity. Combined with the results of this study, this can also provide insights for using amino acids as lipid antioxidants.

Unveiling the importance of the C-terminus in the sugar acid dehydratase of the IlvD/EDD superfamily.

Microbial non-phosphorylative oxidative pathways present promising potential in the biosynthesis of platform chemicals from the hemicellulosic fraction of lignocellulose. An L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii catalyzes the rate-limiting step in the non-phosphorylative oxidative pathways, that is, converts sugar acid to 2-dehydro-3-deoxy sugar acid. We have shown earlier that the enzyme forms a dimer of dimers, in which the C-terminal histidine residue from one monomer participates in the formation of the active site of an adjacent monomer. The histidine appears to be conserved across the sequences of sugar acid dehydratases. To study the role of the C-terminus, five variants (H579A, H579F, H579L, H579Q, and H579W) were produced. All variants showed decreased activity for the tested sugar acid substrates, except the variant H579L on D-fuconate, which showed about 20% increase in activity. The reaction kinetic data showed that the substrate preference was slightly modified in H579L compared to the wild-type enzyme, demonstrating that the alternation of the substrate preference of sugar acid dehydratases is possible. In addition, a crystal structure of H579L was determined at 2.4 Å with a product analog 2-oxobutyrate. This is the first enzyme-ligand complex structure from an IlvD/EDD superfamily enzyme. The binding of 2-oxobutyrate suggests how the substrate would bind into the active site in the orientation, which could lead to the dehydration reaction. KEY POINTS: • Mutation of the last histidine at the C-terminus changed the catalytic activity of L-arabinonate dehydratase from R. leguminosarum bv. trifolii against various C5/C6 sugar acids. • The variant H579L of L-arabinonate dehydratase showed an alteration of substrate preferences compared with the wild type. • The first enzyme-ligand complex crystal structure of an IlvD/EDD superfamily enzyme was solved.

ALTERATIONS IN HISTIDINE METABOLISM IS A FEATURE OF EARLY AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD).

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by epithelial proliferation and progressive cyst enlargement. Using a non-targeted high-resolution metabolomics approach, we analyzed biofluids from 36 ADPKD and 18 healthy controls with estimated glomerular filtration rate (eGFR) > 60 ml/min to identify features specific to ADPKD or that associate with disease severity [eGFR or height-corrected total kidney volume (htTKV)]. Multiple pathways differed between ADPKD subjects and controls, with the histidine pathway being the most highly represented. Plasma histidine, urinary N-methylhistamine, methylimidazole-acetaldehyde, and imidazole-acetaldehyde, as well as 3-methylhistidine and anserine were increased, while plasma N-acetylhistamine and urinary imidazole-acetic acid were decreased in ADPKD compared to controls. In ADPKD, urinary histidine and a histidine derivative, urocanate (a precursor of glutamate), were significantly associated. HtTKV and eGFR were inversely associated with urinary glutamine and plasma 4-imidazolone-5-propionic acid, respectively. Supernatant from cultured human ADPKD renal cystic epithelia demonstrated increased aspartate and glutamate levels at 8 and 24 hours compared to primary tubular epithelia (p < 0.001). Following exposure over 48 hours to α-fluromethylhistidine, an inhibitor of histamine production, primary human PKD1 cyst epithelia proliferation increased significantly from baseline (p < 0.01) and greater than non-cystic epithelia (p < 0.05). The histidine ammonia lyase inhibitor nitromethane reversed α-fluromethylhistidine-induced cyst epithelia proliferation indicating a role for glutamate in cyst growth. In conclusion, histidine metabolism is altered preferentially leading to glutamate production and epithelial proliferation in ADPKD and associates with disease severity.

Proton Transfer Kinetics in Histidine Side Chains Determined by pH-Dependent Multi-Nuclear NMR Relaxation.

Histidine is a key amino-acid residue in proteins with unique properties engendered by its imidazole side chain that can exist in three different states: two different neutral tautomeric forms and a protonated, positively charged one with a pKa value close to physiological pH. Commonly, two or all three states coexist and interchange rapidly, enabling histidine to act as both donor and acceptor of hydrogen bonds, coordinate metal ions, and engage in acid/base catalysis. Understanding the exchange dynamics among the three states is critical for assessing histidine's mechanistic role in catalysis, where the rate of proton exchange and interconversion among tautomers might be rate limiting for turnover. Here, we determine the exchange kinetics of histidine residues with pKa values representative of the accessible range from 5 to 9 by measuring pH-dependent 15N, 13C, and 1H transverse relaxation rate constants for 5 nuclei in each imidazole. Proton exchange between the imidazole and the solvent is mediated by hydronium ions at acidic and neutral pH, whereas hydroxide mediated exchange becomes the dominant mechanism at basic pH. Proton transfer is very fast and reaches the diffusion limit for pKa values near neutral pH. We identify a direct pathway between the two tautomeric forms, likely mediated by a bridging water molecule or, in the case of high pH, hydroxide ion. For histidines with pKa 7, we determine all rate constants (lifetimes) involving protonation over the entire pH range. Our approach should enable critical insights into enzymatic acid/base catalyzed reactions involving histidines in proteins.

A pH-Responsive Protein Assembly through Clustering of a Charge-Tunable Single Amino Acid Repeat.

Specific targeting of tumor cells is a key to achieving high therapeutic efficacy while minimizing off-target side effects. As a general approach to targeting diverse tumor cells, considerable attention has been paid to the tumor microenvironment, particularly its slightly acidic pH (6.5-6.8). However, existing pH-sensitive nanomaterials, based on organic polymers and proteins, often lack sufficient pH sensitivity and specificity. Here, we demonstrate a strategy to construct a pH-responsive protein assembly through clustering of a single amino acid repeat as a charge-tunable moiety. As a proof of concept, a histidine peptide with varying lengths was displayed on the surface of a ferritin assembly composed of 24 subunits by genetic fusion to a subunit. The resulting self-assembled ferritin particles, termed "pHerricle (pH-responsive ferritin particle)", were shown to exhibit a specific binding to tumor cells in response to pH changes through cooperative effects of histidine peptides. Increasing the histidine peptide length from 0 to 12 residues increased the pHerricle's cell-binding capacity by 21-fold and allowed modulation of the targetable pH range. General applicability as a tumor cell-targeting platform was shown by specific delivery of a cytotoxic cargo by the pHerricle into tumor cells of various origins in a pH-dependent manner.