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1.
Methods Mol Biol ; 2792: 97-111, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38861081

RESUMO

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.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Escherichia coli , Hidroxipiruvato Redutase , Monoéster Fosfórico Hidrolases , Arabidopsis/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxipiruvato Redutase/genética , Hidroxipiruvato Redutase/metabolismo , Hidroxipiruvato Redutase/química , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Monoéster Fosfórico Hidrolases/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/química , Histidina/metabolismo , Histidina/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/isolamento & purificação , Oxirredutases do Álcool/química , Cromatografia de Afinidade/métodos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
2.
Protein Sci ; 33(7): e5069, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38864740

RESUMO

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.


Assuntos
Proteínas Luminescentes , Cinética , Proteínas Luminescentes/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Concentração de Íons de Hidrogênio , Processos Fotoquímicos , Proteína Vermelha Fluorescente , Histidina/química , Deutério/química , Luz
3.
Structure ; 32(6): 647-649, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38848680

RESUMO

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.


Assuntos
Trifosfato de Adenosina , Humanos , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Microscopia Crioeletrônica , RNA de Transferência/metabolismo , RNA de Transferência/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Catálise , Modelos Moleculares , Histidina/química , Histidina/metabolismo
4.
J Chromatogr A ; 1729: 465057, 2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-38857565

RESUMO

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.


Assuntos
Ensaios de Triagem em Larga Escala , Maleatos , Receptores Histamínicos H1 , Ligantes , Maleatos/química , Ensaios de Triagem em Larga Escala/métodos , Receptores Histamínicos H1/química , Receptores Histamínicos H1/metabolismo , Humanos , Histidina/química , Animais , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Células CHO , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Antagonistas dos Receptores Histamínicos H1/química , Poliestirenos/química , Cricetulus , Oligopeptídeos/química
5.
AAPS PharmSciTech ; 25(5): 102, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38714592

RESUMO

Freezing of biological drug substance (DS) is a critical unit operation that may impact product quality, potentially leading to protein aggregation and sub-visible particle formation. Cryo-concentration has been identified as a critical parameter to impact protein stability during freezing and should therefore be minimized. The macroscopic cryo-concentration, in the following only referred to as cryo-concentration, is majorly influenced by the freezing rate, which is in turn impacted by product independent process parameters such as the DS container, its size and fill level, and the freezing equipment. (At-scale) process characterization studies are crucial to understand and optimize freezing processes. However, evaluating cryo-concentration requires sampling of the frozen bulk, which is typically performed by cutting the ice block into pieces for subsequent analysis. Also, the large amount of product requirement for these studies is a major limitation. In this study, we report the development of a simple methodology for experimental characterization of frozen DS in bottles at relevant scale using a surrogate solution. The novel ice core sampling technique identifies the axial ice core in the center to be indicative for cryo-concentration, which was measured by osmolality, and concentrations of histidine and polysorbate 80 (PS80), whereas osmolality revealed to be a sensitive read-out. Finally, we exemplify the suitability of the method to study cryo-concentration in DS bottles by comparing cryo-concentrations from different freezing protocols (-80°C vs -40°C). Prolonged stress times during freezing correlated to a higher extent of cryo-concentration quantified by osmolality in the axial center of a 2 L DS bottle.


Assuntos
Embalagem de Medicamentos , Congelamento , Gelo , Embalagem de Medicamentos/métodos , Concentração Osmolar , Polissorbatos/química , Histidina/química , Produtos Biológicos/química
6.
Protein Sci ; 33(6): e5021, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38747394

RESUMO

While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.


Assuntos
Histidina , Polifosfatos , Histidina/química , Polifosfatos/química , Polifosfatos/metabolismo , Ácido Nitrilotriacético/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Humanos , Proteínas/química , Proteínas/isolamento & purificação
7.
Nat Commun ; 15(1): 4204, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38760374

RESUMO

Life continuously transduces energy to perform critical functions using energy stored in reactive molecules like ATP or NADH. ATP dynamically phosphorylates active sites on proteins and thereby regulates their function. Inspired by such machinery, regulating supramolecular functions using energy stored in reactive molecules has gained traction. Enzyme-free, synthetic systems that use dynamic phosphorylation to regulate supramolecular processes have not yet been reported, to our knowledge. Here, we show an enzyme-free reaction cycle that consumes the phosphorylating agent monoamidophosphate by transiently phosphorylating histidine and histidine-containing peptides. The phosphorylated species are labile and deactivate through hydrolysis. The cycle exhibits versatility and tunability, allowing for the dynamic phosphorylation of multiple precursors with a tunable half-life. Notably, we show the resulting phosphorylated products can regulate the peptide's phase separation, leading to active droplets that require the continuous conversion of fuel to sustain. The reaction cycle will be valuable as a model for biological phosphorylation but can also offer insights into protocell formation.


Assuntos
Peptídeos , Fosforilação , Peptídeos/metabolismo , Peptídeos/química , Histidina/metabolismo , Histidina/química , Trifosfato de Adenosina/metabolismo , Hidrólise
8.
J Med Chem ; 67(10): 8172-8185, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38695666

RESUMO

Several novel and effective cysteine targeting (Cys) covalent drugs are in clinical use. However, the target area containing a druggable Cys residue is limited. Therefore, methods for creating covalent drugs that target different residues are being looked for; examples of such ligands include those that target the residues lysine (Lys) and tyrosine (Tyr). Though the histidine (His) side chain is more frequently found in protein binding locations and has higher desirable nucleophilicity, surprisingly limited research has been done to specifically target this residue, and there are not many examples of His-targeting ligands that have been rationally designed. In the current work, we created novel stapled peptides that are intended to target hMcl-1 His 252 covalently. We describe the in vitro (biochemical, NMR, and X-ray) and cellular design and characterization of such agents. Our findings further suggest that the use of electrophiles to specifically target His residues is warranted.


Assuntos
Histidina , Peptídeos , Histidina/química , Humanos , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica em alfa-Hélice , Cristalografia por Raios X , Modelos Moleculares , Desenho de Fármacos , Ligantes
9.
J Vis Exp ; (206)2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38738876

RESUMO

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.


Assuntos
Cromatografia de Afinidade , Cromatografia de Afinidade/métodos , Endonucleases Flap/química , Endonucleases Flap/isolamento & purificação , Endonucleases Flap/metabolismo , Cromatografia Líquida/métodos , Histidina/química , Escherichia coli/genética , Escherichia coli/química , Escherichia coli/metabolismo , Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
10.
Anal Bioanal Chem ; 416(15): 3605-3617, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38713223

RESUMO

The analysis of dietary supplements is far less regulated than pharmaceuticals, leading to potential quality issues. Considering their positive effect, many athletes consume supplements containing L-histidine and ß-alanine. A new microfluidic method for the determination of L-histidine and ß-alanine in dietary supplement formulations has been developed. For the first time, capacitively coupled contactless conductivity detection was employed for the microchip electrophoresis of amino acids in real samples. A linear relationship between detector response and concentration was observed in the range of 10-100 µmol L-1 for L-histidine (R2 = 0.9968) and ß-alanine (R2 = 0.9954), while achieved limits of detection (3 × S/N ratio) were 4.2 µmol L-1 and 5.2 µmol L-1, respectively. The accuracy of the method was confirmed using recovery experiments as well as CE-UV-VIS and HPLC-UV-VIS techniques. The developed method allows unambiguous identification of amino acids in native form without chemical derivatization and with the possibility of simultaneous analysis of amino acids with metal cations.


Assuntos
Suplementos Nutricionais , Condutividade Elétrica , Eletroforese em Microchip , Histidina , beta-Alanina , Eletroforese em Microchip/métodos , Suplementos Nutricionais/análise , beta-Alanina/análise , beta-Alanina/química , Histidina/análise , Histidina/química , Limite de Detecção , Química Verde/métodos , Vidro/química
11.
Molecules ; 29(10)2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38792029

RESUMO

In this study, Cu2+ modulated silver nanoclusters were constructed for the turn-on, label-free detection of L-histidine. Six Ag NCs protected by oligonucleotides (DNA-Ag NCs) were tested in a series of experiments. Finally, A-DAN-Ag NCs were chosen as the best candidate due to their excellent fluorescent properties. The fluorescence of A-DAN-Ag NCs was quenched using Cu2+ through energy or electron transfer. However, quenched fluorescence could be restored dramatically in the presence of L-histidine due to Cu2+ liberation from A-DAN-Ag NCs and because of the chelation between the imidazole group of L-histidine and Cu2+. The proposed sensor exhibited high selectivity towards L-histidine over other amino acids, with a limit of detection (LOD) of 0.096 µM ranging from 0 to 8 µM. The proposed sensor succeeded in detecting L-histidine in diluted human urine. Therefore, the sensor has promising practical applications in biological systems.


Assuntos
Cobre , Histidina , Nanopartículas Metálicas , Prata , Espectrometria de Fluorescência , Histidina/química , Histidina/urina , Histidina/análise , Cobre/química , Cobre/análise , Prata/química , Nanopartículas Metálicas/química , Espectrometria de Fluorescência/métodos , Humanos , Limite de Detecção , Técnicas Biossensoriais/métodos , Fluorescência , Íons , Corantes Fluorescentes/química
12.
Molecules ; 29(10)2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38792033

RESUMO

Copper(II), nickel(II) and zinc(II) complexes of various peptide fragments of tau protein were studied by potentiometric and spectroscopic techniques. All peptides contained one histidyl residue and represented the sequences of tau(91-97) (Ac-AQPHTEI-NH2), tau(385-390) (Ac-KTDHGA-NH2) and tau(404-409) (Ac-SPRHLS-NH2). Imidazole-N donors of histidine were the primary metal binding sites for all peptides and all metal ions, but in the case of copper(II) and nickel(II), the deprotonated amide groups were also involved in metal binding by increasing pH. The most stable complexes were formed with copper(II) ions, but the presence of prolyl residues resulted in significant changes in the thermodynamic stability and speciation of the systems. It was also demonstrated that nickel(II) and especially zinc(II) complexes have relatively low thermodynamic stability with these peptides. The copper(II)-catalyzed oxidation of the peptides was also studied. In the presence of H2O2, the fragmentation of peptides was detected in all cases. In the simultaneous presence of H2O2 and ascorbic acid, the fragmentation of the peptide is less preferred, and the formation of 2-oxo-histidine also occurs.


Assuntos
Complexos de Coordenação , Cobre , Níquel , Fragmentos de Peptídeos , Zinco , Proteínas tau , Níquel/química , Cobre/química , Zinco/química , Proteínas tau/química , Complexos de Coordenação/química , Fragmentos de Peptídeos/química , Oxirredução , Histidina/química , Concentração de Íons de Hidrogênio , Peróxido de Hidrogênio/química , Termodinâmica
13.
Int J Biol Macromol ; 270(Pt 2): 132393, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38761898

RESUMO

Light chain amyloidosis is a conformational disease caused by the abnormal proliferation and deposition of antibody light chains as amyloid fibers in organs and tissues. The effect of Cu(II) binding to the model recombinant protein 6aJL2-R24G was previously characterized in our group, and we found an acceleration of the aggregation kinetics of the protein. In this study, in order to confirm the Cu(II) binding sites, histidine variants of 6aJL2-R24G were prepared and the effects of their interaction with Cu(II) were analyzed by circular dichroism, fluorescence spectroscopy, isothermal calorimetry titrations, and molecular dynamics simulations. Confirming our earlier work, we found that His8 and His99 are the highest affinity Cu(II) binding sites, and that Cu(II) binding to both sites is a cooperative event.


Assuntos
Cobre , Histidina , Ligação Proteica , Cobre/metabolismo , Cobre/química , Histidina/química , Histidina/metabolismo , Humanos , Sítios de Ligação , Simulação de Dinâmica Molecular , Cadeias Leves de Imunoglobulina/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/química , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose/metabolismo , Amiloidose/genética , Cinética
14.
Photochem Photobiol Sci ; 23(5): 997-1010, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38693447

RESUMO

Firefly luciferases emit yellow-green light and are pH-sensitive, changing the bioluminescence color to red in the presence of heavy metals, acidic pH and high temperatures. These pH and metal-sensitivities have been recently harnessed for intracellular pH indication and toxic metal biosensing. However, whereas the structure of the pH sensor and the metal binding site, which consists mainly of two salt bridges that close the active site (E311/R337 and H310/E354), has been identified, the specific role of residue H310 in pH and metal sensing is still under debate. The Amydetes vivianii firefly luciferase has one of the lowest pH sensitivities among the group of pH-sensitive firefly luciferases, displaying high bioluminescent activity and special spectral selectivity for cadmium and mercury, which makes it a promising analytical reagent. Using site-directed mutagenesis, we have investigated in detail the role of residue H310 on pH and metal sensitivity in this luciferase. Negatively charged residues at position 310 increase the pH sensitivity and metal sensitivity; H310G considerably increases the size of the cavity, severely impacting the activity, H310R closes the cavity, and H310F considerably decreases both pH and metal sensitivities. However, no substitution completely abolished pH and metal sensitivities. The results indicate that the presence of negatively charged and basic side chains at position 310 is important for pH sensitivity and metals coordination, but not essential, indicating that the remaining side chains of E311 and E354 may still coordinate some metals in this site. Furthermore, a metal binding site search predicted that H310 mutations decrease the affinity mainly for Zn, Ni and Hg but less for Cd, and revealed the possible existence of additional binding sites for Zn, Ni and Hg.


Assuntos
Vaga-Lumes , Histidina , Luciferases de Vaga-Lume , Mutagênese Sítio-Dirigida , Concentração de Íons de Hidrogênio , Animais , Luciferases de Vaga-Lume/metabolismo , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Vaga-Lumes/enzimologia , Histidina/química , Histidina/metabolismo , Cor , Metais Pesados/química , Metais Pesados/metabolismo , Mercúrio/química , Mercúrio/metabolismo , Cádmio/química , Cádmio/metabolismo
15.
Mikrochim Acta ; 191(6): 307, 2024 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-38713296

RESUMO

An assay that integrates histidine-rich peptides (HisRPs) with high-affinity aptamers was developed enabling the specific and sensitive determination of the target lysozyme. The enzyme-like activity of HisRP is inhibited by its interaction with a target recognized by an aptamer. In the presence of the target, lysozyme molecules progressively assemble on the surface of HisRP in a concentration-dependent manner, resulting in the gradual suppression of enzyme-like activity. This inhibition of HisRP's enzyme-like activity can be visually observed through color changes in the reaction product or quantified using UV-visible absorption spectroscopy. Under optimal conditions, the proposed colorimetric assay for lysozyme had a detection limit as low as 1 nM and exhibited excellent selectivity against other nonspecific interferents. Furthermore, subsequent research validated the practical applicability of the developed colorimetric approach to saliva samples, indicating that the assay holds significant potential for the detection of lysozymes in samples derived from humans.


Assuntos
Colorimetria , Muramidase , Saliva , Muramidase/análise , Muramidase/química , Muramidase/metabolismo , Colorimetria/métodos , Humanos , Saliva/química , Saliva/enzimologia , Limite de Detecção , Peptídeos/química , Aptâmeros de Nucleotídeos/química , Proteínas/análise , Técnicas Biossensoriais/métodos , Histidina/análise , Histidina/química
16.
J Chromatogr A ; 1727: 465011, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-38776604

RESUMO

Chiral enantiomers, especially the enantiomers of chiral drugs often exhibit different pharmacological activity, metabolism and toxicity, thus it is of great research significance to scientifically and reasonably develop single chiral drugs with low toxicity and high efficiency. Among them, high performance liquid chromatographic techniques based on chiral stationary phases (CSPs) has become one of the most attractive methods used to evaluate the enantiomeric purity of single-enantiomers compound of pharmacological relevance. In this work, pillar[5]arene functionalized with L- and D-histidine, respectively, were modified on the surface of mesoporous silica as novel chiral stationary phases called L/DHis-BP5-Sil. Notably, L/D-histidine had the characteristics of low steric hindrance and easy derivatization. Although the π-π interaction of imidazole group was weaker than that of benzene ring, the benzene ring bonding imidazole-conjugated ring in the structure produced better enantioseparation effect. The results showed that L/DHis-BP5-Sil can separate a variety of complex structural enantiomers with excellent reproducibility, thermal stability and separation performance. Hence, the unique advantage of the highly selective separation of L/DHis-BP5-Sil provides new insights into the enantioseparation field.


Assuntos
Calixarenos , Histidina , Dióxido de Silício , Estereoisomerismo , Dióxido de Silício/química , Calixarenos/química , Histidina/química , Cromatografia Líquida de Alta Pressão/métodos , Porosidade , Reprodutibilidade dos Testes , Compostos de Amônio Quaternário/química
17.
Nat Commun ; 15(1): 3975, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38729930

RESUMO

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.


Assuntos
Proteínas de Bactérias , Oxigenases de Função Mista , Oxirredução , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Domínio Catalítico , Triptofano/metabolismo , Polissacarídeos/metabolismo , Mutação , Estresse Oxidativo , Tirosina/metabolismo , Modelos Moleculares , Histidina/metabolismo , Histidina/genética
18.
Org Lett ; 26(18): 3991-3996, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38691578

RESUMO

Peptide modification by C(sp3)-H functionalization of residues at the internal positions remains underdeveloped due to the inhibitory effect of backbone amides. In this study, using histidine (His) as an endogenous directing group, we developed a novel method for the ß-C(sp3)-H functionalization of alanine (Ala) at diverse positions of peptides. Through this approach, a wide range of linear peptides were modified on the side-chain of Ala adjacent to His to afford the functionalized peptides in moderate to good yield and excellent position selectivity. Furthermore, conjugation of peptides with functional molecules such as glucuronide, oleanolic acid, dipeptide, and fluorophore derivatives was achieved.


Assuntos
Alanina , Histidina , Peptídeos , Alanina/química , Histidina/química , Peptídeos/química , Estrutura Molecular
19.
Life Sci ; 350: 122672, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38705456

RESUMO

Non-esterified fatty acids (NEFAs), key to energy metabolism, may become pathogenic at elevated levels, potentially eliciting immune reactions. Our laboratory's findings of reduced L-histidine in ketotic states, induced by heightened NEFA concentrations, suggest an interrelation with NEFA metabolism. This observation necessitates further investigation into the mitigating role of L-histidine on the deleterious effects of NEFAs. Our study unveiled that elevated NEFA concentrations hinder the proliferation of Bovine Mammary Epithelial Cells (BMECs) and provoke inflammation in a dose-responsive manner. Delving into L-histidine's influence on BMECs, RNA sequencing revealed 2124 genes differentially expressed between control and L-histidine-treated cells, with notable enrichment in pathways linked to proliferation and immunity, such as cell cycle and TNF signaling pathways. Further analysis showed that L-histidine treatment positively correlated with an increase in EdU-555-positive cell rate and significantly suppressed IL-6 and IL-8 levels (p < 0.05) compared to controls. Crucially, concurrent treatment with high NEFA and L-histidine normalized the number of EdU-555-positive cells and cytokine expression to control levels. Investigating the underlying mechanisms, Gab2 (Grb2-associated binder 2) emerged as a central player; L-histidine notably reduced Gab2 expression, while NEFA had the opposite effect (p < 0.05). Gab2 overexpression escalated nitric oxide (NO) production and IL6 and IL8 expression. However, L-histidine addition to Gab2-overexpressing cells resulted in NO concentrations indistinguishable from controls. Our findings collectively indicate that L-histidine can counteract NEFA-induced inflammation in BMECs by inhibiting Gab2 expression, highlighting its therapeutic potential against NEFA-related metabolic disturbances.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ácidos Graxos não Esterificados , Histidina , Inflamação , Animais , Ácidos Graxos não Esterificados/metabolismo , Bovinos , Inflamação/metabolismo , Histidina/farmacologia , Histidina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Feminino , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo
20.
Dalton Trans ; 53(20): 8692-8708, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38700377

RESUMO

Selective recognition of fructosyl amino acids in water by arylboronic acid-based receptors is a central field of modern supramolecular chemistry that impacts biological and medicinal chemistry. Fructosyl valine (FV) and fructosyl glycyl histidine (FGH) occur as N-terminal moieties of human glycated hemoglobin; therefore, the molecular design of biomimetic receptors is an attractive, but very challenging goal. Herein, we report three novel cationic Zn-terpyridine complexes bearing a fluorescent N-quinolinium nucleus covalently linked to three different isomers of strongly acidified phenylboronic acids (ortho-, 2Zn; meta-, 3Zn and para-, 4Zn) for the optical recognition of FV, FGH and comparative analytes (D-fructose, Gly, Val and His) in pure water at physiological pH. The complexes were designed to act as fluorescent receptors using a cooperative action of boric acid and a metal chelate. Complex 3Zn was found to display the most acidic -B(OH)2 group (pKa = 6.98) and exceptionally tight affinity for FV (K = 1.43 × 105 M-1) with a strong quenching analytical response in the micromolar concentration range. The addition of fructose and the other amino acids only induced moderate optical changes. On the basis of several spectroscopic tools (1H, 11B NMR, UV-Vis, and fluorescence titrations), ESI mass spectrometry, X-ray crystal structure, and DFT calculations, the interaction mode between 3Zn and FV is proposed in a 1 : 1 model through a cooperative two-point recognition involving a sp3 boronate-diol esterification with simultaneous coordination bonding of the carboxylate group of Val to the Zn atom. Fluorescence quenching is attributed to a static complexation photoinduced electron transfer mechanism as evidenced by lifetime experiments. The addition of FGH to 3Zn notably enhanced its emission intensity with micromolar affinity, but with a lower apparent binding constant than that observed for FV. FGH interacts with 3Zn through boronate-diol complexation and coordination of the imidazole ring of His. DFT-optimized structures of complexes 3Zn-FV and 3Zn-FGH show a picture of binding which shows that the Zn-complex has a suitable (B⋯Zn) distance to the two-point recognition with these analytes. Molecular recognition of fructosyl amino acids by transition-metal-based receptors has not been explored until now.


Assuntos
Ácidos Borônicos , Complexos de Coordenação , Corantes Fluorescentes , Piridinas , Água , Zinco , Zinco/química , Ácidos Borônicos/química , Complexos de Coordenação/química , Complexos de Coordenação/síntese química , Piridinas/química , Água/química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Valina/química , Estrutura Molecular , Histidina/química
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