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1.
Phys Chem Chem Phys ; 22(5): 3008-3016, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957772

RESUMO

Infrared (IR) spectroscopy is commonly utilized for the investigation of protein structures and protein-mediated processes. While the amide I band provides information on protein secondary structures, amino acid side chains are used as IR probes for the investigation of protein reactions, such as proton pumping in rhodopsins. In this work, we calculate the IR spectra of the solvated aspartic acid, with both zwitterionic and protonated backbones, and of a capped form, i.e. mimicking the aspartic acid residue in proteins, by means of molecular dynamics (MD) simulations and the perturbed matrix method (PMM). This methodology has already proved its good modeling capabilities for the amide I mode and is here extended to the treatment of protein side chains. The computed side chain vibrational signal is in very good agreement with the experimental one, well reproducing both the peak frequency position and the bandwidth. In addition, the MD-PMM approach proposed here is able to reproduce the small frequency shift (5-10 cm-1) experimentally observed between the protonated and zwitterionic forms, showing that such a shift depends on the excitonic coupling between the modes localized on the side chain and on the backbone in the protonated form. The spectrum of the capped form, in which the amide I band is also calculated, agrees well with the corresponding experimental spectrum. The reliable calculation of the vibrational bands of carboxyl-containing side chains provides a useful tool for the interpretation of experimental spectra.


Assuntos
Aminoácidos/química , Simulação de Dinâmica Molecular , Proteínas/química , Espectrofotometria Infravermelho , Ácido Aspártico/química , Ácido Glutâmico/química , Teoria Quântica
2.
Phys Chem Chem Phys ; 22(3): 1092-1096, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31894209

RESUMO

Mechanical force plays a critical role in the relationship between protein structure and function. Force manipulation by Atomic Force Microscope can be significant and trigger chemical and biological activities of proteins. Previously we have reported that Apo-CaM undergoes through a spontaneous tertiary structural rupture under a piconewton compressive force. Here we have observed that the ruptured Apo-CaM molecules can be available to bind with C28W peptide, a typical protein signalling activity that only a Ca2+-activated CaM has. This behaviour is both unexpected and profound, as CaM in its Ca2+-non-activated form has a closed structure which does not presumably allow the molecule to bind to target peptides. In this experiment, we demonstrate that both chemical activation and force activation can play a vital role in biology, such as the cell-signalling protein dynamics and function.


Assuntos
Calmodulina/química , Proteínas/química , Transdução de Sinais , Fenômenos Biomecânicos , Ligação Proteica , Estrutura Terciária de Proteína
3.
Phys Chem Chem Phys ; 22(3): 1107-1114, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31895350

RESUMO

Super-resolution imaging techniques have largely improved our capabilities to visualize nanometric structures in biological systems. Their application further permits the quantitation relevant parameters to determine the molecular organization and stoichiometry in cells. However, the inherently stochastic nature of fluorescence emission and labeling strategies imposes the use of dedicated methods to accurately estimate these parameters. Here, we describe a Bayesian approach to precisely quantitate the relative abundance of molecular aggregates of different stoichiometry from segmented images. The distribution of proxies for the number of molecules in a cluster, such as the number of localizations or the fluorescence intensity, is fitted via a nested sampling algorithm to compare mixture models of increasing complexity and thus determine the optimum number of mixture components and their weights. We test the performance of the algorithm on in silico data as a function of the number of data points, threshold, and distribution shape. We compare these results to those obtained with other statistical methods, showing the improved performance of our approach. Our method provides a robust tool for model selection in fitting data extracted from fluorescence imaging, thus improving the precision of parameter determination. Importantly, the largest benefit of this method occurs for small-statistics or incomplete datasets, enabling an accurate analysis at the single image level. We further present the results of its application to experimental data obtained from the super-resolution imaging of dynein in HeLa cells, confirming the presence of a mixed population of cytoplasmic single motors and higher-order structures.


Assuntos
Imagem Molecular , Proteínas/química , Teorema de Bayes , Modelos Químicos , Proteínas/ultraestrutura
4.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(1): 57-59, 2020 Jan 10.
Artigo em Chinês | MEDLINE | ID: mdl-31922598

RESUMO

OBJECTIVE: To explore the genetic basis of a child with idiopathic mental retardation. METHODS: Clinical data and peripheral blood sample of the child were collected. Genomic DNA was extracted and subjected to copy number analysis using single nucleotide polymrophism array comparative genome hybridization (SNP-aCGH) and targeted capture and next generation sequencing (NGS). RESULTS: No microdeletion/microduplication were detected by SNP-aCGH. NGS has detected homozygous c.722delA (p.Asp241fs) variant of the LISN1 gene, which is known to underlie autosomal recessive mental retardation-27 (MRT 27). Both parents are carriers of the variant, conforming to the autosomal recessive inheritance. CONCLUSION: A novel pathogenic variant of the LINS1 gene has been identified, which probably underlies the MRT 27 in the patient.


Assuntos
Deficiência Intelectual , Proteínas , Criança , Hibridização Genômica Comparativa , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Deficiência Intelectual/genética , Proteínas/genética
5.
Science ; 367(6473): 26, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31896705
6.
Hum Genet ; 139(2): 257-271, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31942643

RESUMO

Severe asthenozoospermia is a common cause of male infertility. Recent studies have revealed that SPEF2 mutations lead to multiple morphological abnormalities of the sperm flagella (MMAF) without primary ciliary dyskinesia (PCD) symptoms in males, but PCD phenotype was also found in one female individual. Therefore, whether there is a phenotypic continuum ranging from infertile patients with PCD to MMAF patients with no or low noise PCD manifestations remains elusive. Here, we performed whole-exome sequencing in 47 patients with severe asthenozoospermia from 45 unrelated Chinese families. We identified four novel biallelic mutations in SPEF2 (8.9%, 4/45) in six affected individuals (12.8%, 6/47), while no deleterious biallelic variants in SPEF2 were detected in 637 controls, including 219 with oligoasthenospermia, 195 with non-obstructive azoospermia, and 223 fertile controls. Notably, all six patients exhibited PCD-like symptoms, including recurrent airway infections, bronchitis, and rhinosinusitis. Ultrastructural analysis revealed normal 9 + 2 axonemes of respiratory cilia but consistently abnormal 9 + 0 axoneme or disordered accessory structures of sperm flagella, indicating different roles of SPEF2 in sperm flagella and respiratory cilia. Subsequently, a Spef2 knockout mouse model was used to validate the PCD-like phenotype and male infertility, where the subfertility of female Spef2-/- mice was found unexpectedly. Overall, our data bridge the link between MMAF and PCD based on the association of SPEF2 mutations with both infertility and PCD in males and provide basis for further exploring the molecular mechanism of SPEF2 during spermiogenesis and ciliogenesis.


Assuntos
Anormalidades Múltiplas/patologia , Proteínas de Ciclo Celular/genética , Cílios/patologia , Transtornos da Motilidade Ciliar/patologia , Infertilidade Masculina/patologia , Proteínas/fisiologia , Cauda do Espermatozoide/patologia , Anormalidades Múltiplas/genética , Animais , Cílios/genética , Transtornos da Motilidade Ciliar/genética , Feminino , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Motilidade Espermática , Cauda do Espermatozoide/metabolismo
7.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(1): 81-86, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-31950794

RESUMO

Objective: To preliminarily investigate the differences of protein composition between immature dendritic cells (DC2.4) and their derived exosomes (DC-Exo) using a relatively rapid and sample-saving method based on nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). Methods: The supernatant of DC2.4 cells culture medium was collected and gradient centrifugation was applied to primarily extract and isolate DC-Exo; then sucrose density gradient ultracentrifugation was adopted to purify the DC-Exo. Bradford protein assay was used to determine the total protein content of the purified DC-Exo, and dynamic light scattering and transmission electron microscope were conducted to characterize the morphology and size distribution of the DC-Exo. Afterwards, protein samples including DC2.4 cells and DC-Exo were prepared by FASP enzymolysis method. Samples were performed nanoLC-MS/MS assay. The µLPickUp sample loading mode was used and only 1 µg of protein sample was required for each assay. The phase of Transport liquid and Micro A were both 0.05% trifluoroacetic acid-2% acetonitrile (ACN) aq. ( V/ V). Acclaim ® PepMap RSLC column was used to separate sample compositions and the gradient elute was adopted where the mobile phase consisted of (A) 0.1% formic acid (FA) and (B) 0.08% FA-80% ACN aq. ( V/ V) with flow rate of 0.3 µL/min. Positive APCI nanospray interface was used and "one-drive-ten" schema was set to collect primary information. The collected data was then searched and matched based on Uniport Mouse Fasta file as protein database in this case, and the re-annotated data was further sorted out and analyzed. Results: In the current study, relatively high yield of DC-Exo samples with sizes of 40-200 nm were obtained. The lyophilized protein samples prepared by FASP method could be loaded directly after redissolution, and only 1 µg of protein sample is required. The annotated results showed that DC2.4 cells contained 998 kinds of proteins, among which 227 were highly expressed and 535 were unique; while DC-Exo contained only 348 types of proteins, among which 18 were uniquely and highly expressed. There were 306 kinds of consensus proteins in both DC2.4 cells and DC-Exo, among them 7 kinds were highly expressed. Conclusion: The nanoLC-MS/MS method developed in this study only requires very small amount of protein samples, and it could primarily differentiate the protein compositions between DC2.4 cells and their derived exosomes rapidly.


Assuntos
Cromatografia Líquida , Células Dendríticas , Exossomos , Proteínas , Espectrometria de Massas em Tandem , Animais , Células Dendríticas/química , Exossomos/química , Camundongos , Proteínas/química
8.
J Chem Phys ; 152(2): 025101, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31941320

RESUMO

Many fundamental biological processes are regulated by protein-DNA complexes called synaptosomes, which possess multiple interaction sites. Despite the critical importance of synaptosomes, the mechanisms of their formation are not well understood. Because of the multisite nature of participating proteins, it is widely believed that their search for specific sites on DNA involves the formation and breaking of DNA loops and sliding in the looped configurations. In reality, DNA in live cells is densely covered by other biological molecules that might interfere with the formation of synaptosomes. In this work, we developed a theoretical approach to evaluate the role of obstacles in the target search of multisite proteins when the formation of DNA loops and the sliding in looped configurations are possible. Our theoretical method is based on analysis of a discrete-state stochastic model that uses a master equations approach and extensive computer simulations. It is found that the obstacle slows down the search dynamics in the system when DNA loops are long-lived, but the effect is minimal for short-lived DNA loops. In addition, the relative positions of the target and the obstacle strongly influence the target search kinetics. Furthermore, the presence of the obstacle might increase the noise in the system. These observations are discussed using physical-chemical arguments. Our theoretical approach clarifies the molecular mechanisms of formation of protein-DNA complexes with multiple interactions sites.


Assuntos
DNA/química , Simulação de Dinâmica Molecular , Proteínas/química , Método de Monte Carlo
9.
Chem Commun (Camb) ; 56(1): 74-77, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31790117

RESUMO

We developed a new method for the de novo formation of fluorophores based on citrate (DNFC) in biological samples. Use of an amide coupling reagent and microwave irradiation greatly facilitates the fluorophore formation on peptides and proteins with N-terminal cysteine or serine. Since N-terminal cysteine and serine can form thiazolopyridone- or oxazolopyridone-based fluorophores emitting blue and green fluorescence, respectively, by the DNFC staining, each organelle, cell and tissue exhibited a characteristic fluorescence distribution. The DNFC staining is able to provide a new potential protocol for future cell imaging, histology and diagnosis.


Assuntos
Corantes Fluorescentes/metabolismo , Sondas Moleculares/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Linhagem Celular Tumoral , Ácido Cítrico/metabolismo , Cisteína/química , Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Humanos , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/química , Células NIH 3T3 , Peptídeos/química , Estudo de Prova de Conceito , Proteínas/química , Piridonas/química , Piridonas/metabolismo , Serina/química , Tiazóis/química , Tiazóis/metabolismo
10.
J Sci Food Agric ; 100(3): 1072-1079, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31650550

RESUMO

BACKGROUND: Acid-induced hydrolysis of proteins has been used to improve the solubility and functional properties of various proteins, and could be a promising tool to facilitate the use of currently underutilized insoluble microalgae protein-rich fractions in food applications. However, the results of a prior study showed an unusual resistance of an insoluble microalgae protein-rich fraction to acid hydrolysis at room temperature. RESULTS: In the present study, the insoluble protein-rich fraction extracted from microalgae Chlorella prothothecoides was treated with 0.5 mol L-1 hydrochloric acid at 25, 45, 65 or 85 °C for 0-4 h. The results showed that hydrolysis of the fraction at 85 °C for 4 h led to decreases in the amount of insoluble protein-rich aggregates and the formation of fragments with a lower molecular weight, as well as an increase in protein solubility by approximately 40%. Nevertheless, some aggregated insoluble protein-rich particles remained, even after hydrolysis at 85 °C for 4 h. CONCLUSION: The higher temperature improved the efficiency of the acid hydrolysis of the insoluble protein fraction from microalgae Chlorella prothothecoides, which is highly acid-resistant. Overall, an erosion-based mechanism was suggested for the acid hydrolysis of insoluble microalgae protein fraction. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


Assuntos
Chlorella/química , Proteínas/química , Fracionamento Químico , Temperatura Alta , Ácido Clorídrico/química , Hidrólise , Microalgas/química , Peso Molecular , Proteínas/isolamento & purificação , Solubilidade
11.
J Chem Theory Comput ; 16(1): 553-563, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31738552

RESUMO

Proteins are exposed to various mechanical loads that can lead to covalent bond scissions even before macroscopic failure occurs. Knowledge of these molecular breakages is important to understand mechanical properties of the protein. In regular molecular dynamics (MD) simulations, covalent bonds are predefined, and reactions cannot occur. Furthermore, such events rarely take place on MD time scales. Existing approaches that tackle this limitation either rely on computationally expensive quantum calculations (e.g., QM/MM) or complex bond order formalisms in force fields (e.g., ReaxFF). To circumvent these limitations, we present a new reactive kinetic Monte Carlo/molecular dynamics (KIMMDY) scheme. Here, bond rupture rates are calculated based on the interatomic distances in the MD simulation and then serve as an input for a kinetic Monte Carlo step. This easily scalable hybrid approach drastically increases the accessible time scales. Using this new technique, we investigate bond ruptures in a multimillion atom system of tensed collagen, a structural protein found in skin, bones, and tendons. Our findings show a clear concentration of bond scissions near chemical cross-links in collagen. We also examine subsequent dynamic relaxation steps. Our method exhibits only a minor slowdown compared to classical MD and is straightforwardly applicable to other complex (bio)materials under load and related chemistries.


Assuntos
Proteínas/química , Animais , Colágeno/química , Dipeptídeos/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Método de Monte Carlo , Conformação Proteica , Teoria Quântica , Estresse Mecânico
12.
J Chem Theory Comput ; 16(1): 528-552, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31714766

RESUMO

Molecular dynamics (MD) simulations have become increasingly popular in studying the motions and functions of biomolecules. The accuracy of the simulation, however, is highly determined by the molecular mechanics (MM) force field (FF), a set of functions with adjustable parameters to compute the potential energies from atomic positions. However, the overall quality of the FF, such as our previously published ff99SB and ff14SB, can be limited by assumptions that were made years ago. In the updated model presented here (ff19SB), we have significantly improved the backbone profiles for all 20 amino acids. We fit coupled φ/ψ parameters using 2D φ/ψ conformational scans for multiple amino acids, using as reference data the entire 2D quantum mechanics (QM) energy surface. We address the polarization inconsistency during dihedral parameter fitting by using both QM and MM in aqueous solution. Finally, we examine possible dependency of the backbone fitting on side chain rotamer. To extensively validate ff19SB parameters, and to compare to results using other Amber models, we have performed a total of ∼5 ms MD simulations in explicit solvent. Our results show that after amino-acid-specific training against QM data with solvent polarization, ff19SB not only reproduces the differences in amino-acid-specific Protein Data Bank (PDB) Ramachandran maps better but also shows significantly improved capability to differentiate amino-acid-dependent properties such as helical propensities. We also conclude that an inherent underestimation of helicity is present in ff14SB, which is (inexactly) compensated for by an increase in helical content driven by the TIP3P bias toward overly compact structures. In summary, ff19SB, when combined with a more accurate water model such as OPC, should have better predictive power for modeling sequence-specific behavior, protein mutations, and also rational protein design. Of the explicit water models tested here, we recommend use of OPC with ff19SB.


Assuntos
Aminoácidos/química , Peptídeos/química , Proteínas/química , Água/química , Simulação de Dinâmica Molecular , Conformação Proteica , Estabilidade Proteica , Teoria Quântica , Termodinâmica
13.
Chemistry ; 26(1): 33-48, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31599057

RESUMO

The ability to modify biologically active molecules such as antibodies with drug molecules, fluorophores or radionuclides is crucial in drug discovery and target identification. Classic chemistry used for protein functionalisation relies almost exclusively on thermochemically mediated reactions. Our recent experiments have begun to explore the use of photochemistry to effect rapid and efficient protein functionalisation. This article introduces some of the principles and objectives of using photochemically activated reagents for protein ligation. The concept of simultaneous photoradiosynthesis of radiolabelled antibodies for use in molecular imaging is introduced as a working example. Notably, the goal of producing functionalised proteins in the absence of pre-association (non-covalent ligand-protein binding) introduces requirements that are distinct from the more regular use of photoactive groups in photoaffinity labelling. With this in mind, the chemistry of thirteen different classes of photoactivatable reagents that react through the formation of intermediate carbenes, electrophiles, dienes, or radicals, is assessed.


Assuntos
Preparações Farmacêuticas/química , Proteínas/química , Animais , Anticorpos/química , Linhagem Celular Tumoral , Radioisótopos de Cobre/química , Reação de Cicloadição , Humanos , Marcação por Isótopo , Ligantes , Metano/análogos & derivados , Metano/química , Camundongos , Camundongos Nus , Neoplasias/diagnóstico por imagem , Tomografia Computadorizada de Emissão de Fóton Único , Transplante Heterólogo , Raios Ultravioleta
14.
Exp Parasitol ; 208: 107802, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31730782

RESUMO

In insects, diet plays an important role in growth and development. Insects can vary their diet composition based on their physiological needs. In this study we tested the influence of diet composition involving varying concentrations of macronutrients and zinc on the immune-tolerance following parasite and pathogen exposure in Spodoptera litura larvae. We also tested the insecticidal potential of Mesorhabditis belari, Enterobacter hormaechei and its secondary metabolites on Spodoptera litura larvae. The results shows macronutrient composition does not directly affect the larval tolerance to nematode infection. However, Zinc supplemented diet improved the immune tolerance. While larvae exposed to bacterial infection performed better on carbohydrate rich diet. Secondary metabolites from bacteria produced an immune response in dose dependent mortality. The study shows that the larvae maintained on different diet composition show varied immune tolerance which is based on the type of infection.


Assuntos
Enterobacter/fisiologia , Controle Biológico de Vetores , Rhabditoidea/fisiologia , Spodoptera/imunologia , Análise de Variância , Animais , Bioensaio , Carboidratos/administração & dosagem , Cromatografia Líquida de Alta Pressão , Dieta , Enterobacter/imunologia , Enterobacter/patogenicidade , Cromatografia Gasosa-Espectrometria de Massas , Tolerância Imunológica , Larva/imunologia , Dose Letal Mediana , Proteínas/administração & dosagem , Rhabditoidea/imunologia , Rhabditoidea/patogenicidade , Espectroscopia de Infravermelho com Transformada de Fourier , Spodoptera/fisiologia , Simbiose , Virulência , Zinco/administração & dosagem
15.
Biomed Chromatogr ; 34(1): e4633, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31257628

RESUMO

Bioanalysis assays that reliably quantify biotherapeutics and biomarkers in biological samples play pivotal roles in drug discovery and development. Liquid chromatography coupled with mass spectrometry (LC-MS), owing to its superior specificity, faster method development and multiplex capability, has evolved as one of the most important platforms for bioanalysis of biotherapeutics, particularly new scaffolds such as half-life extension platforms for proteins and peptides, as well as antibody drug conjugates. Intact LC-MS analysis is orthogonal to bottom-up surrogate peptide approach by providing whole molecule quantitation and high-level sequence and structure information. Here we review the latest development in LC-MS bioanalysis of intact proteins and peptides by summarizing recent publications and discussing the important topics such as the comparison between top-down intact analysis and bottom-up surrogate peptide approach, as well as simultaneous quantitation and catabolite identification. Key bioanalytical issues around intact protein bioanalysis such as sensitivity, data processing strategies, specificity, sample preparation and LC condition are elaborated. For peptides, topics including quantitation of intact peptide vs. digested surrogate peptide, metabolites, sensitivity, LC condition, assay performance, internal standard and sample preparation are discussed.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Proteínas/análise , Animais , Biomarcadores/análise , Humanos , Camundongos
16.
Food Chem ; 302: 125199, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31400699

RESUMO

Animal bones are a high-quality source of protein and comprehensive nutrients and improper handling can cause resource wasting and environmental issues. Pretreatment before enzymatic hydrolysis of bone could significantly improve the enzymolytic efficiency, which is an essential step to achieve high value-added utilization of bones. This study investigated the effect of lipase pretreatment on the enzymatic hydrolysis of bones. The degree of hydrolysis after lipase pretreatment was 12.58%, which was 8.19% higher than that without pretreatment. Lipase pretreatment was optimal at 9% substrate concentration and initial pH 7.5, with 0.08% lipase, followed by 4 h incubation at 40 °C. Mechanism analysis indicated that lipase pretreatment improved the enzymolytic efficiency by significantly decreasing the lipid content, and changing the surface structure and surface element content of C, N, and O, promoting the attachment of alkaline protease onto the sample. Overall, lipase pretreatment was an effective method to reduce the costs of production.


Assuntos
Osso e Ossos/metabolismo , Lipase/metabolismo , Proteínas/metabolismo , Animais , Bovinos , Hidrólise
17.
Food Chem ; 302: 125334, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31419773

RESUMO

Carotenoids, natural pigments, are a group of chemically heterogeneous molecules, present in numerous taxonomical clusters. Because of their various bioactivities, carotenoids are day-by-day applied in numerous fields. The present work aimed to investigate an efficient extraction process of carotenoids from blue crab shells and their identification by HR-ESI-MS technique. In this context, different methods (enzymatic, maceration, Soxhlet, etc.) and solvents (variable polarity index) were tested. Maceration using the binary system hexane/isopropanol (50/50) was found to be the most efficient process, producing high carotenoids content and low total phenolic and soluble protein amounts (p < 0.05). When combined with an enzymatic pretreatment, this procedure was found to be remarkably (p < 0.05) more efficient and selective especially towards astaxanthin (p < 0.05). The HR-ESI-MS identified 23 compounds, depending on the adopted extraction approach. The compounds identified may have potential for applications in food or pharmaceutical industries.


Assuntos
Exoesqueleto/química , Braquiúros/química , Carotenoides/isolamento & purificação , Fracionamento Químico/métodos , 2-Propanol/química , Animais , Carotenoides/análise , Hexanos/química , Fenóis/análise , Fenóis/química , Proteínas/análise , Proteínas/isolamento & purificação , Solventes/química , Espectrometria de Massas por Ionização por Electrospray , Espectroscopia de Infravermelho com Transformada de Fourier , Xantofilas/análise , Xantofilas/isolamento & purificação
18.
J Chem Theory Comput ; 16(1): 601-611, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31841332

RESUMO

Extensive benchmarking calculations are presented to assess the accuracy of the standard approximate coupled cluster singles and doubles method (CC2) in studying ππ* excited states properties of model protein chains containing a phenylalanine residue, namely capped peptides, whose ground state conformers adopt the prototypical secondary structural features of proteins. First, the dependence with the basis set of the CC2 excitation energies, CC2 geometry optimizations, and amide A region frequencies of the lowest ππ* excited state in a reference system, the N-acetylphenylalaninylamide, are investigated, and the results are compared with experimental data. Second, at the best level of theory determined, the CC2/aug(N,O,π)-cc-pVDZ//CC2/cc-pVDZ level, a series of capped peptides of increasing size and containing residues of different nature are investigated. Along the series, compared to the experimental values, a mean absolute error of 0.10 eV is achieved for the 0-0 transition energies with a systematic overestimation. In addition, mode-dependent linear scaling functions for the calculated frequencies of the amide A region have been determined from the set of 95 experimental frequencies available; they lead to a quantitative simulation of the observed shifts of the amide A region frequencies upon ππ* excitation (root-mean-square deviation of 5 cm-1). These results confirm the reliability of the CC2 method to characterize the lowest ππ* excited state of such medium-sized systems, emphasizing this class of theoretical approaches as a relevant spectroscopic tool, including for tasks as difficult as conformational assignment.


Assuntos
Peptídeos/química , Fenilalanina/química , Proteínas/química , Algoritmos , Modelos Moleculares , Teoria Quântica , Termodinâmica
19.
Food Chem ; 308: 125592, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-31648086

RESUMO

This paper investigated the conformational and functional properties of myofibrillar protein modified by five phenolic compounds, including cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, caffeic acid, quercetin and rutin, dominantly presented in mulberry polyphenols-enriched sausage. These phenolic compounds significantly affected the structure of myofibrillar protein as indicated by the remarkable losses of carbonyl and ε-NH2 and the obviously fluorescence quenching effect (P < 0.05). Modified myofibrillar protein increased antioxidative activity but decreased thermal stability. Myofibirllar protein modified with rutin had no change in thermal stability but improved emulsifying properties. Quercetin has little effect on secondary structure of myofibirlliar protein. Caffeic acid triggered the conversion of α-helix to ß-sheet in myofibrillar protein, and the resulted protein exhibited the strongest fluorescence quenching, solubility and antioxidant activity among all samples. Overall, the results suggested that all phenolic compounds involved in the changes of meat product quality, with caffeic acid and rutin being the most critical ones.


Assuntos
Morus/química , Extratos Vegetais/química , Polifenóis/química , Proteínas/química , Frutas/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
20.
Bioelectrochemistry ; 131: 107371, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31513986

RESUMO

Single-channel conductance measurements in biological pores have demonstrated the importance of interfacial effects in nanopores, particularly in protein channels with low aspect ratio (length over aperture radius). Access resistance (AR), the contribution to the total measured resistance arising from the electrodiffusive limitation that ions experience in passing from bulk solution to confinement within the pore, becomes essential in the description of ionic transport across these biological channels. Common analytical estimates of AR are based on idealized nanopore models, cylindrical in shape, electrically neutral and embedded in a neutral substrate. Here we calculate the AR of five protein channels by using their atomic structure and a mean-field approach based on solving 3D Poisson and Nernst-Planck equations. Our approach accounts for the influence of the protein charged ionizable residues, the geometry of the pore mouth and the ion concentration gradients near the pore. We compare numerical calculations with the few available AR measurements and show for several protein channels that analytical predictions tend to overestimate AR for physiological concentrations and below. We also discuss the relationship between AR and the size of the channel aperture in single-pore channels and three-pore channels and demonstrate that in the latter case, there is an enhancement of AR.


Assuntos
Biologia Computacional , Nanoporos , Proteínas/química , Transporte de Íons , Eletricidade Estática
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