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1.
Food Chem ; 338: 128017, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32927203

RESUMO

This study aims at providing new insight on protein denaturation in freezing-thawing. Freezing-thawing minced pork reduced water-holding of myofibrils and increased surface hydrophobicity. One additional freezing-thawing cycle at slow freezing rate caused appearance of a 160 kDa myosin-4 fragment in SDS-PAGE, further decreased water-holding of myofibrils and increased surface hydrophobicity. Fresh minced pork was exposed to either high salt (2 M KCl) only or high salt with lower pH to mimic conditions in freezing. Exposure to high salt only increased water-holding of myofibrils and hence did not reproduce myofibrillar protein changes in freezing. Exposure to combinations of lower pHs and high salt decreased water-holding and increased surface hydrophobicity, suggesting myofibrillar protein denaturation occurred by a comparable mechanism as in freezing-thawing. We propose that exposure to decreased pH combined with high solute concentrations in the unfrozen water of frozen meat is the primary cause of myofibrillar protein denaturation in frozen-thawed meat.


Assuntos
Proteínas de Carne/química , Miofibrilas/química , Carne de Porco , Animais , Eletroforese em Gel de Poliacrilamida , Congelamento , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Miosinas/química , Concentração Osmolar , Carne de Porco/análise , Desnaturação Proteica , Cloreto de Sódio/química , Soluções , Água
2.
Food Chem ; 339: 127823, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32829242

RESUMO

Quinoa protein possesses great amino acid profiles and can be a potential food ingredient with broad applications. The objective of this study was to investigate the effect of different drying methods, namely freeze drying, spray drying, and vacuum drying on the functional and physicochemical properties of quinoa protein isolate, e.g., morphology, amino acid composition, SDS-PAGE profile, sulfhydryl/disulfide content, secondary structure, surface hydrophobicity, and thermal stability. The freeze-dried protein exhibited the highest emulsification capacity and stability and oil binding capacity, which was contributed to its higher surface hydrophobicity, while the spray-dried sample had the highest solubility and water absorption capacity at pH 7. Gels (8%) prepared with the freeze-dried protein had higher elastic and viscous modulus than that from others. The freeze-dried protein had the highest maximal denaturation temperature but lowest enthalpy, which may be attributed to its higher amount of random coil but lower percent of regular α-helix and ß-sheet structures. Overall, quinoa protein isolate from different processing methods demonstrated distinct functional properties. This information will be useful to optimize quinoa protein production and benefit its applications.


Assuntos
Chenopodium quinoa/química , Dessecação/métodos , Proteínas de Plantas/química , Aminoácidos/análise , Eletroforese em Gel de Poliacrilamida , Emulsões/química , Liofilização , Géis/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Plantas/isolamento & purificação , Proteínas de Vegetais Comestíveis/química , Proteínas de Vegetais Comestíveis/isolamento & purificação , Desnaturação Proteica , Estrutura Secundária de Proteína , Solubilidade , Temperatura , Vácuo , Viscosidade
3.
J Vis Exp ; (165)2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33226031

RESUMO

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.


Assuntos
Proteômica/métodos , Alquilação , Contagem de Células , Fracionamento Celular , Linhagem Celular , Cromatografia Líquida , Análise de Dados , Receptores ErbB/metabolismo , Citometria de Fluxo , Humanos , Sistema de Sinalização das MAP Quinases , Espectrometria de Massas/métodos , Desnaturação Proteica , Tripsina/metabolismo
4.
Phys Chem Chem Phys ; 22(43): 25165-25176, 2020 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-33124623

RESUMO

This work reports the experimental measurements of solvent acidity (SA), basicity (SB), and solvent dipolarity and polarizability (SPP) for water solutions with urea (U) and its molecular derivatives, monomethyl-urea (MU), 1,3-dimethyl-urea (DMU) and tetramethyl-urea (TMU). These solvatochromic parameters are applied to understanding the variation of indexes of refraction and densities and other physico-chemical properties reported for these solutions. These properties are well correlated to the SA, SB, and SPP solvent parameters of these solutions. As a result, from the characterization of the physico-chemical properties, one can infer that urea and its molecular derivatives are mainly modifiers in the structure of liquid water. The solvatochromic parameters indicate the possible existence of different mechanisms in the denaturation process of proteins in these urea/water solutions.


Assuntos
Desnaturação Proteica , Solventes/química , Ureia/química , Água/química , Concentração de Íons de Hidrogênio , Ureia/análogos & derivados
5.
Arch Biochem Biophys ; 695: 108484, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-32883513

RESUMO

The event of protein folding is associated with essential biological functionalities and unfolding of protein native state can cause intra-cellular toxicity leading to biological dysfunctions and even cell death. The present study discusses the folding-unfolding equilibrium of the small globular protein Trp-cage in presence of denaturing and protecting osmolytes urea and choline-O-sulfate (COS), respectively, employing Replica Exchange Molecular Dynamics (REMD), extensive free energy calculations and temperature scanned free energy landscapes. It is shown that, while 6 M urea quite easily denatures the protein, 0.5 M and 1 M COS is able to protect the protein from urea induced denaturation at room temperature. However, REMD simulations reveal that while the protein in pure water can withstand a simulation temperature as high as 420 K without melting, the protecting effect of 0.5 M and 1 M COS is operative up to 300 and 340 K respectively. This study furnishes evidences to shed light into the protecting mechanism of COS regarding urea induced protein unfolding, thereby putting forward the use of COS as a proper protecting osmolyte towards different types of proteins.


Assuntos
Colina/química , Simulação de Dinâmica Molecular , Desnaturação Proteica , Desdobramento de Proteína , Proteínas/química , Ureia/química , Termodinâmica
6.
Sci Rep ; 10(1): 15165, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938971

RESUMO

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of [Formula: see text]-helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/ .


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/genética , Engenharia de Proteínas , Estabilidade Proteica , Software , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clostridium/química , Clostridium/genética , Simulação por Computador , Transportador Equilibrativo 1 de Nucleosídeo/química , Transportador Equilibrativo 1 de Nucleosídeo/genética , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Conformação Proteica em alfa-Hélice/genética , Desnaturação Proteica , Pirofosfatases/química , Pirofosfatases/genética , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia Estrutural de Proteína
7.
Biochim Biophys Acta Gen Subj ; 1864(12): 129709, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32858085

RESUMO

BACKGROUND: In the endoplasmic reticulum (ER), folding of glycoproteins is assisted by a combined action of enzymes and chaperones that leads them to biologically functional structures. In this system, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) plays an essential role as the "folding sensor" by virtue of its ability to discriminate folding states of client glycoproteins. However, besides its transferase activity, whether UGGT1 possesses any chaperone activity that facilitates protein folding is yet to be addressed. METHODS: We prepared oligomannose-type glycan modified RNase (M9GN2-RNase) by chemoenzymatic means using M9GN-oxazoline and glycan truncated RNase B and analyzed the effect of human UGGT1 (HUGT1) for refolding of the denatured M9GN2-RNase. Refolding was evaluated based on the RNase activity which was measured by the cleavage of the RNA substrate. RESULTS: HUGT1 slightly accelerated the folding of M9GN2-RNase and non-glycosylated RNase A as the same extent. However, HUGT1 remarkably accelerated the folding of M9GN2-RNase in the presence of UDP-Glc. In contrast, neither UDP nor UDP-Gal was effective in enhancing the folding. Additionally, an HUGT1 mutant which lacks the glucosyltransferase activity did not accelerate the protein folding of M9GN2-RNase. CONCLUSIONS: HUGT1has the ability to promote the refolding of denatured protein and the effect would be enhanced when HUGT1 tightly interacts with the client protein via glycan recognition. GENERAL SIGNIFICANCE: Our study provides a possibility that HUGT1 play a role not only in sensing the misfolded glycoprotein but also in promoting folding of glycoproteins in the endoplasmic reticulum glycoprotein quality control.


Assuntos
Glucosiltransferases/metabolismo , Polissacarídeos/metabolismo , Redobramento de Proteína , Ribonucleases/metabolismo , Glicosilação , Humanos , Manose/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Especificidade por Substrato
8.
Plant Mol Biol ; 104(4-5): 451-465, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32803477

RESUMO

KEY MESSAGE: The first biochemical characterization of a chloroplastic disaggregase is reported (Arabidopsis thaliana ClpB3). ClpB3 oligomerizes into active hexamers that resolubilize aggregated substrates using ATP and without the aid of partners. Disaggregases from the Hsp100/Clp family are a type of molecular chaperones involved in disassembling protein aggregates. Plant cells are uniquely endowed with ClpB proteins in the cytosol, mitochondria and chloroplasts. Chloroplastic ClpB proteins have been implicated in key processes like the unfolded protein response; however, they have not been studied in detail. In this study, we explored the biochemical properties of a chloroplastic ClpB disaggregase, in particular, ClpB3 from A. thaliana. ClpB3 was produced recombinantly in Escherichia coli and affinity-purified to near homogeneity. ClpB3 forms a hexameric complex in the presence of MgATP and displays intrinsic ATPase activity. We demonstrate that ClpB3 has ATPase activity in a wide range of pH and temperature values and is particularly resistant to heat. ClpB3 specifically targets unstructured polypeptides and mediates the reactivation of heat-denatured model substrates without the aid of the Hsp70 system. Overall, this work represents the first in-depth biochemical description of a ClpB protein from plants and strongly supports its role as the putative disaggregase chaperone in chloroplasts.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Magnésio/metabolismo , Chaperonas Moleculares/metabolismo , Desnaturação Proteica , Temperatura
9.
Arch Biochem Biophys ; 692: 108547, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32828796

RESUMO

Anthrax lethal factor (LF) is a critical component of the anthrax toxin, and functions intracellularly as a zinc-dependent endopeptidase targeting proteins involved in maintaining critical host signaling pathways. To reach the cytoplasm, LF requires to be unfolded and guided through the narrow protective antigen pore in a pH-dependent process. The current study sought to address the question as to whether LF is capable of retaining its metal ion when exposed to a low-pH environment (similar to that found in late endosomes) and an unfolding stress (induced by urea). Using a combination of tryptophan fluorescence spectroscopy and chelation studies, we show that a decrease in the pH value (from 7.0 to 5.0) leads to a pronounced shift in the onset of structural alterations in LF to lower urea concentrations. More importantly, the enzyme was found to retain its Zn2+ ion beyond the unfolding transitions monitored by Trp fluorescence, a finding indicative of tight metal binding to LF in a non-native state. In addition, an analysis of red-edge excitation shift (REES) spectra suggests the protein to maintain residual structure (a feature necessary for metal binding) even at very high denaturant concentrations. Furthermore, studies using the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) revealed LF's Zn2+ ion to become accessible to complexation at urea concentrations in between those required to cause structural changes and metal dissociation. This phenomenon likely originates from the conversion of a PAR-inaccessible (closed) to a PAR-accessible (open) state of LF at intermediate denaturant concentrations.


Assuntos
Antígenos de Bactérias/química , Bacillus anthracis/química , Toxinas Bacterianas/química , Quelantes/química , Zinco/química , Concentração de Íons de Hidrogênio , Desnaturação Proteica
10.
PLoS One ; 15(8): e0237888, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813716

RESUMO

Norovirus, the leading cause of non-bacterial food poisoning, is responsible for several outbreaks associated with bivalves and ready-to-eat food products worldwide. As norovirus is resistant to alcohol, which is commonly used in food manufacturing processes, sodium hypochlorite is used for its inactivation. However, sodium hypochlorite has two disadvantages: it cannot be added to foods, and its effect is significantly reduced in the presence of organic compounds. Thus, a novel disinfectant against norovirus is urgently required for food hygiene. Thermally denatured egg white lysozyme inactivates norovirus; however, the optimal inactivating conditions and the underlying mechanism are unclear. In the present study, the inactivating mechanism of heat-denatured lysozyme against norovirus was analyzed using murine norovirus strain 1 (MNV-1). We found that the inactivating effect was enhanced by adjusting the pH of the lysozyme solution before thermal denaturation to 6.5 or higher. The reaction of heat-denatured lysozyme and MNV-1 was irreversible, and norovirus was completely inactivated after exposure to heat-denatured lysozyme. Furthermore, it was found that lysozyme residues 5-39 contributed to the norovirus-inactivating effect. Notably, the hydrophobicity and positive charges in this region contributed to the norovirus-inactivating effect, as evidenced by the norovirus inactivation test using mutated residues 5-39. These findings are novel and highlight the possible application of heat-denatured lysozyme as a disinfectant against norovirus in a wide range of food processes.


Assuntos
Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Muramidase/metabolismo , Norovirus/fisiologia , Desnaturação Proteica , Inativação de Vírus , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Macrófagos/virologia , Camundongos , Muramidase/química , Peptídeos/química , Domínios Proteicos , Células RAW 264.7
11.
Sci Rep ; 10(1): 11118, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632120

RESUMO

CEST-MRI of the rNOE signal has been demonstrated in vitro to be closely linked to the protein conformational state. As the detectability of denaturation and aggregation processes on a physiologically relevant scale in living organisms has yet to be verified, the aim of this study was to perform heat-shock experiments with living cells to monitor the cellular heat-shock response of the rNOE CEST signal. Cancer cells (HepG2) were dynamically investigated after a mild, non-lethal heat-shock of 42 °C for 20 min using an MR-compatible bioreactor system at 9.4 T. Reliable and fast high-resolution CEST imaging was realized by a relaxation-compensated 2-point contrast metric. After the heat-shock, a substantial decrease of the rNOE CEST signal by 8.0 ± 0.4% followed by a steady signal recovery within a time of 99.1 ± 1.3 min was observed in two independent trials. This continuous signal recovery is in coherence with chaperone-induced refolding of heat-shock induced protein aggregates. We demonstrated that protein denaturation processes influence the CEST-MRI signal on a physiologically relevant scale. Thus, the protein folding state is, along with concentration changes, a relevant physiological parameter for the interpretation of CEST signal changes in diseases that are associated with pathological changes in protein expression, like cancer and neurodegenerative diseases.


Assuntos
Carcinoma Hepatocelular/patologia , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Neoplasias Hepáticas/patologia , Imagem por Ressonância Magnética/métodos , Imagem Molecular/métodos , Monitorização Fisiológica , Algoritmos , Carcinoma Hepatocelular/metabolismo , Proteínas de Choque Térmico/química , Células Hep G2 , Humanos , Interpretação de Imagem Assistida por Computador , Neoplasias Hepáticas/metabolismo , Agregados Proteicos , Desnaturação Proteica
12.
Phys Chem Chem Phys ; 22(28): 16258-16266, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32643726

RESUMO

Data indicate that small globular proteins (consisting of less than about 70 residues) tend to have high denaturation temperatures. This finding is analysed by comparing experimental denaturation enthalpy and entropy changes of a selected set of small proteins with values calculated on the basis of average and common properties of globular proteins. The conclusion is that the denaturation entropy change is smaller than expected, leading to an increase in denaturation temperature. The proposed molecular rationalization considers the existence of long-wavelength, low-frequency vibrational modes in the native state of small proteins due to their large surface-to-interior ratio. The effect of decreasing the conformational entropy gain associated with denaturation on thermal stability is directly verified by means of an already devised theoretical model [G. Graziano, Phys. Chem. Chem. Phys. 2010, 12, 14245-14252; 2014, 16, 21755-21767].


Assuntos
Proteínas/química , Termodinâmica , Desnaturação Proteica
13.
Food Chem ; 333: 127514, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32683259

RESUMO

This study investigated the effect of pH on the denaturation extent, the surface chemical composition, the water sorption isotherm and the glass transition temperature of camel and bovine whey protein's powders. The LC-MS analysis indicated that the ß-Lactoglobulin was the most denatured protein in bovine whey powders regardless the pH value, while this protein was totally absent in camel whey. The α-Lactalbumin was relatively heat stable after drying and predominated the powder surface (X-ray photoelectron spectroscopy results) in both camel and bovine whey powders regardless the pH (neutral (6.7) or acidic (4.3 and 4.6)). Analysis of the water sorption isotherms indicated that decreasing the pH induced the increase of the water activity of lactose crystallization for camel and bovine whey powders. Finally, decreasing the pH led to the decrease of the glass transition temperature of camel and bovine whey powder (at 0.13, 0.23, and 0.33 of water activity).


Assuntos
Pós/química , Proteínas do Soro do Leite/química , Adsorção , Animais , Calorimetria , Camelus , Bovinos , Cromatografia Líquida de Alta Pressão , Cristalização , Concentração de Íons de Hidrogênio , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Lactose/química , Espectrometria de Massas , Desnaturação Proteica , Propriedades de Superfície , Temperatura de Transição , Água/química , Proteínas do Soro do Leite/metabolismo
14.
Sci Rep ; 10(1): 9562, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32533020

RESUMO

Knots are remarkable topological features in nature. The presence of knots in crystallographic structures of proteins have stimulated considerable research to determine the kinetic and thermodynamic consequences of threading a polypeptide chain. By mechanically manipulating MJ0366, a small single domain protein harboring a shallow trefoil knot, we allow the protein to refold from either the knotted or the unknotted denatured state to characterize the free energy profile associated to both folding pathways. By comparing the stability of the native state with reference to the knotted and unknotted denatured state we find that knotting the polypeptide chain of MJ0366 increase the folding energy barrier in a magnitude close to the energy cost of forming a knot randomly in the denatured state. These results support that a protein knot can be formed during a single cooperative step of folding but occurs at the expenses of a large increment on the free energy barrier.


Assuntos
Dobramento de Proteína , Desdobramento de Proteína , Dicroísmo Circular , Cinética , Methanocaldococcus/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Pinças Ópticas , Conformação Proteica , Desnaturação Proteica , Proteínas Recombinantes/química , Imagem Individual de Molécula , Termodinâmica
15.
Sci Rep ; 10(1): 9817, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32555390

RESUMO

Molecular chaperones assist proteins in achieving a functional structure and prevent them from misfolding into aggregates, including disease-associated deposits. The BRICHOS domain from familial dementia associated protein Bri2 (or ITM2B) probably chaperones its specific proprotein region with high ß-sheet propensity during biosynthesis. Recently, Bri2 BRICHOS activity was found to extend to other amyloidogenic, fibril forming peptides, in particular, Alzheimer's disease associated amyloid-ß peptide, as well as to amorphous aggregate forming proteins. However, the biological functions of the central nervous system specific homologue Bri3 BRICHOS are still to be elucidated. Here we give a detailed characterisation of the recombinant human (rh) Bri3 BRICHOS domain and compare its structural and functional properties with rh Bri2 BRICHOS. The results show that rh Bri3 BRICHOS forms more and larger oligomers, somewhat more efficiently prevents non-fibrillar protein aggregation, and less efficiently reduces Aß42 fibril formation compared to rh Bri2 BRICHOS. This suggests that Bri2 and Bri3 BRICHOS have overlapping molecular mechanisms and that their apparently different tissue expression and processing may result in different physiological functions.


Assuntos
Peptídeos beta-Amiloides/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/química , Agregados Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Humanos , Cinética , Modelos Moleculares , Desnaturação Proteica , Domínios Proteicos
16.
Complement Ther Med ; 51: 102453, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32507419

RESUMO

OBJECTIVES: There is a growing body of evidence supporting the role of whole-body cryostimulation (WBC) and sauna - bathing as treatments for relaxation, mental well-being and several health problems. Despite their polar opposite temperatures, both of these treatments come with a dose of similar health benefits. This study is designed to compare effects of WBC and sauna application on the athletes' response to exercise. DESIGN: The blood samples were collected from 10 professional cross-country skiers at four stages: before exercise, after exercise, at 1-h recovery and after 24 h of rest in sessions before and after 10 thermal treatments. Differential scanning calorimetry (DSC) was used to examine the process of serum denaturation. The parameters of endothermic transition were compared at various stages of each exercise session. RESULTS: Post-exercise changes in DSC profiles of athlete's blood serum are similar in character but clearly stronger in the session held after sauna treatments and slightly weaker after WBC than those in the session not preceded by treatments. These changes can be, at least in part, explained by the exercise induced increase in the concentration of oxidized albumin. A return of serum denaturation transition to pre-exercise shape has been observed within a few hours of rest. It suggests relatively quick restoration of a fraction of non-oxidized albumin molecules during the recovery period. CONCLUSIONS: An exercise performed by athletes after a series of sauna treatments leads to temporary greater modification of the blood serum proteome than the similar exercise during the session preceded by WBC treatments.


Assuntos
Atletas , Proteínas Sanguíneas/química , Crioterapia/métodos , Exercício Físico/fisiologia , Banho a Vapor/métodos , Adulto , Varredura Diferencial de Calorimetria , Humanos , Desnaturação Proteica , Proteoma/química , Soro/química , Esqui
17.
Food Chem ; 329: 126775, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32512387

RESUMO

Fish products are a promising source of collagen; however, these extracts are biochemically unstable. Acid-soluble collagen (ASC) was isolated from the skin of eleven fish species at various physiological temperatures (Tp). Structural features of these samples were analysed in detail using Circular Dichroism (CD) and compared to their biochemical characteristics. Positive correlation (r = 0.74, p < 0.01) between the Tp and ratio of positive peak intensity to negative peak intensity (Rpn) in CD analysis suggested a higher thermal stability of ASC from warm-water fish, owing to a higher content of cyclic imino acids, such as proline and hydroxyproline (Hyp). Conversely, cold-water fish ASCs contain significantly higher levels of acyclic, hydroxyl groups carrying Ser. These results indicated that CD spectrum techniques including Rpn measurement are concise and helpful for direct detection of the triple helix structure of fish collagens, and that this structure is tightly linked to thermostability of this molecule.


Assuntos
Colágeno Tipo I/química , Hidroxiprolina/química , Prolina/química , Serina/química , Animais , Dicroísmo Circular , Peixes , Desnaturação Proteica , Temperatura
18.
Nucleic Acids Res ; 48(W1): W36-W40, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32459331

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy data provides valuable information on the behaviour of proteins in solution. The primary data to determine when studying proteins are the per-atom NMR chemical shifts, which reflect the local environment of atoms and provide insights into amino acid residue dynamics and conformation. Within an amino acid residue, chemical shifts present multi-dimensional and complexly cross-correlated information, making them difficult to analyse. The ShiftCrypt method, based on neural network auto-encoder architecture, compresses the per-amino acid chemical shift information in a single, interpretable, amino acid-type independent value that reflects the biophysical state of a residue. We here present the ShiftCrypt web server, which makes the method readily available. The server accepts chemical shifts input files in the NMR Exchange Format (NEF) or NMR-STAR format, executes ShiftCrypt and visualises the results, which are also accessible via an API. It also enables the "biophysically-based" pairwise alignment of two proteins based on their ShiftCrypt values. This approach uses Dynamic Time Warping and can optionally include their amino acid code information, and has applications in, for example, the alignment of disordered regions. The server uses a token-based system to ensure the anonymity of the users and results. The web server is available at www.bio2byte.be/shiftcrypt.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Software , Aminoácidos/química , Redes Neurais de Computação , Desnaturação Proteica , Dobramento de Proteína , Desdobramento de Proteína
19.
PLoS Comput Biol ; 16(5): e1007767, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365068

RESUMO

Many proteins have the potential to aggregate into amyloid fibrils, protein polymers associated with a wide range of human disorders such as Alzheimer's and Parkinson's disease. The thermodynamic stability of amyloid fibrils, in contrast to that of folded proteins, is not well understood: the balance between entropic and enthalpic terms, including the chain entropy and the hydrophobic effect, are poorly characterised. Using a combination of theory, in vitro experiments, simulations of a coarse-grained protein model and meta-data analysis, we delineate the enthalpic and entropic contributions that dominate amyloid fibril elongation. Our prediction of a characteristic temperature-dependent enthalpic signature is confirmed by the performed calorimetric experiments and a meta-analysis over published data. From these results we are able to define the necessary conditions to observe cold denaturation of amyloid fibrils. Overall, we show that amyloid fibril elongation is associated with a negative heat capacity, the magnitude of which correlates closely with the hydrophobic surface area that is buried upon fibril formation, highlighting the importance of hydrophobicity for fibril stability.


Assuntos
Amiloide/química , Amiloide/fisiologia , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/fisiologia , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Teóricos , Simulação de Dinâmica Molecular , Desnaturação Proteica , Dobramento de Proteína , Temperatura , Termodinâmica
20.
Nat Commun ; 11(1): 2330, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393818

RESUMO

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cß) that increase the unfolding temperature of Cα/Cß by 20 °C, improve the expression of four separate α/ß TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cß mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro.


Assuntos
Anticorpos Biespecíficos/imunologia , Biologia Computacional/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/química , Varredura Diferencial de Calorimetria , Citotoxicidade Imunológica , Imunoglobulina G/metabolismo , Camundongos , Mutação/genética , Polissacarídeos/metabolismo , Desnaturação Proteica , Estabilidade Proteica , Subunidades Proteicas/metabolismo , Receptores de Antígenos de Linfócitos T/química , Proteínas Recombinantes/metabolismo , Solubilidade , Temperatura
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