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1.
Redox Biol ; 53: 102332, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35598378

RESUMO

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli's proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidAHOCl's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Chaperonas Moleculares , Animais , Arginina , Cromatografia Líquida , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Halogenação , Interações Hidrofóbicas e Hidrofílicas , Ácido Hipocloroso/química , Iminas/metabolismo , Lisina , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteômica , Espectrometria de Massas em Tandem
2.
Methods Mol Biol ; 2228: 53-62, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950483

RESUMO

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH-gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures such as Coomassie, silver staining, or fluorescence labeling. This article gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.


Assuntos
Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Proteínas/análise , Proteoma , Proteômica , Animais , Corantes Fluorescentes , Humanos , Concentração de Íons de Hidrogênio , Medições Luminescentes , Projetos de Pesquisa , Coloração e Rotulagem
3.
Methods Mol Biol ; 2228: 77-84, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950485

RESUMO

Classical 2D-PAGE allows comparison and quantitation of proteomes by visualization of protein patterns using gel stains and comparative image analysis. The introduction of fluorescent reagents for protein labeling (difference in-gel electrophoresis or DIGE) has brought substantial improvement in this field. It provides multiplexing of up to three samples in one gel, higher sensitivity compared to normal protein staining methods, and a higher linear range for quantitation. This article gives detailed protocols for 2D-DIGE, including both minimal and saturation labeling.


Assuntos
Eletroforese em Gel de Poliacrilamida , Proteínas/análise , Proteoma , Proteômica , Eletroforese em Gel Diferencial Bidimensional , Animais , Corantes Fluorescentes , Humanos , Medições Luminescentes , Projetos de Pesquisa , Coloração e Rotulagem
4.
Proteins ; 63(2): 322-6, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16372358

RESUMO

Pressure perturbation calorimetry quantifies the temperature dependence of a solute's thermal expansion coefficient, providing information about solute-solvent interactions. We tested the idea that pressure perturbation calorimetry can provide information about solvent-accessible surface area by studying peptides with different secondary structures. The peptides comprised two host-guest series: one predominately an alpha-helix, the other predominately a polyproline II helix. In aqueous buffer, we find a correlation between the amount of secondary structure as assessed by circular dichroism spectropolarimetry and the pressure perturbation calorimetry data. We conclude that pressure perturbation calorimetry can provide information about the exposure of polar and nonpolar surface area. Data acquired in a buffered urea solution, however, are not as easily interpreted.


Assuntos
Peptídeos/química , Calorimetria , Dicroísmo Circular , Pressão , Estrutura Secundária de Proteína , Soluções , Temperatura , Ureia
5.
Mol Divers ; 9(4): 295-9, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16311805

RESUMO

A general method was used to prepare an array of unsymmetric sulfamides. This was accomplished by the stepwise addition of CSI to tert-butanol followed by the addition of amines. To increase diversity, nitrogen group of Boc-sulfamides was alkylated with alcohols using Mitsunobu reaction and Boc-group was removed using Si-TsOH. Microwave heating was used in all the steps. The final sulfamides were released from Si-TsOH using NH3 in MeOH.


Assuntos
Benzenossulfonatos/química , Ésteres do Ácido Fórmico/química , Ésteres do Ácido Fórmico/isolamento & purificação , Temperatura Alta , Micro-Ondas , Silicatos/química , Sulfonamidas/química , Alquilação , Técnicas de Química Combinatória , Sulfonamidas/síntese química
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