Strategie badań tiolowych oksydoreduktaz

Strategies for the analysis of thioloxidorductases
E. K. Jagusztyn-Krynicka, A. M. Banaś, M. J. Grzeszczuk

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1. Wprowadzenie. 2. Analizy funkcjonowania białek Dsb in vivo. 2.1. Wyznaczanie stanu redoks białek. 2.2. Analiza fenotypowa zmutowanych szczepów. 3. Analizy funkcjonalne białek Dsb in vitro. 3.1. Test redukcji insuliny. 3.2 Określanie potencjału redoks. 3.3. Analiza aktywności oksydacyjnej i izomeryzacyjnej. 3.4. Określanie wartości pKa nukleofilowej cysteiny motywu CXXC. 3.5. Analizy oddziaływań pomiędzy DsbA a DsbB. 3.6. Struktury białek. 3.7. Identyfikacja substratów białek Dsb. 4. Podsumowanie

Abstract: Bacterial Dsb (disulfide bond) enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between thiol groups of cysteine residues. This process is critical for the correct folding and structural stability of many secreted and membrane proteins. The rapidly expanding number of sequenced bacterial genomes has revealed the enormous diversity among bacterial Dsb systems. While the Escherichia coli oxidative protein folding has been studied in great details, the mechanism of the Dsb systems functioning in other bacteria are rather poorly understood. Herein, we present the current methodology, both in vivo and in vitro experimental techniques, which allow us to understand the functioning of the Dsb proteins and has broaden our knowledge in the field of biochemistry and microbiology of this posttranslational protein modification. Many bacterial virulence factors are extracytoplasmic Dsb-dependent proteins. Thus, this system plays an important role in bacterial pathogenesis and the proteins of the Dsb network represent possible targets for new drugs.

1. Introduction. 2. Analysis of the Dsb functioning in vivo. 2.1. Determination of the in vivo redox state. 2.2. Phenotypic assay of the mutated strains. 3. Analysis of the Dsb functioning in vitro. 3.1. Insulin reduction assay. 3.2. Determination of the redox potential. 3.3. Assay of the oxidative and isomerase activity. 3.4. Determination of the pKa value of the cysteine residue 3.5. Determination of the interaction between DsbA and DsbB. 3.6. Protein structures. 3.7. Searching for Dsb protein substrates. 4. Conclusions