Abstract
The study of protein interactions constitutes an important domain to understand the physiology and pathogenesis of microorganisms. The two-dimensional blue native/SDS-PAGE was initially reported to analyze membrane protein complexes. In this study, both cytoplasmic and membrane complexes of a bacterium, the strain J99 of the gastric pathogen Helicobacter pylori, were analyzed by this method. It was possible to identify 34 different proteins grouped in 13 multiprotein complexes, 11 from the cytoplasm and two from the membrane, either previously reported partially or totally in the literature. Besides complexes involved in H. pylori physiology, this method allowed the description of interactions involving known pathogenic factors such as (i) urease with the heat shock protein GroEL or with the putative ketol-acid reductoisomerase IlvC and (ii) the cag pathogenicity island CagA protein with the DNA gyrase GyrA as well as insight on the partners of TsaA, a peroxide reductase/stress-dependent molecular chaperone. The two-dimensional blue native/SDS-PAGE combined with mass spectrometry is a potential tool to study the differences in complexes isolated in various situations and also to study the interactions between bacterial and eucaryotic cell proteins.
Highlights
The study of protein interactions constitutes an important domain to understand the physiology and pathogenesis of microorganisms
One of the strategies of functional proteomics, a method used to identify gene function at the protein level, is the comprehensive analysis of protein-protein interactions related to the functional linkage among proteins and the analysis of functional cellular machinery to better understand the basis of the organism’s functions
We found that the best multiprotein complex separation was obtained with a linear 4 –9% acrylamide gradient
Summary
Helicobacter pylori Proteomics studies focused on the protein complexes of the respiratory chain (20 –27). Because this method is reproducible, it was successfully used to study the detection of protein complex deficiencies of mitochondria (28 –32). The anionic dye used in this method (Coomassie Brilliant Blue G-250) binds to the surface of all proteins, on aromatic residues and on arginines This binding of a large number of negatively charged dye molecules to proteins facilitates the multiprotein complex migration in a first dimension native electrophoresis (BN-PAGE), and the tendency for protein aggregation is reduced considerably. A dialysis must be performed on cytoplasmic extracts to eliminate salt and small molecules [43] This method was applied to analyze both H. pylori cytoplasmic and membrane complexes. Purification steps such as liquid IEF or chromatography fractionation and enrichment were used to improve the multiprotein complex separation from the cytoplasm
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