Detection and analysis of protein-protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes.
It is an essential and challenging task to unravel protein-protein interactions in their actual in vivo context. Native gel systems provide a separation platform allowing the analysis of protein complexes on a rather proteome-wide scale in a single experiment. This review focus on blue-native (BN)-PAGE as the most versatile and successful gel-based approach to separate soluble and membrane protein complexes of intricate protein mixtures derived from all biological sources. BN-PAGE is a charge-shift method with a running pH of 7.5 relying on the gentle binding of anionic CBB dye to all membrane and many soluble protein complexes, leading to separation of protein species essentially according to their size and superior resolution than other fractionation techniques can offer. The closely related colorless-native (CN)-PAGE, whose applicability is restricted to protein species with intrinsic negative net charge, proved to provide an especially mild separation capable of preserving weak protein-protein interactions better than BN-PAGE. The essential conditions determining the success of detecting protein-protein interactions are the sample preparations, e.g. the efficiency/mildness of the detergent solubilization of membrane protein complexes. A broad overview about the achievements of BN- and CN-PAGE studies to elucidate protein-protein interactions in organelles and prokaryotes is presented, e.g. the mitochondrial protein import machinery and oxidative phosphorylation supercomplexes. In many cases, solubilization with digitonin was demonstrated to facilitate an efficient and particularly gentle extraction of membrane protein complexes prone to dissociation by treatment with other detergents. In general, analyses of protein interactomes should be carried out by both BN- and CN-PAGE.
Krause
Frank
F
Department of Chemistry, Physical Biochemistry, Darmstadt University of Technology, Germany. f_krause@pop.tu-darmstadt.de
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D015388:Organelles / Q000737:chemistry*; D011387:Prokaryotic Cells / Q000737:chemistry*; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis*
D008565:Membrane Proteins; D020543:Proteome