Vladivostok, Vladivostok, Russian Federation
Yersinia pseudotuberculosis porin OmpF is a transmembrane protein that has an antiparallel β-structure, packed as a β-barrel. In this study conformational transformations of the porin were studied during its transition from a fully unfolded to a native-like state in aqueous media. The porin folding was monitored by light scattering, size- exclusion chromatography (SEC), and optical spectroscopy. SEC analysis showed, that immediately after removal of the main part of the denaturant the partially folded forms of the porin, with a predominance of one type folding intermediates are formed. These intermediates are more compact than the completely unfolded protein and they aggregate to form the soluble multimers. The chaperone Skp addition to unfolded porin solution prevents the aggregation of folding intermediates. According to circular dichroism and fluorescence spectra, OmpF porin folding intermediates have a substantial secondary structure and sufficiently compact, but not tightly packed tertiary structure. These folding intermediates structurally resemble a molten globule. It was estimated macromolecular crowding effect on folding of the porin. These results contribute to the understanding of the mechanisms of the membrane proteins folding and aggregation in vivo and promote the development of methods for the efficient expression of the recombinant proteins in the form of "non-classical" inclusion bodies.
recombinant porin OmpF, Yersinia pseudotuberculosis, structure of denaturated proteins, aggregation of proteins, protein folding
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