Abstract
Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8 M urea and exists in random coil conformation. In aqueous solutions, rOmpF undergoes conformational changes, reversible self-association, and aggregation. When transferred from 8M urea into water, PBS (containing 0.15 M NaCl, pH 7.4), or buffer containing 0.8 M urea (pH 8.0), fully unfolded rOmpF forms relatively compact monomeric intermediates prone to self-association with formation of multimers. The oligomeric intermediates have high content of native protein-like secondary structure and pronounced tertiary structure. In acidic media (pH 5.0, close to the protein isoelectric point), rOmpF undergoes rapid irreversible aggregation. Therefore, we found that medium composition significantly affects both porin folding and processes of its self-association and aggregation.
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Abbreviations
- AI:
-
aggregation index
- 8-ANS:
-
8-anilino-1-naphthalenesulfonic acid
- DLS:
-
dynamic light scattering
- IBs:
-
inclusion bodies
- MALDI-TOF MS:
-
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
- PEG:
-
polyethylene glycol
- RH:
-
hydrodynamic radius
- rOmpF:
-
recombinant OmpF
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Original Russian Text © E. V. Sidorin, V. A. Khomenko, N. Yu. Kim, P. S. Dmitrenok, A. M. Stenkova, O. D. Novikova, T. F. Solov’eva, 2017, published in Biokhimiya, 2017, Vol. 82, No. 11, pp. 1657–1669.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM17-313, October 9, 2017.
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Sidorin, E.V., Khomenko, V.A., Kim, N.Y. et al. Self-Organization of Recombinant Membrane Porin OmpF from Yersinia pseudotuberculosis in Aqueous Environments. Biochemistry Moscow 82, 1304–1313 (2017). https://doi.org/10.1134/S0006297917110086
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DOI: https://doi.org/10.1134/S0006297917110086