Formation of Amyloid-Like Conformational States of β-Structured Membrane Proteins on the Example of the OmpF Porin from the Yersinia pseudotuberculosis Outer Membrane

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Abstract

The work presents the results of an in vitro and in silico study of the formation of amyloid-like structures under harsh denaturing conditions by the nonspecific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with a β-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with an acidic pH value at elevated temperature, followed by long-term incubation at room temperature is necessary. After heating at 95 °C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of the tertiary and secondary structure of the protein, which are accompanied by an increase in the content of the total β-structure and a sharp decrease in the value of the characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to the formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of changes in intrinsic fluorescence during protein aggregation suggest that during the formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only areas with an internally disordered structure corresponding to the external loops of the porin, but also the main framework of the molecule, which has a rigid spatial structure inherent to β-barrel.

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O. D. Novikova

G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

Author for correspondence.
Email: novolga_05@mail.ru
Russian Federation, Vladivostok

T. V. Rybinskaya

G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

Email: novolga_05@mail.ru
Russian Federation, Vladivostok

E. A. Zelepuga

G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

Email: novolga_05@mail.ru
Russian Federation, Vladivostok

V. N. Uversky

University of South Florida

Email: novolga_05@mail.ru
United States, Tampa

N. Yu. Kim

G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

Email: novolga_05@mail.ru
Russian Federation, Vladivostok

E. A. Chingizova

G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

Email: novolga_05@mail.ru
Russian Federation, Vladivostok

E. S. Menchinskaya

G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

Email: novolga_05@mail.ru
Russian Federation, Vladivostok

V. A. Khomenko

G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

Email: novolga_05@mail.ru
Russian Federation, Vladivostok

D. K. Chistyulin

G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

Email: novolga_05@mail.ru
Russian Federation, Vladivostok

O. Yu. Portnyagina

G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

Email: odd64@mail.ru
Russian Federation, Vladivostok

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2. Fig. 1. Fluorescence spectra of thioflavin T complex with porins. a - YpOmpFt after incubation in phosphate-citrate buffer solution (pH 4.5) for 2 and 4 weeks at 42 °C. b - YpOmpFt after heating at 95 °C for 5 h in Tris-HCl-buffer (pH 7.4) and subsequent exposure at 25 °C for 10 days. c - Recombinant porins, full-length (RP) and mutant c deletion of outer loop 6 (RR_del6), samples were stored in phosphate-salt buffer solution (pH 7.4) in the presence of 0.01% Zw 3-14 at 4 °C for 6 months. d - YpOmpFt after incubation in phosphate-citrate buffer solution (pH 4.5) for 2 weeks at 42 °C, followed by heating at 95 °C for 5 h and subsequent exposure of the sample at 25 °C for 10 days. Fluorescence was excited at 412 nm. Data are presented as mean ± standard deviation from three experimental repeats

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3. Fig. 2. Fluorescence microscopy of thioflavin-stained T aggregates. a - YpOmpFt after incubation in phosphate-citrate buffer solution (pH 4.5) for 4 weeks at 42 °C. b - YpOmpFt after heating at 95 °C for 5 h in Tris-HCl-buffer (pH 7.4) followed by holding the sample at 25 °C for 10 days. c - Recombinant mutant porin with deletion of outer loop 6 (RR_del6), the sample was stored in phosphate-salt buffer solution (pH 7.4) in the presence of 0.01% Zw 3-14 at 4 °C for 6 months. d - YpOmpFt after incubation in phosphate-citrate buffer solution (pH 4.5) for 2 weeks at 42 °C and heating at 95 °C for 5 h. e - YpOmpFt after incubation under the conditions indicated for panel (d) and subsequent incubation at 25 °C for 10 days. Images were obtained using an AXIO Imager microscope. A1 (Zeiss); objective was ECPlan-NEOFLUAR 40 × 0.75. Scale: 50 µm

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4. Fig. 3. Cytotoxicity of YpOmpFt against mammalian cell culture (mouse neuroblastoma Neuro-2aCCL-131™ (‘ATCC’, USA)). Cytotoxic activity was expressed as the effective concentration (EC50) at which the metabolic activity of cells was inhibited by 50%. The proportion of dead cells was normalised in each case relative to the negative control (phosphate-salt buffer). Data are presented as mean ± standard deviation from three experimental repeats; the significance of differences between experimental and control groups was assessed using the Stjudent's t-criterion (* p ≤ 0.05)

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5. Fig. 4. Physicochemical characterisation of different molecular forms of porin from Y. pseudotuberculosis. a - Electrophoregram of different molecular forms of porin from Y. pseudotuberculosis: 1 - YpOmpFm_10 kDa, denatured porin trimer, after treatment at pH 4.5 for 5 h at 95 °C followed by incubation at 25 °C for 10 days; 2 - YpOmpFm; 3 - YpOmpFt; 4 - marker proteins. b - Peptide maps of tryptic hydrolysis products of heat denatured monomer, YpOmpFm and polypeptide YpOmpFm_10 kDa. c - Determination of characteristic viscosity of different molecular forms of porin OmpF from Y. pseudotuberculosis. d - ER-MALDI spectra of YpOmpFt and polypeptide YpOmpFm_10 kDa

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6. Fig. 5. Characterisation of the spatial structure of different molecular forms of OmpF porin of Y. pseudotuberculosis. a - CD spectra in the aromatic region. Protein intrinsic fluorescence spectra of YpOmpFt and YpOmpFm_10 kDa porin samples at 280 nm (b) and 296 nm (c) excitation

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7. Fig. 6. Spatial structure model and analysis of the susceptibility of Y. pseudotuberculosis OmpF porin (IB serovar O-serotype, strain IP 31758; UniProtIDA0A0U1QUP9) to internal disorder. a - Results of the analysis of the amino acid sequence of YpOmpF porin obtained using bioinformatic tools. High values of the internal disorder probability (>0.5) on the graph correspond to regions of the amino acid sequence with intrinsically disordered structure, whereas values of the internal disorder probability from 0.15 to 0.5 are inherent to regions of the amino acid sequence with increased structural flexibility. The curves in different colours correspond to calculations made by different prediction programs: PONDR® VLS2 [45, 46], PONDR® VL3 [47], PONDR® VLXT [48], PONDR® FIT [49], IUPred-Long and IUPred-Short [50]. The web application Rapid Insorder Analysis Online (RIDAO) was used to summarize the results obtained with each prediction program [51]. b - The theoretical model of porin YpOmpF is presented as a ribbon diagram and coloured according to the predisposition of these structure sites to internal disorder: ordered sites are marked in brown and structurally plastic sites in blue.Trp residues are given in the spherical rod representation, and Tyr residues are given in the rod representation.The sequences of OmpF porins from Y. pseudotuberculosis strains 1b IP 31758, UniProtIDA0A0U1QUP9 and 1b 598 are identical

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