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10Measurement of conformational changes in target protein in the presence of highly diluted antibody solution
Judith Klein-Seetharaman
College of Health Solutions & School of Molecular Sciences, Arizona State University, USA
jkleinse@asu. edu
The physicochemical properties ofhighly diluted antibodies to IFNg (hd-anti-IFNg) have been characterized spectroscopically [1]. These results have suggested that hd-anti-IFNg are self-organizing, dispersed systems in which nano -objects are formed. Self-organization of hd-anti-IFNg may be accompanied by a change in their properties as compared with similarly treated water. Additionallythe hd-anti-IFNg solutions might contain aggregates of initial antibodies (Abs) associated with gas nanobubbles, which remain in a multiple times -diluted liquid because of the flotation effect [2]. In addition, piezoelectric immunosensor assay s investigating the interaction between IFNg and IFNg -specific Abs suggested a biological effect of the hd-anti-IFNg on its target IFNg [3]. The presence of biological activity of a substance after a process of serial dilutions has been termed "released-activity" to emphasize the technogenic source of this activity[4]. The goal of the present study was to investigate if there is a conformational change in IFNg in the presence of hd-anti-IFNg that might contribute mechanistically to released-activity.
To this end, high-resolution solution NMR spectroscopy using a Bruker Avance 900 MHz spectrometer was used to detect protein conformational changes in IFNg. Hd-anti-IFNg (the theoretical concentration of initial Abs might be 2.5x1024 mg/ml) or placebo (hd-water) were added to NMR tubes containing 15N-labeled IFNg. Two-dimensional 1H-15N spectra were acquired using TopSpin version 3.0 software, processed and analyzed using NMR View and Sparky software as described [5]. A total of 13 peaks had shifted after addition of the hd -anti-IFNg to IFNg. The corresponding alterations were not observed in IFNg in the presence of placebo. The amino acid positions affected were A9, E39, E40, D42, I45, Q47, I50, F82, F83, and S85, as well a s V117, A119, and E120 at the C -terminus of the protein. The data support the conclusion that there is a change in structure of IFNg focused on the interface between the two monomers Likely, such steric changes in the structure could potentiallyffect the monomer-dimer equilibrium of IFNg, alter the protein oligomerization equilibria and dyiamics, and influence stoichiometry of IFNg/IFNg receptor complex formation.
The results of the current study provide support for the hypothesis that unlike traditional Ab-based drugs, the highly diluted Abs (i.e. "released-active" Abs) act by inducing conformational modifications in their targets. The hd-anti-IFNg induced conformational changes in IFNg may serve as the basis for downstream effects in the target-dependent biological pathway.
[1] I.S. Ryzhkina, L.I. Murtazina, Yu.V. Kiseleva, A.I. Konovalov, Self-organization and physicochemical properties of aqueous solutions of the antibodies to interferon gamma at ultrahigh dilution, Dokl. Phys. Chem, 462, 110-114, (2015).
[2] N.F. Bunkin, A.V. Shkirin, N.V. Penkov, S.N. Chirikov, Pi. Ignatiev, VA. Kozlov, The physical nature of mesoscopic inhomogeneities in highly diluted aqueous suspensions of protein particles, Phys. Wave Phenom, 27, 102-112, (2019).
[3] E. Don, O. Farafonova, S. Pokhil, D. Barykina, M. Nikiforova, D. Shulga, A. Borshcheva, S. Tarasov, T. Ermolaeva, and O. Epstein Use of piezoelectric immunosensors for detection of interferon-gamma interaction with specific antibodies in the presence of released-active forms of antibodies to interferon-gamma, Sensors (Basel), 16, 96, (2016).
[4] O. Epstein, The spatial homeostasis hypothesis, Symmetry, 10, 103, (2018).
[5] S.A. Tarasov, E .A. Gorbunov, E .S. Don, A .G. Emelyanova, A .L. Kovalchuk, N . Yanamala, S. Schleker, J. Klein -Seetharaman, R. Groenestein, J.-P. Tafani, P van der Meide, and O. Epstein, Insights into the Mechanism of Action of Highly-Diluted Biologics, The Journal of Immunology, 205, 1345-1354, (2020).
