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6New insights into the microscopic interactions affected by highly
diluted protein solutions
K. Woods
Lehrstuhl für BioMolekulare Optik, Ludwig-Maximilians-Universitat, Oettingenstr, 67, 80538 München,
Deutschland
A number of crystal structures [1] of antigens in the bound state have revealed an essential role of the solvent molecules in the hydration shell in stabilizing their complexes. But they have also provided a detailed molecular basis for understanding the thermodynamical forces that drive the association between the antigen-antibody interactions. Hydration of molecules depends on the properties of the solution. Recently, it has been shown that the protein conformation can be significanly changed by the highly diluted (HD) antibodies [3]. Such HD antibodies were received as a result of using the technology of sequential dilutions with intensive vibration treatment between the dilution steps. Previously, the term "released-activity" had been proposed to emphasize the technogenic source of such activity [2].
We have performed experimental terahertz (THz) spectroscopy measurements on HD antibodies of interferon gamma (anti-IFN-g), HD antibodies to the interferon gamma receptor 1 (anti-IFNgR1) and HD of interferon gamma (IFN-g). The results of our investigation have uncovered distinct differences in the experimental THz properties of the HD samples when contrasted with the same samples but at a higher concentration. Specifically, we have found that HD sample preparation promotes long-range correlations on the protein surface that alter the conformational populations that influence both ligand-binding affinity and ligand recognition. It is interesting to point out that the water and HD-water spectra exhibit nearly identical peak structures, but the intensity of the two samples differs considerably. It is possible that the differences in spectral intensity reflect a modification in the H-bonding structure or H-bonding network of the water molecules that somehow stems from the HD sample preparation process.
Experimentally we have detected distinct bands in the spectra associated with specific intermolecular protein (inter-protein and protein-solvent) dynamics that correlate with the reorganization of the surface residue dynamics at the solvent-protein interface of the HD samples (HD-IFN-g and HD-anti-IFNgR1) that determine both structural and kinetic heterogeneous dynamics that induce interactions that enhance the binding probability of the antigen binding site.
Further, by use of computational modeling we are also able to distinguish the underlying microscopic dynamics that promote spatial heterogeneity within the macromolecule that alters the conformational populations that ultimately govern ligand-binding associations and ligand recognition in the studied HD samples. In the molecular dynamics simulation of the HD-anti-IFN-g dimer, we have found that the greatest modifications in dynamics take place at the dimer interface and in the C-terminal surface loop, where both regions play a critical role in binding IFN-g receptors and signaling. Our findings are in line with recent experimental measurements of the HD-prepared antibodies to IFN-g that have confirmed that interaction with IFN-g induces conformational changes in the cytokine that are conjectured to alter its biological activity as well as its interactions with other macromolecules [3].
In summary, the results of our work uncover distinct differences in the experimental THz properties of HD samples of proteins when compared with either liquid water or higher concentration protein equivalents. Moreover, with the aid of MD simulation we are able to pinpoint the physical mechanism(s) that correlate these detected differences with observable alterations in protein dynamics and dynamical interactions that directly transmit novel binding associations and enhanced functional activity caused by HD (released-active) samples influence.
[1] L. Esser, S. Shukla, F. Zhou, S.V. Ambudkar, D. Xia, Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein, Acta Crystallographica Section F: Structural Biology Communications, 72, 636-641, (2016).
[2] O. Epstein, The spatial homeostasis hypothesis, Symmetry, 10, 103, (2018).
[3] S.A. Tarasov, E.A. Gorbunov, E.S. Don, A.G. Emelyanova, A.L. Kovalchuk, N. Yanamala, A.S.S. Schleker, J. Klein-Seetharaman, R. Groenestein, J.-P. Tafani, P. van der Meide, O.I. Epstein, Insights into the Mechanism of Action of Highly Diluted Biologics, The Journal of Immunology, 205, 1345-1354, (2020).
