RETRACTED ARTICLE: New insights into the microscopic interactions associated with the physical mechanism of action of highly diluted biologics

In this investigation, we report the effect on the microscopic dynamics and interactions of the cytokine interferon gamma (IFN-γ) and antibodies to IFN-γ (anti-IFN-γ) and to the interferon gamma receptor 1 (anti-IFNGR1) prepared in exceptionally dilute solutions of initial proteins. Using both THz spectroscopy and molecular dynamics simulations we have uncovered that the high dilution method of sample preparation results in the reorganization of the sample surface residue dynamics at the solvent–protein interface that leads to both structural and kinetic heterogeneous dynamics that ultimately create interactions that enhance the binding probability of the antigen binding site. Our results indicate that the modified interfacial dynamics of anti-IFN-γ and anti-IFGNR1 that we probe experimentally are directly associated with alterations in the complementarity regions of the distinct antibodies that designate both antigen–antibody affinity and recognition.


Supplementary
: van Hove self correlation function of (a) water in the pure water IFN-g solution and (b) water in the IFN-g mixed water -ethanol solution.

Conformational analyses and transition probabilities of IFNGR2 in the IFN-g complex
The biggest changes that we observe from the FCA analysis of IFNGR2 when transitioning from conformational substate 1 to 1a is (i) the position (angle) of the N-terminal surface loop that is involved with binding IFN-g and (ii) the C-terminal loop region, that is far more rigid in the intermediate state when compared with the same region in conformation 1 as seen in Supplementary Figure S3a . From our analyses of the MD simulation trajectories, we find that both structural regions are strongly influenced by direct H-bonding with the solvent (water) in the hydration shell. It is likely that the strong H-bonds with the solvent stabilize the conformational intermediate (conformational substate 1a) and as a consequence also modulate the energy barrier that promotes the transition to conformational substate 2 and hence the dynamical transition of the entire receptor complex.
A clustering analysis of the MD simulation of the conformational substates of the receptor has revealed details about the dwell times within the conformational substates as well as the transition probabilities among them. Using this analysis, we have determined that the majority of the time (an average of 86.9 % of the simulation time) the receptor is in conformation 1. The receptor fluctuates within this energy basin until the receptor is able to overcome the barrier separating conformation 1 and conformation 1a. The receptor spends on average only 5.5 % of the MD simulation time in conformation 1a before rapidly transitioning to conformation 2. The receptor spends again only a short period of time in conformation 2 (3.8% of the MD simulation time) before transitioning back to conformation 1. After this time period, we see arbitrary fluctuations between the two major conformational substates (conformation 1 and conformation 2), but on average the receptor spends more time in conformation 1.
In Supplementary Figure S3b, a plot of the clustering of the conformational states as a function of MD simulation time allows one to clearly discern the "jump" from conformation 1 to conformation 2. The inset of Supplementary Figure S3b shows the close-up of the time region preceding the transition from conformation 1 to conformation 2, where one can also distinguish the transition into the intermediate state (conformation 1a) that is populated briefly before transitioning to conformation 2.
The influence of the hydration water molecules in the relaxation pathway of IFNGR2 is also apparent from the plot of the RMSD of the hydrogen atoms in the hydration shell in Supplementary Figure S3c. Here we find that the dynamics of the water molecule hydrogen atoms in the hydration shell are directly tied with the relaxation dynamics of the protein. The change in the RMSD of the hydrogen atoms from the initial population in conformation 1 to the population when returning to conformation 1 (after the transition from conformation 2) reflects the more heterogeneous population of interactions after the (dynamical) transition has taken place. For comparison, we also plot the RMSD of the hydrogen atoms of the ethanol molecules in the receptor hydration shell. In the latter case, we determine that the ethanol molecules are not closely coupled with the conformational substate dynamics of the receptor. The changes that we see in the RMSD of the ethanol H-atoms from before the transition to after the transition reflect to a greater extent, a more heterogeneous environment of protein -solvent molecule interactions when compared with the interactions taking place before the transition..