The release of host-derived antibodies bound to the variant surface glycoprotein (VSG) of

Background: Methods: We used size exclusion chromatography, where molecules are separated according to their hydrodynamic radius to see if the VSG is present as a homodimer at both pH values. To gain information about the structural integrity of the protein we used circular dichroism spectroscopy by exposing the VSG in solution to a mixture of right- and left-circularly polarized light and analysing the absorbed UV spectra. Evaluation of protein stability and molecular dynamics simulations at different pH values was performed using different computational methods. Results: We show, for an A2-type VSG, that the dimer size is only slightly larger at pH 5.2 than at pH 7.4. Moreover, the dimer was marginally more stable at lower pH due to the higher affinity (ΔG = 353.37 kcal/mol) between the monomers. Due to the larger size, the predicted epitopes were more exposed to the solvent at low pH. Moderate conformational changes (ΔRMSD = 0.35 nm) in VSG were detected between the dimers at pH 5.2 and pH 7.4 in molecular dynamics simulations, and no significant differences in the protein secondary structure were observed by circular dichroism spectroscopy. Conclusions: Thus, the dissociation of anti-VSG-antibodies in endosomes cannot be explained by changes in pH.