Abstract
Understanding protein folding in different environmental conditions is fundamentally important for predicting protein structures and developing innovative antibody formulations. While the thermodynamics and kinetics of folding and unfolding have been extensively studied by computational methods, experimental methods for determining antibody conformational transition pathways are lacking. Motivated to fill this gap, we prepared a series of unique formulations containing a high concentration of a chimeric immunoglobin G4 (IgG4) antibody with different excipients in the presence and absence of the ionic liquid (IL) choline dihydrogen phosphate. We determined the effects of different excipients and IL on protein thermal and structural stability by performing variable temperature circular dichroism and bio-layer interferometry analyses. To further rationalise the observations of conformational changes with temperature, we carried out molecular dynamics simulations on a single antibody binding fragment from IgG4 in the different formulations, at low and high temperatures. We developed a methodology to study the conformational transitions and associated thermodynamics of biomolecules, and we showed IL-induced conformational transitions. We showed that the increased propensity for conformational change was driven by preferential binding of the dihydrogen phosphate anion to the antibody fragment. Finally, we found that a formulation containing IL with sugar, amino acids and surfactant is a promising candidate for stabilising proteins against conformational destabilisation and aggregation. We hope that ultimately, we can help in the quest to understand the molecular basis of the stability of antibodies and protein misfolding phenomena and offer new candidate formulations with the potential to revive lost therapeutic candidates.
Highlights
Accumulation of misfolded proteins can cause diseases, including the highly prevalent degenerative diseases Alzheimer's and type II diabetes mellitus.[1]
We developed a series of unique ionic liquid (IL) containing formulations and introduced a comprehensive methodology to examine the structural and thermal stability of antibodies in solution, as well as the conformational transitions, energetic properties, and thermodynamic behaviours of complex systems
Variable temperature circular dichroism (CD) spectroscopy experiments showed secondary structural changes of immunoglobin G4 (IgG4) in relation to thermal denaturation, and enabled an experimental conformational search on antibodies, which can be applied to other proteins and biomolecules
Summary
Accumulation of misfolded proteins can cause diseases, including the highly prevalent degenerative diseases Alzheimer's and type II diabetes mellitus.[1]. Edge Article appropriate experimental data, the current view of energy landscapes for protein misfolding and aggregation is generally qualitative rather than quantitative.[5,6] Primarily, computational simulations are used to sample the conformational landscape of proteins.[7,8] Yet, to date, we are limited by the experimental techniques that can be used for determining the conformational diversity of proteins, since structural characterisation of conformers, other than the most prevalent one, in solution is intensely challenging.[9] there is a growing need to develop novel methods to study protein folding, conformational transitions and sample the potential energy landscape of complex systems
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