| We have used hydrogen-deuterium exchange (HX) and electrospray ionization mass spectrometry to examine structural, kinetic, and thermodynamic features of protein aggregation in four contexts. Unfolding during salt-induced precipitation of hen egg white lysozyme was investigated first. Precipitation with (NH4)SO4 and NaCl did not unfold lysozyme. KSCN was an effective precipitant, but also disrupted lysozyme structure and caused the formation of insoluble precipitate fractions. Biphasic unfolding kinetics (from a native state to a molten globule-like state) were thought to reflect a smaller propensity for lysozyme to unfold in the precipitated state. The stability and structural changes of recombinant human interferon-gamma (IFN-γ) during aggregation induced by guanidine hydrochloride (GdnHCl) and KSCN was also investigated. Insoluble IFN-γ aggregates formed in either 1.0 M GdnHCl or 0.3 M KSCN showed a HX pattern consistent with a partially unfolded state with an intact Helix C. 1.0 M GdnHCl destabilized the protein in solution, whereas KSCN first precipitated native IFN-γ, and then facilitated partial unfolding of the precipitated protein in the solid phase. These results show that Helix C, which forms the hydrophobic core of the IFN-γ dimer, is highly protected from HX under native conditions, unfolds more slowly in GdnHCl than the rest of the protein, and remains intact in both GdnHCl- and KSCN-induced aggregates. In addition, HX-MS and dynamic light scattering experiments were used to investigate the effect of benzyl alcohol, a pharmaceutical formulation preservative, on IFN-γ. Only ∼1% of the protein was aggregated after a few days. Longer incubation times (up to eight days) led to larger fractions of unfolded, irreversibly aggregated protein. A fourth project was initiated to determine the structural aspects of fibril formation for β-amyloid, the peptide found in fibril deposits in the brains of Alzheimer's disease patients. The studies suggest that native state HX patterns may presage regions of the protein susceptible to unfolding during aggregation. The HX-MS methods developed and employed here could aid in the rational control of pharmaceutical protein aggregation during purification and delivery as well as other applications where aggregation plays a crucial role. |
