Conformational behavior of alpha-2-macroglobulin: Aggregation and inhibition induced by TFE

Abstract

Alpha-2-macroglobulin (α₂M), a pan-proteinase inhibitor, inhibits a variety of endogenous and exogenous proteinases and constitutes an important part of body's innate defense system. In the present study, we explored how trifluoroethanol (TFE) may modulate the structure, antiproteinase activity and aggregation of α₂M. TFE was sequentially added over a range of 0-20% (v/v) and the effects induced were studied by activity assay, intrinsic fluorescence, ANS fluorescence, circular dichroism, turbidity assay, Rayleigh scattering measurement and ThT fluorescence measurement. Decrease in activity and increase in fluorescence intensity of α₂M upon addition of TFE shows structural deviation from the native structure and suggests aggregation of protein upon solvent addition. Increase in turbidity and Rayleigh scattering of modified α₂M confirms the formation of aggregates. Insignificant ThT fluorescence intensity of TFE treated α₂M is indicative of amorphous or non-amyloid aggregation. Further, circular dichroism results indicate the changes in secondary structure of native α₂M as negative ellipticity decreased on addition of the polar solvent to the inhibitor. The turbidometric analysis, Rayleigh scattering, ThT fluorescence intensity of modified α₂M suggests that the protein might be driven towards non-amyloid or amorphous aggregation. Our studies provide important mechanistic insight how α₂M undergoes conformational and functional changes when exposed to TFE.

Keywords: Aggregation; Alpha-2-macroglobulin; Protein folding; Proteinase inhibitor; TFE.