To assess the impact of PEG 550 Da conjugation on the thermal stability of Gal3C, we recorded circular dichroism (CD) spectra from 200 to 280 nm (Figure ). CD spectra of Gal3C-PEG 550 Da at 30 ºC which were consistent with a well-folded protein containing mostly -sheet secondary structure and are at most only marginally different from CD spectra of Gal3C and Gal3C-PEG 5 kDa recorded at the same temperature (Figure ). Temperature-dependent CD spectra recorded with Gal3C-PEG 550 Da showed a single cooperative unfolding transition with an apparent Tm of ~58 ºC with a min of 225 nm. The thermal unfolding behavior of Gal3C-PEG 550 Da was highly similar to that of Gal3C but contrasted starkly to the thermal unfolding behavior of Gal3C-PEG 5 kDa. Consistent with earlier findings, thermal unfolding of Gal3C-PEG 5 kDa showed two separate unfolding transitions and the formation of an intermediate conformation, as evidenced by a shift in min from 225 to 215 nm (Figure ). This stark contrast in the thermal unfolding behavior between Gal3C-PEG 550 Da and Gal3C-PEG 5 kDa motivated a deeper investigation into the molecular mechanisms underlying these distinct unfolding pathways.