To examine the impact of PEG on the conformational dynamics and solvent organization of conjugated Gal3C, we compared unconjugated Gal3C with two Gal3C conjugates, each linked to poly (ethylene glycol) (PEG) of molecular weight of either 550 Da or 5 kDa, referred to here as Gal3C-PEG 550 Da or Gal3C-PEG 5 kDa, respectively. Gal3C PEGylation was performed using maleimide-functionalized PEG, which reacted specifically with a single extrinsic cysteine at position 243 in Gal3C[T243C]. We refer to this modified protein as Gal3C throughout the text. Gal3C-PEG 5 kDa was prepared according to previously reported protocols , while Gal3C-PEG 550 Da was prepared using a similar protocol, substituting maleimide-PEG 5K with a functionalized maleimide-PEG starting material of 550 Da (Section S1). values for lactose to Gal3C, Gal3C-PEG 550 Da and Gal3C-PEG 5 kDa were essentially identical, confirming that Gal3C-PEG 550 Da retained native activity (Figure ).