Changes in protein conformation and surface hydrophobicity upon peroxidase-catalyzed cross-linking of apo-a-lactalbumin

In this study, we explore the effect of peroxidase-catalyzed cross-linking on the molecular conformation of apo-a-lactalbumin (apo-a-LA) and the resulting changes in protein surface hydrophobicity. In studying conformational changes, we distinguish between early stages of the reaction (“partial cross-linking”), in which only protein oligomers (106 Da > Mw = 104 Da) are formed, and a later stage (“full cross-linking”), in which larger protein particles (Mw = 106 Da) are formed. Partial cross-linking induces a moderate loss of a-helical content. Surprisingly, further cross-linking leads to a partial return of a-helices that are lost upon early cross-linking. At the same time, for partially and fully cross-linked apo-a-LA, almost all tertiary structure is lost. The protein surface hydrophobicity first increases for partial cross-linking, but then decreases again at full cross-linking. Our results highlight the subtle changes in protein conformation and surface hydrophobicity of apo-a-LA upon peroxidase-catalyzed cross-linking