Molecular mechanisms of the anomalous thermal aggregation of green fluorescent protein

► Thermal denaturation behavior of GFP is remarkably different from that of BCA-II. ► The inside of the GFP β-can is involved in intensive interactions with water molecules. ► At high temperatures, the GFP structure does not unfold but breaks. ► Irreversible thermal denaturation GFP is accompanied by massive aggregation. ► No accumulation of soluble denatured protein is detected.