Structure-Guided Discovery of a Cold-Responsive Antifreeze-like Protein from Antarctic Flavobacterium sp. PL002

Antarctic microorganisms experience persistent subzero temperatures and repeated freeze-thaw cycles that require specialized mechanisms for survival. Although transcriptomic studies have identified numerous cold-responsive genes, many remain annotated as hypothetical proteins with unknown functions. In this study, we investigated PL002-1792, a strongly upregulated hypothetical protein from Antarctic Flavobacterium sp. PL002 identified under severe cold stress (-20 °C versus -6 °C; log₂ fold change = 5.61, adjusted p = 1.89 × 10^-138). Sequence analysis revealed a 402-amino-acid protein containing a predicted Sec/SPII lipoprotein signal peptide. AlphaFold3 prediction generated a high-confidence structural model (pTM = 0.96) with an elongated β-sheet-rich architecture resembling bacterial ice-binding proteins. Comparative analysis with the ice-binding protein from Flavobacterium frigoris (FfIBP) identified conserved glycine-rich and TXT-like motifs associated with putative ice-binding surfaces. Recombinant PL002-1792 was expressed in Escherichia coli, recovered from inclusion bodies, and successfully refolded into a predominantly β-sheet-rich conformation as confirmed by circular dichroism spectroscopy. Functional assays demonstrated moderate ice recrystallisation inhibition activity, reducing relative ice crystal mean grain size to approximately 90% of the control, and significantly enhanced freeze-thaw survival, with recombinant cells retaining 67% viability after three freeze-thaw cycles compared with 27% for the empty-vector control. These findings identify PL002-1792 as a novel antifreeze-like protein and highlight the utility of structure-guided approaches for uncovering previously uncharacterized cold-adaptation mechanisms in polar microorganisms.