The structure and function of the Antarctic marine diatom Chaetoceros neogracile antifreeze protein (Cn-AFP), as well as its expression levels and characteristics of the ice-binding site, were analyzed in the present study. In silico analysis revealed that the Cn-AFP promoter contains both light- and temperature-responsive elements. Northern and Western blot analyses demonstrated that both Cn-AFP transcript and protein expression were strongly and rapidly stimulated by freezing, as well as temperature and high light stress. Immunogold labeling revealed that Cn-AFP is preferentially localized to the intracellular space near the chloroplast membrane. Recombinant Cn-AFP had clear antifreeze activity. Protein-folding simulation was used to predict the putative ice-binding sites in Cn-AFP, and site-directed mutagenesis of the Cn-AFP b-face confirmed their identification.
Keywords: Chaetoceros neogracile; ice-binding site; immunogold labeling.
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