Antifreeze Proteins from the Sea Ice Diatom Fragilariopsis cylindrus

Abstract

A molecular and crystallographic characterization of antifreeze proteins from the sea ice diatom Fragilariopsis cylindrus (fcAFPs) is presented here. The molecular study relies on sequence analyses, phylogenetic studies, gene and protein expression analyses. These AFPs constitute a multigene family of isoforms with a conserved antifreeze domain. The exposure of F. cylindrus to subzero conditions typical for sea ice brine caused differential regulation of the isoforms, as revealed by gene expression analysis. Some fcAFP genes were downregulated to nearly zero, whereas selected isoforms were strongly upregulated (up to 200-fold). Differential gene regulation was reflected in protein expression pattern. The widespread occurrence of fcAFPs and fcAFP-like proteins in polar or cold-tolerant organisms further underlines their importance as elements of adaptation to subzero conditions. Several events of horizontal gene transfer seem to have contributed to the distribution of the fcAFP genes. These results suggest a strong relevance of these proteins for F. cylindrus, probably contributing to its dominat position within sea ice assemblages. Studies with a recombinant isoform of fcAFP, chosen based on the molecular results, confirmed its antifreeze activity. The protein caused moderate freezing point depression and strong recrystallization inhibition. These effects were enhanced in concentrated saline solutions. In order to shed light on the physical antifreeze mechanism of this protein, its interaction with ice is presented in more detail here. Several crystal planes are suggested for preferential protein-ice interaction. Habit modification in the presence of fcAFPs, with inhibited ice growth on the lateral (prismatic) planes, indicates binding on the primary and secondary prismatic faces. Pitting observed on the basal plane of a pure crystal submerged in an fcAFP solution suggests further protein binding on non-lateral planes of the ice crystal. The most prominent feature shown here is the influence of fcAFPs on ice microstructure. The proteins clearly affected ice texture, causing a marked pattern of oriented striations, interpreted as ice fibers with inclusions and not observed without fcAFPs. Analysis of c-axis orientation revealed a strong influence on axis distribution within individual crystals and further indicates an inhibiting effect of fcAFPs on ice nucleation. Considered in the frame of the natural environment of F. cylindrus, results on antifreeze activity of fcAFPs and crystallographic analyses suggest that the effect on ice texture is the most relevant biological role of fcAFPs. Modifications of the microstructure of brine inclusions change physical properties of sea ice, which determine its habitability.