Saturn-Shaped Ice Burst Pattern and Fast Basal Binding of an Ice-Binding Protein from an Antarctic Bacterial Consortium

Abstract

Ice-binding proteins (IBPs) bind to ice crystals and control their growth, enabling host organisms to adapt to subzero temperatures. By binding to ice, IBPs can affect the shape and recrystallization of ice crystals. The shapes of ice crystals produced by IBPs vary and are partially due to which ice planes the IBPs are bound to. Previously, we have described a bacterial IBP found in the metagenome of the symbionts of Euplotes focardii ( EfcIBP). EfcIBP shows remarkable ice recrystallization inhibition activity. As recrystallization inhibition of IBPs and other materials are important to the cryopreservation of cells and tissues, we speculate that the EfcIBP can play a future role as an ice recrystallization inhibitor in cryopreservation applications. Here we show that EfcIBP results in a Saturn-shaped ice burst pattern, which may be due to the unique ice-plane affinity of the protein that we elucidated using the fluorescent-based ice-plane affinity analysis. EfcIBP binds to ice at a speed similar to that of other moderate IBPs (5 ± 2 mM s); however, it is unique in that it binds to the basal and previously unobserved pyramidal near-basal planes, while other moderate IBPs typically bind to the prism and pyramidal planes and not basal or near-basal planes. These insights into EfcIBP allow a better understanding of the recrystallization inhibition for this unique protein.

Ice‐binding proteins and the ‘domain of unknown function’ 3494 family

“…Basal plane binding is a requirement for hyperactivity, but some IBPs with weak TH activity, like the Lp IBP from grass, also bind the basal plane . Indeed, FIPA analysis of Tis IBP6 and Euplotes focardii bacterial consortium IBP ( Efc IBP) showed that these moderate IBPs are able to bind basal and other planes . This binding pattern to ice crystal planes was confirmed also for the moderate IBP isoform 11 from the sea‐ice diatom Fragilariopsis cylindrus ( fc IBP11) by laser confocal fluorescence microscopy of a single ice crystal in a solution of the fluorescently labelled protein .…”

“…Experiments on ice‐binding kinetics might help to understand the differences between hyperactive and moderate basal‐binder IBPs. Kinetics experiments carried out on the moderately active Efc IBP indicate it binds ice crystals very fast when compared to the hyperactive sbwAFP from spruce budworm .…”

“…On the other hand, in the presence of the moderate Efc IBP, ice crystals assume a hexagonal truncated trapezohedron shape and they burst perpendicular to the c ‐axis (Fig. A,C) . Moreover, detailed studies on single crystals in the presence of the moderate fc IBP11 (Fig.…”

“…Studies on single crystals of 10–50 μm in diameter show that DUF3494 IBPs also produce different patterns of ice growth and burst (i.e. the rapid growth of ice crystals at temperature below the non‐equilibrium freezing point) . For instance, in the presence of the hyperactive Sf IBP_1 within the TH gap, ice crystals present hexagonal shape, (Fig.…”

Peptidic Antifreeze Materials: Prospects and Challenges

“…Yet, the physico-chemical principles of the interaction between AF(G)Ps and ice remain elusive, as both experimental and theoretical studies of protein binding to ice are challenging. In recent years, computational studies [29,33,34,35,36,37] and microscopy experiments [38,39,40] have been particularly illuminating in this area. The simulations of Midya, and Bandyopadhyay [41] beautifully illustrate how AF(G)Ps can arrest growth via the Kelvin effect as originally proposed by Knight and DeVries [42,43].…”