Functional Analysis of a Bacterial Antifreeze Protein Indicates a Cooperative Effect between Its Two Ice-Binding Domains

Abstract

Antifreeze proteins make up a class of ice-binding proteins (IBPs) that are possessed and expressed by certain cold-adapted organisms to enhance their freezing tolerance. Here we report the biophysical and functional characterization of an IBP discovered in a bacterium recovered from a deep glacial ice core drilled at Vostok Station, Antarctica (IBPv). Our study showed that the recombinant protein rIBPv exhibited a thermal hysteresis of 2 °C at concentrations of >50 μM, effectively inhibited ice recrystallization, and enhanced bacterial viability during freeze-thaw cycling. Circular dichroism scans indicated that rIBPv mainly consists of β strands, and its denaturing temperature was 53.5 °C. Multiple-sequence alignment of homologous IBPs predicted that IBPv contains two ice-binding domains, a feature unique among known IBPs. To examine functional differences between the IBPv domains, each domain was cloned, expressed, and purified. The second domain (domain B) expressed greater ice binding activity. Data from thermal hysteresis and gel filtration assays supported the idea that the two domains cooperate to achieve a higher ice binding effect by forming heterodimers. However, physical linkage of the domains was not required for this effect.

Structure of a bacterial ice binding protein with two faces of interaction with ice

“…role of the cap subdomain in the overall stability of IBP [24] seem to be scaled down. This conclusion is further supported by the structure of the recently published two-domain protein IBPv [28], where each b-helix domain misses the cap subdomain but the protein has a Tm of 53.5°C [34], similar to that of capped IBP-1 fold proteins [23][24][25][26][27]. Overall, the low hydrophobicity of B and C faces might explain the only moderate TH activity of the protein.…”

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

“…Other DUF3494‐containing proteins were also found in yeasts, algae, fungi and even archaea from regions that experience cold temperatures. In the last decade, increasing numbers of proteins containing the DUF3494 were demonstrated to bind ice crystals and, hence, were classified as IBPs . The current number of DUF3494 proteins confirmed as IBPs is so large that it raises the question of whether or not there are DUF3494s that do not bind to ice.…”

“…Currently, only two IBPs belonging to different DUF3494 architectures have been biochemically characterized. The first is IBPv, from the Flavobacteriaceae strain 3519–10 isolated from the Vostok lake . This protein contains two consecutive DUF3494 domains, connected by a 17‐residue linker and ending with a short C‐terminal domain (Fig.…”

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

“…Third, functionalized AFPs should be engineered and developed to overcome the limitations of natural counterparts. Mother nature has suggested the use of RD3 and an IBP from Vostok glacial bacterium [137,209]. In both cases, connecting two almost homologous domains increases the TH value cooperatively compared with their monomeric AFPs [139,209].…”

“…Mother nature has suggested the use of RD3 and an IBP from Vostok glacial bacterium [137,209]. In both cases, connecting two almost homologous domains increases the TH value cooperatively compared with their monomeric AFPs [139,209]. Studies from the laboratories of Tsuda, Davies, and Holland have demonstrated that the multimerization of native type III AFP can increase TH activity [210,211,212].…”