Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Abstract

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

An ice‐binding and tandem beta‐sandwich domain‐containing protein in Shewanella frigidimarina is a potential new type of ice adhesin

“…Similar shaping and burst patterns have been seen for other hyperactive IBPs able to bind to the basal plane of ice [40]. To confirm basal-plane binding of SfIBP_1 IBD, Fluorescence-based Ice Plane Affinity (FIPA) analysis was undertaken, incubating macro-scale hemispheres of single ice crystals with FITC-labelled IBP [21,23,41]. Even after scraping off the outer most surface of the ice to remove any nonspecific binding, the entire ice hemisphere fluoresced, indicating that the SfIBP_1 IBD binds to multiple planes of ice, including the basal plane ( Fig.…”

“…3A), which showed that TH activity increased steeply at low IBP concentrations and approached a plateau below 2°C at 80 lM. To confirm basal-plane binding of SfIBP_1 IBD, Fluorescence-based Ice Plane Affinity (FIPA) analysis was undertaken, incubating macro-scale hemispheres of single ice crystals with FITC-labelled IBP [21,23,41]. The level of activity displayed by SfIBP_1 IBD falls somewhere in between the easily identifiable moderate and hyperactive designations, as seen through comparison with the moderately active LeIBP, which demonstrates activity well below 0.5°C even at 100 lM, and the hyperactive FfIBP, which approaches a plateau in TH activity at 3°C, a full degree higher than that obtained with SfIBP_1 IBD, and at a much lower protein concentration (Fig.…”

“…Fluorescence-based Ice Plane Affinity analysis of the SfIBP_1 IBD was undertaken as previously described [41]. Briefly, macro-scale single ice crystals were grown using degassed water and mounted onto a copper cold finger (À5°C) with the c-axis aligned with the cold finger.…”

Intermediate activity of midge antifreeze protein is due to a tyrosine‐rich ice‐binding site and atypical ice plane affinity

“…Midge AFP was labelled with FITC as described previously for labelling with tetramethylrhodamine isothiocyanate (TRITC) . FIPA analysis was performed using 1.5 mg of midge AFP in 30 mL of working buffer as previously described .…”

Interaction of ice binding proteins with ice, water and ions

“…85,99 The regions where IBPs accumulate appear upon . 100 Prior to such a fluorescence based ice-plane affinity (FIPA) assay, IBPs are either coupled to a fluorescent dye or to a fluorescent protein in a fusion protein construct.…”