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

Abstract

An antifreeze protein (AFP) from a midge (Chironomidae) was recently discovered and modelled as a tightly wound disulfide-braced solenoid with a surface-exposed rank of stacked tyrosines. New isoforms of the midge AFP have been identified from RT-PCR and are fully consistent with the model. Although they differ in the number of 10-residue coils, the row of tyrosines that form the putative ice-binding site is conserved. Recombinant midge AFP has been produced, and the properly folded form purified by ice affinity. This monomeric AFP has a distinct circular dichroism spectrum, a melting temperature between 35 and 50°C and is fully renaturable on cooling. Mutagenesis of the middle tyrosine in the rank of seven eliminates antifreeze activity, whereas mutation of a tyrosine off this predicted icebinding face had no such effect. This AFP has unusual properties compared to other known AFPs. First, its freezing-point depression activity is intermediate between that of the hyperactive and moderately active AFPs. As with hyperactive AFPs, when midge AFP-bound ice crystals exceed their freezing-point depression, ice grows explosively perpendicular to the c-axis. However, midge AFP does not bind to the basal plane of ice as do hyperactive AFPs, but rather to a pyramidal plane that is at a shallower angle relative to the basal plane than binding planes of moderate AFPs. These properties distinguish midge AFP from all other ice-binding proteins and the intermediate activity level fits well to the modest challenge of protecting newly emerged adult insects from late spring frosts. DatabaseNucleotide sequences of new midge AFP isoforms are available in the GenBank database under accession numbers KU094814-8. Sequences will be released after publication.

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

“…Specifically, moderately active IBPs bind to prism and/or pyramidal planes, while hyperactive IBPs are also able to bind to the basal plane of ice. Similar rows of Thr, Asn, Ala and Tyr residues have been seen in several other b-solenoid IBPs [27][28][29][30]. For example, Leucosporidium (Le)IBP, with 0.35°C at 370 lM is moderately active, but Flavobacterium frigoris (Ff)IBP and Colwellia sp.…”

“…For example, the hyperactive ice-binding protein from the Antarctic bacterium Marinomonas primoryensis is able to order water to match several planes of ice (including the basal plane and primary prims plane), using a GTGND repeat to align two rows of ice-binding Thr and Asn along one face of its b-solenoid structure [26]. Similar rows of Thr, Asn, Ala and Tyr residues have been seen in several other b-solenoid IBPs [27][28][29][30]. However, most DUF3494 IBPs typically lack a repetitive motif of any kind, with their ice-binding faces populated by residues that vary from coil to coil of the b-solenoid, and even more so between different IBPs.…”

Origin of an antifreeze protein gene in response to Cenozoic climate change

“…Similar convergence, albeit with a different structural framework, was seen with arthropod AFPs that adopt a β-helical conformation. A beetle (yellow mealworm) and a fly (midge) produce tight, disulfide-stabilized solenoids, with an ice-binding surface composed of a double row of Thr residues or a single row of Tyr residues, respectively 58 , 59 . The looser solenoid of the moth (spruce budworm) is more triangular and lacks bisecting disulfide bonds, but like the beetle AFP, its ice-binding surface consists of a double row of Thr residues 60 .…”

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

“…Several lines of evidence support the idea that the extent of TH activity may depend on the specific plane of ice crystals to which the IBPs adsorb. Most hyperactive IBPs bind to the basal plane of ice, in addition to the prismatic and pyramidal crystal planes to which moderate IBPs associate . Data of IRI activity have been reported only for a subset of IBPs.…”