Compound Ice-Binding Site of an Antifreeze Protein Revealed by Mutagenesis and Fluorescent Tagging

Abstract

By binding to the surface of ice crystals, type III antifreeze protein (AFP) can depress the freezing point of fish blood to below that of freezing seawater. This 7-kDa globular protein is encoded by a multigene family that produces two major isoforms, SP and QAE, which are 55% identical. Disruptive mutations on the ice-binding site of type III AFP lower antifreeze activity but can also change ice crystal morphology. By attaching green fluorescent protein to different mutants and isoforms and by examining the binding of these fusion proteins to single-crystal ice hemispheres, we show that type III AFP has a compound ice-binding site. There are two adjacent, flat, ice-binding surfaces at 150° to each other. One binds the primary prism plane of ice; the other, a pyramidal plane. Steric mutations on the latter surface cause elongation of the ice crystal as primary prism plane binding becomes dominant. SP isoforms naturally have a greatly reduced ability to bind the prism planes of ice. Mutations that make the SP isoforms more QAE-like slow down the rate of ice growth. On the basis of these observations we postulate that other types of AFP also have compound ice-binding sites that enable them to bind to multiple planes of ice.

Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice

“…All proteins were labeled with TRITC except for nfeAFP8 (row viii), which was labeled with Pacific Blue. [12]. The difference in binding ability can be traced to amino acid changes on the portion of the IBS that binds to the primary prism plane of ice.…”

“…1). Fluorescence-based ice plane affinity (FIPA) analysis has demonstrated that nfeAFP6 adsorbs solely to a pyramidal plane of ice and is incapable of arresting ice growth at undercooling temperatures [12].…”

Interaction of ice binding proteins with ice, water and ions

“…Amino acid residues of the IBS have frequently been identified via mutagenesis studies. 50,66,67 Mutations of amino acids that are important for ice binding result in a large reduction of thermal hysteresis activity. For example, the Thr and Ala residues that are important for ice binding of wfAFP-I are all located on one relatively flat and hydrophobic side of the a-helix.…”

“…69 While for most IBPs the IBS is localized on one face of the protein that can bind to either one or several ice crystal plane(s), in some cases, a "compound" IBS is identified. 59,67 Type III AFP from notched fin eel pout and ocean pout have an IBS composed of two regions positioned at an angle of roughly 150 with respect to each other. One binds the primary prism plane of ice; the other a pyramidal plane [ Fig.…”

NMR structure note: a defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongates Kner

“…That is, the QAE-2 isoform nfeAFP11 can only shape ice crystals, but has no TH activity, while its triple mutant nfeAFP11-V9Q/V19L/G20V (denoted nfeAFP11-tri) perfectly exhibits both activities. These residues were thought to construct a "compound" ice-binding site (IBS) that consists of two adjacent flat surfaces inclined at an angle of approximately 150° to each other (Garnham et al 2010). To determine if the triple mutations in nfeAFP11 led to changes in the structure of the IBS that could affect its icebinding activity, the multidimensional NMR spectra of both nfeAFP11 and nfeAFP11-tri were examined.…”