# Activity of ice-binding proteins can be markedly enhanced by protein tags

## TL;DR

This study shows that protein tags can enhance the activity of ice-binding proteins, which help cold-adapted organisms survive.

## Contribution

The study reveals that specific protein tags can significantly boost the ice-recrystallization inhibition activity of ice-binding proteins.

## Key findings

- Most protein tags do not affect the ice-recrystallization inhibition (IRI) activity of ice-binding proteins (IBPs).
- N-terminal HaloTag attachment to QAE increases IRI activity by an order of magnitude.
- HaloTag itself has moderate IRI activity and induces hexagonal prism ice crystal morphology.

## Abstract

Ice-binding proteins (IBPs) are crucial for the survival of cold-adapted organisms, as they regulate ice crystal formation and growth. To understand their molecular mode of action, fluorescence microscopy of IBPs bound to ice-crystal surfaces has been shown to be very helpful, although it is unknown whether the (fluorescent) tags typically used in these studies affect the activities of the IBPs. Here, we evaluate the impact of mEos3.2, SNAP-tag, and HaloTag on the ice-recrystallization inhibition (IRI) activity of IBPs. We find that most tags, in most orientations, do not affect the IRI activity of IBPs. These tags are promising candidates for investigating the binding mechanisms of IBPs in their native form with fluorescence microscopy. A surprising exception is the N-terminal attachment of HaloTag to QAE, an isoform of AFP type III: for this case, we find an order of magnitude higher IRI activity. Additionally, we show that HaloTag also has moderate IRI activity by itself and induces the formation of ice crystals with hexagonal prism morphology, suggesting binding affinity for the primary prism plane of ice. Our findings indicate that moderately IRI-active proteins may synergistically enhance the IRI activity of IBPs, when attached in the proper orientation.

Ice-binding proteins (IBPs) regulate ice crystal growth, in particular through ice recrystallization inhibition (IRI). IRI activity can be synergistically enhanced when IBPs are fused to protein tags with moderate activity.