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

Summary

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

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.

Introduction

The production of antifreeze proteins (AFPs) is an important survival mechanism of some organisms that live in ice-laden environments. Until recently, it was thought that the sole function of AFPs was to prevent or slow the growth of internal ice crystals that would block circulation, cause tissue damage, and osmotic stress. Organisms that cannot tolerate any degree of freezing, such as fish, express AFPs to completely inhibit ice crystal growth¹. Others, such as grass, are freeze tolerant and express AFPs to inhibit ice recrystallization which reduces the formation of large ice crystals in their tissues². Stabilization of membranes in low temper....

Protocol

1. Growing Single Ice Crystals

1. Take a clean metal pan (15 cm diameter, 4.5 cm high) that fits into, and can float on, an ethylene glycol cooling bath.
2. Prepare polyvinyl chloride (PVC) cylindrical molds, (4.5 cm diameter, 3-4 cm high, 4 mm thick), by sawing sections from a pipe.
   1. Cut a notch, (1 mm wide, 2 mm high) on one side (Figure 1A).
   2. Prepare as many molds that can fit comfortably into the pan (Figure 1B).
      Note: A study showed that polyvinyl alcohol (PVA) can affect ice nucleation²². However, in our open mold PVC system we have not encountered problems of aty....

Results

Preparation and mounting of the single ice crystal are the two steps of the FIPA procedure where errors are most commonly made. Determining if the prepared ice crystal is single is done by examining it through crossed polaroids (Figure 1D), as outlined in step 2.1 of the protocol section. If a multicrystalline ice crystal is used for the FIPA analysis, the result will be discontinuous binding of the AFPs on the hemisphere without a coherent binding pattern (Figure 6B). If the ice crystal.......

Discussion

Development of the ice-etching method by Charles Knight for determination of AFP-bound ice planes greatly advanced studies on the mechanism of ice binding by AFPs. Whereas structures of AFPs could be solved by X-ray crystallography^26,27 there was no obvious method for deducing the complementary surface on ice to which the AFP bound. When type I AFP from winter flounder was initially characterized, it was hypothesized to bind to the primary prism planes of ice²⁸. However, Knight's ground bre.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NESLAB RTE Refrigerating Bath/Circulators	Thermo Scientific	RTE7
Ethylene glycol, Premixed Antifreeze/Coolant	Certified	29-3037-0	Common automotive antifreeze
Cold finger	not available	not available	Custom made with brass (9 cm long, 1.5 cm outer diameter)
Hemispherical cup	not available	not available	Custom made with resin (8 cm outer diameter, 6 cm inner diameter)
High Dual Output Lighting System	Lightools Research	LT-99D2, Illumatools DLS 120 volts AC, LT-9470FX, LT-9549FX	Additional and custom excitation filters can be purchased from Lightools Research
Camera	Canon	EOS 50D
Emission Filters	Lightools Research	LT-9EFPVG, LT-9GFPVG, LT-9RFPVG	Filter ring adapter may be required to fit filter onto camera lens