Dataset Information

Microfluidic Cold-Finger Device for the Investigation of Ice-Binding Proteins.

ABSTRACT: Ice-binding proteins (IBPs) bind to ice crystals and control their structure, enlargement, and melting, thereby helping their host organisms to avoid injuries associated with ice growth. IBPs are useful in applications where ice growth control is necessary, such as cryopreservation, food storage, and anti-icing. The study of an IBP's mechanism of action is limited by the technological difficulties of in situ observations of molecules at the dynamic interface between ice and water. We describe herein a new, to our knowledge, apparatus designed to generate a controlled temperature gradient in a microfluidic chip, called a microfluidic cold finger (MCF). This device allows growth of a stable ice crystal that can be easily manipulated with or without IBPs in solution. Using the MCF, we show that the fluorescence signal of IBPs conjugated to green fluorescent protein is reduced upon freezing and recovers at melting. This finding strengthens the evidence for irreversible binding of IBPs to their ligand, ice. We also used the MCF to demonstrate the basal-plane affinity of several IBPs, including a recently described IBP from Rhagium inquisitor. Use of the MCF device, along with a temperature-controlled setup, provides a relatively simple and robust technique that can be widely used for further analysis of materials at the ice/water interface.

SUBMITTER: Haleva L

PROVIDER: S-EPMC5034346 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Microfluidic Cold-Finger Device for the Investigation of Ice-Binding Proteins.

Haleva Lotem L Celik Yeliz Y Bar-Dolev Maya M Pertaya-Braun Natalya N Kaner Avigail A Davies Peter L PL Braslavsky Ido I

Biophysical journal 20160901 6

Ice-binding proteins (IBPs) bind to ice crystals and control their structure, enlargement, and melting, thereby helping their host organisms to avoid injuries associated with ice growth. IBPs are useful in applications where ice growth control is necessary, such as cryopreservation, food storage, and anti-icing. The study of an IBP's mechanism of action is limited by the technological difficulties of in situ observations of molecules at the dynamic interface between ice and water. We describe he ...[more]

PMID: 27653473

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Project description:Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above -30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins.

Dataset Information

Microfluidic Cold-Finger Device for the Investigation of Ice-Binding Proteins.

Publications

Microfluidic Cold-Finger Device for the Investigation of Ice-Binding Proteins.

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