Haleva Lotem, Celik Yeliz, Bar-Dolev Maya, Pertaya-Braun Natalya, Kaner Avigail, Davies Peter L, Braslavsky Ido
Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.
Department of Physics and Astronomy, Ohio University, Athens, Ohio; Department of Physics and Physical Sciences, Marshall University, Huntington, West Virginia.
Biophys J. 2016 Sep 20;111(6):1143-1150. doi: 10.1016/j.bpj.2016.08.003.
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.
冰结合蛋白（IBPs）与冰晶结合并控制其结构、生长和融化，从而帮助其宿主生物体避免与冰生长相关的损伤。IBPs在需要控制冰生长的应用中很有用，例如冷冻保存、食品储存和防冰。对IBP作用机制的研究受到在冰与水的动态界面原位观察分子的技术困难的限制。在此，我们描述了一种据我们所知的新型装置，该装置设计用于在微流控芯片中产生可控的温度梯度，称为微流控冷指（MCF）。该装置允许生长稳定的冰晶，无论溶液中有无IBPs，都可以轻松对其进行操作。使用MCF，我们发现与绿色荧光蛋白偶联的IBPs的荧光信号在冷冻时降低，并在融化时恢复。这一发现加强了IBPs与其配体冰不可逆结合的证据。我们还使用MCF证明了几种IBPs的基面亲和力，包括最近描述的来自长角窃蠹的一种IBP。使用MCF装置以及温度控制装置，提供了一种相对简单且强大的技术，可广泛用于进一步分析冰/水界面的材料。
