Mechanism of Antifreeze Proteins for Ice Growth Inhibition

描述(由申请人提供)：抗冻蛋白(AFP)因其抑制种子冰晶生长的功能而保护生物免受冰冻损害。在生物医学研究中，AFP在哺乳动物细胞、组织和器官的防寒保护以及在冷冻外科中加强肿瘤细胞的破坏方面得到了应用。例如，实验证明，AFP可以帮助保护低温保存后的整个大鼠肝脏的生物功能，这对提高人体器官移植的质量和保质期具有重大影响。虽然AFP的结构和功能已经得到了广泛的研究，但其确切的抗冻作用机制仍不完全清楚。本研究的长期目标是发现防冻机理，发展防冻理论，为后续的生物医学研究和应用寻找特定的防冻材料和设计复杂的防冻方法。 为了理解AFP的防冻机理，本研究将检验以下假设：(1)由于Gibbs能量的降低，AFP倾向于扩散到水-冰界面形成水-AFP-冰(WAI)界面区，抑制冰的生长主要是由于界面AFP浓度增加的综合作用；(2)AFP与冰面的结构匹配以及AFP疏水侧链与冰面的van der Waals相互作用是AFP倾向于停留在WAI界面区的最重要驱动力，AFP亲水侧链与液态水的相互作用提高了AFP的溶解度，也平衡了AFP与冰面的相互作用。本研究将采取以下补充方法：(1)我们将继续发展热力学理论模型和实验方法，包括对吉布斯能量、热焓和熵变化的体积和热分析，以了解AFP的防冻作用。(2)我们将继续通过使用特定的侧链核磁共振活性同位素标记的AFP，以及开发和应用包括双共振和自旋晶格驰豫在内的前沿核磁共振技术，继续探索WAI界面区I型AFP的结构相互作用和动力学的方向。(3)将进行分子模拟，以了解I型AFP的特定残基的功能作用，并输入实验确定的结构和动态数据。

DESCRIPTION (provided by applicant): Antifreeze proteins (AFP) afford protection for organisms from freezing damage due to their function to inhibit the growth of seed-ice crystals. In biomedical research, AFPs find applications in cold protection of mammalian cells, tissues and organs, and in enhancement of tumor cell destruction for cryosurgery. For example, experiments demonstrated that AFPs could help protect biological functions of whole rat livers following hypothermic cryogenic storage, which has significant impact on improving the quality and shelf time for human organ transplantations. Although the structures and function of AFPs have been extensively studied, the precise mechanism of antifreeze action is still not fully understood. The long-term goal of this research is to find the antifreeze mechanism and to develop an antifreeze theory for the purpose of finding particular antifreeze materials and designing sophisticated antifreeze methods for the subsequent biomedical research and applications. To understand the antifreeze mechanism, this research will test the following hypotheses: (1) AFPs tend to diffuse to the water-ice interface to form a water-AFP-ice (WAI) interfacial region due to the decrease in Gibbs energy, and the ice growth inhibition arises primarily from the colligative effect of the enhanced interfacial AFP concentration; (2) the structural match of AFPs with ice surfaces and the van der Waals interactions of AFPs' hydrophobic side chains with ice surfaces are the most important driving forces for AFPs to tend to stay in the WAI interfacial region, and the interactions of AFPs' hydrophilic side chains with liquid water enhance the solubility and also balance the their interacting sides with ice surfaces. The following complementary approaches will be carried out for this study: (1) We will continue to develop thermodynamic theoretical model and perform experimental approaches, including volumetric and thermal analyses of Gibbs energy, enthalpy and entropy changes, to understand AFPs' antifreeze action. (2) We will continue the study in the direction of probing structural interactions and dynamics of type I AFPs in the WAI interfacial region via using specific side-chain NMR-active isotope-labeled AFPs, and developing and applying cutting-edge NMR techniques including double resonance NMR and spin lattice relaxation NMR. (3) Molecular modeling will be carried out to understand the functional roles of specific residues of type I AFPs with the input of the experimentally determined structural and dynamic data.