NMR Studies of Type I Antifreeze Protein HPLC6 Function

抗冻蛋白(AFP)具有抑制冰晶生长和重结晶的能力，对生物的冻害具有保护作用。在生物医学研究中，AFP被应用于哺乳动物细胞、组织和器官的防寒，以及在冷冻手术中加强肿瘤细胞的破坏。然而，AFP的抗冻机制尚不清楚。一个问题来自分子水平的检测技术，这些技术可以直接检测AFP分别与冰-水界面和冰水界面内的相互作用和动态。在这项拟议的研究中，最先进的固态 核磁共振技术将用于研究I型HPLC6异构体的抗冻机理 法新社。核磁共振技术使用核自旋的局部偶极耦合。因此，可以在分子水平上更深入地了解AFP的识别、相互作用和动力学。本研究的长期目标是在机理研究的基础上，为生物医学研究和应用寻找更有效的防冻材料提供基础。这项研究除了在抗冻蛋白领域具有重要意义外，还将对蛋白质-蛋白质、蛋白质-膜和蛋白质-药物相互作用的生物医学研究产生广泛的影响，因为核磁共振技术在这些研究中的发展和应用。 为了揭示防冻机理的不同方面，将开展四个方面的研究：(1)通过多量子过滤-自旋交换和蛋白质扩散核磁共振实验研究HPLC6多肽在冰面上的可逆性和吸附动力学；(2)通过1H和t3C多量子核磁共振实验研究HPLC6多肽与冰表面结合的协同性；(3)通过四极回波双共振实验研究AFP对冰重结晶过程中冰水分子动力学和扩散的影响；以及(4)通过自旋回波双共振实验和回声-绝热通道双共振核磁共振实验确定HPLC6多肽与冰表面的结合残基和表面。

Antifreeze proteins (AFPs) afford protection of freezing damage for organisms due to their ability to inhibit the growth and recrystallization of ice crystals. In biomedical research, AFPs found applications in cold protection of mammalian cells, tissues, and organs, and in enhancement of tumor cell destruction during cryosurgery. However, the antifreeze mechanism of AFPs remains unclear. One problem arose from the absemce of molecular-level detection techniques that can directly detect the interaction and dynamics of AFPs with and within, respectively, ice-water interfaces. In this proposed research, state of the art solid-state NMR techniques will be applied to study the antifreeze mechanism of the HPLC6 isoform of type I AFPs. The NMR techniques use local dipolar couplings of nuclear spins. Therefore, close insight into molecular-level recognition, interaction and dynamics of AFPs can be obtained. The long-term goal of this research is to provide a foundation, base on the mechanistic study, for finding more effective antifreeze materials for biomedical research and applications. Besides the significance in the field of antifreeze proteins, this research will also have broad impact on biomedical research of protein- protein, protein-membrane and protein-drug interactions in terms of development and application of NMR techniques for these studies. Four studies will be carded out to reveal the different aspects of antifreeze mechanism: (1) Reversibility and kinetics of ice-surface adsorption of HPLC6 peptides studied by Multiple Quantum Filtering-Spin Exchange and protein diffusion NMR experiments; (2) Cooperativity of HPLC6 peptides binding to ice surfaces studied by 1H and t3C Multiple Quantum NMR experiments; (3) Influence of AFPs on the kinetics and diffusion of ice-water molecules during the recrystallization process of ice studied by Quadrupolar Echo Double Resonance NMR experiment; and (4) Binding residues and surfaces ofHPLC6 peptides to ice surfaces determined by Spin Echo Double Resonance, and Rotational Echo-Adiabatic Passage-Double Resonance NMR experiments.