NMR Studies of Type I Antifreeze Protein HPLC6 Function

I 型抗冻蛋白 HPLC6 功能的 NMR 研究

• 批准号: 7101953
• 负责人: YONG BA
• 金额: $ 14.72万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7101953
• 关键词: binding sites; chemical kinetics; cryoprotective agents; cryoscience; dipole moment; glycoproteins; intermolecular interaction; molecular dynamics; nuclear magnetic resonance spectroscopy; protein structure function; surface property

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.

抗冻蛋白（Antifreeze Proteins，AFP）具有抑制冰晶生长和重结晶的能力，对生物体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、组织和器官的低温保护，以及在冷冻手术中增强肿瘤细胞的破坏。然而，AFP的抗冻机制仍不清楚。一个问题出现了从分子水平的检测技术，可以直接检测AFP的相互作用和动态内，分别与冰水界面的absemce。在这项研究中，最先进的固态 NMR技术将用于研究HPLC 6 I型异构体的抗冻机理 AFP。NMR技术使用核自旋的局部偶极耦合。因此，密切洞察分子水平的识别，相互作用和动力学的AFP可以获得。本研究的长期目标是为寻找更有效的防冻材料，为生物医学研究和应用提供基础。除了在抗冻蛋白领域的意义外，这项研究还将对蛋白质-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，因为这些研究的NMR技术的发展和应用。 本论文从四个方面研究了HPLC 6肽的抗冻机理：（1）利用多量子过滤-自旋交换和蛋白质扩散NMR实验研究了HPLC 6肽在冰表面吸附的可逆性和动力学，（2）利用~ 1H和~（t3）C多量子NMR实验研究了HPLC 6肽与冰表面结合的协同性，（3）利用蛋白质扩散NMR实验研究了HPLC 6肽与冰表面结合的可逆性和动力学，（4）利用蛋白质扩散NMR实验研究了HPLC 6肽与冰表面结合的可逆性和动力学。（3）利用四极回波双共振NMR实验研究了AFP对冰重结晶过程中冰水分子动力学和扩散的影响;（4）用自旋回波双共振测定HPLC 6肽与冰表面的结合残基和表面，和旋转回声-绝热通道-双共振核磁共振实验。