NMR Studies of Type I Antifreeze Protein HPLC6 Function

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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.