Flores Antonia, Quon Justin C, Perez Adiel F, Ba Yong
Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA, 90032, USA.
Eur Biophys J. 2018 Sep;47(6):611-630. doi: 10.1007/s00249-018-1285-3. Epub 2018 Feb 27.
The site-directed spin labeling (SDSL) technique was used to examine the antifreeze mechanisms of type-I antifreeze proteins (AFPs). The effects on the growth of seed ice crystals by the spin-label groups attached to different side chains of the AFPs were observed, and the states of water molecules surrounding the spin-label groups were probed via analyses of variable-temperature (VT) dependent electron paramagnetic resonance (EPR) spectra. The first set of experiments revealed the antifreeze activities of the spin-labeled AFPs at the microscopic level, while the second set of experiments displayed those at the molecular level. The experimental results confirmed the putative ice-binding surface (IBS) of type-I AFPs. The VT EPR spectra indicate that type-I AFPs can inhibit the nucleation of seed ice crystals down to ~ - 20 °C in their aqueous solutions. Thus, the present authors believe that AFPs protect organisms from freezing damage in two ways: (1) inhibiting the nucleation of seed ice crystals, and (2) hindering the growth of seed ice crystals once they have formed. The first mechanism should play a more significant role in protecting against freezing damage among organisms living in cold environments. The VT EPR spectra also revealed that liquid-like water molecules existed around the spin-labeled non-ice-binding side chains of the AFPs frozen within the ice matrices, and ice surrounding the spin-label groups melted at subzero temperatures during the heating process. This manuscript concludes with the proposed model of antifreeze mechanisms of AFPs based on the experimental results.
定点自旋标记（SDSL）技术被用于研究I型抗冻蛋白（AFP）的抗冻机制。观察了连接到AFP不同侧链上的自旋标记基团对籽晶冰晶体生长的影响，并通过分析变温（VT）依赖的电子顺磁共振（EPR）光谱来探测自旋标记基团周围水分子的状态。第一组实验揭示了自旋标记AFP在微观层面的抗冻活性，而第二组实验展示了其在分子层面的抗冻活性。实验结果证实了I型AFP假定的冰结合表面（IBS）。VT EPR光谱表明，I型AFP在其水溶液中能够将籽晶冰晶体的成核抑制至约-20°C。因此，本文作者认为AFP通过两种方式保护生物体免受冻害：（1）抑制籽晶冰晶体的成核，以及（2）在籽晶冰晶体形成后阻碍其生长。第一种机制在保护生活在寒冷环境中的生物体免受冻害方面应发挥更重要的作用。VT EPR光谱还显示，在冰基质中冻结的AFP的自旋标记非冰结合侧链周围存在类似液态的水分子，并且在加热过程中，自旋标记基团周围的冰在零下温度下融化。本文基于实验结果提出了AFP抗冻机制的模型作为结论。
