Analysis of structure-function correlation of oryzacystatin and development of new functional foods

稻谷抑素（Oryzacystatin， OC）是水稻（Oryza sativa）中含有的一种特异性抑制半胱氨酸蛋白酶（CP）的蛋白质。它具有抑制木瓜蛋白酶的化学计量学意义，并具有良好的热稳定性，即使在蒸煮大米的条件下其活性仍保持不变，并且在酸和碱中也很稳定。它通过控制CP的活性来调节种子的代谢，而CP与种子中蛋白质的降解有关。OC还将存在于外源或昆虫、细菌、病毒等感染源中的CP识别为外源靶酶，并抑制其活性，因此在生物防御中具有重要作用。本研究主要采用生物化学方法和三维结构分析对OC进行功能结构相关性分析。通过控制溶液的温度、pH、蛋白质的浓度或添加变性剂，实现了二聚体与OC单体的自由转化。我们发现二聚体的抑制活性低于单体。这一结果表明，在水稻发育阶段，OC作为一种调节剂，根据不同的环境变化，通过其构象的变化来调节蛋白酶的活性。溶液核磁共振结构显示，二聚化时，对抑制活性起重要作用的第一环和第二环区域发生构象变化。这种变化可能是抑制活性降低的原因。此外，我们成功地与OC的同型二聚体进行了结晶，并收集了2.9 A的衍射数据。OC适用于药品、医药和功能食品。本研究表明OC的活性可以通过改变溶液条件来调节，因此我们可以利用这一机制来构建受pH控制的纳米机器，并且具有耐酸和耐热性。

Oryzacystatin (OC) contained within rice (Oryza sativa) is the protein, which inhibits cysteine protease (CP) specifically. It inhibits papain stoichiometrically and has a great thermostability, its activity still remain even if under the conditions of cooking rice, and also is stable with acid and alkali. It regulates the metabolism of seed by controlling the activity of CP, which is concerned with protein degradation in seed. OC also recognizes the CP that exists in foreigners or sources of infection like insect, bacteria and virus as an extrinsic target enzyme and inhibits their activity, so it is said to play an important role in biological defense.This study is focused on function-structure correlation analysis of OC using biochemical method and three-dimensional structural analysis. We succeeded in conversion between dimer and monomer of OC freely by controlling the temperature and pH of solution or concentration of protein or by addition of denaturing agent. We revealed that dimer has less inhibitory activity than monomer. This result suggests that OC acts as a regulator, which regulates protease activity by its conformational change depending on various environmental changes in the stages of development of rice. Solution NMR structure of OC revealed the conformational change occurred with the regions of the first loop and the second loop, which are important for inhibition activity, when it is dimerized. This change would be the cause of the decrease of inhibition activity. In addition, we succeeded in crystallization with homodimer of OC and diffraction data was collected to 2.9 A.OC is applicable to drug medicine and functional food. It became clear that the activity of OC could be regulated by change the solution condition in this study, so we can use this mechanism for construction of nanomachine controlled by pH, and have resistance to acid and heat.