钙调蛋白磷酸酶（Calcineurin，CN）是至今发现的Ser/Thr蛋白磷酸酶家族中唯一一种受钙离子和钙调素调节的蛋白磷酸酶，它由分子量为61kD的催化亚基A（CNA）和19kD 的调节亚基B（CNB）以等摩尔比（1:1）组成异二聚体。目前，已有众多关于CN结构和功能研究的文献报导，但还没有对于其折叠和去折叠机制的研究。本文的主旨是尽量从去折叠途径研究中获得更多关于CN的构象信息。论文第一部分是关于催化亚基CNA和催化核心CNa的去折叠动力学研究。观察二者在不同浓度尿素中的去折叠动力学过程。结果显示CNA在不同浓度的尿素中的构象有不同程度的松散，松散程度随尿素浓度增加而增大，说明CNA本身构象就很紧密。CNa在变性过程中构象先紧缩后松散，说明CNa的原始构象相当松散，尿素对CNa整体构象有影响，但不是单纯的解折叠。酶活力上，CNA受各个尿素浓度的激活，而CNa在任何尿素中都没有观察到激活，说明CNa的构象已经处于对pNPP的最适构象，而CNA的构象并不是催化pNPP的最适构象。论文的第二部分使用稳态变性的方法研究CaM、Ca2+和Mn2+在CNA稳态变性作用的影响。三种调节成分通过不同的组合方式分别加入CNA的稳态变性体系，结果发现三种成分在作用过程中会相互影响，Ca2+可以影响CaM对CNA的作用；CaM可以影响Mn2+对CNA的作用；当CaM与Ca2+结合后，CaM无法影响Mn2+对CNA的作用，但CaM仍然对CNA有作用。论文的第三部分基于第二部分的实验结果，研究Mn2+对CNa、CNA和BA稳态变性作用的影响。结果显示Mn2+在三种蛋白稳态变性作用中的影响都非常明显。Mn2+对三种蛋白均有激活作用，而且它的激活同尿素的激活作用可以叠加，说明Mn2+的激活途径与尿素不同， Mn2+很有可能是通过结合到活性中心来实现其激活作用。 论文的第四部分是关于CNa复性的一些摸索。结果显示在8M尿素变性初始时刻稀释复性可以恢复部分酶活力，如果变性时间过长，则无法通过稀释复性。Ca2+和Mn2+有助于复性。

Calcineurin (CN) is the only known phosphatase regulated by calmodulin (CaM) and Ca2+. It’s a heterodimer composed of catalytic subunit A (CNA, 61KD) and regulatory subunit B (CNB, 19KD). There are many works on structure and function of CN, but little about their folding mechanism. Our aim is to get conformational information as much as possible using unfolding methods.The first part is about the kinetic denaturation of CNA and CNa. Results show that CNA loses its structure gradually in urea. The conformation of CNa becomes tighter in 1M~5M urea and lose its most structure in 6M, 7M urea, which indicates that CNa might have a very loose conformation at the beginning and urea might change them first to a tigher direction. There are big differences in phosphatase activities between CNA and CNa. Urea could activate CNA at the beginning of denaturation but do nothing to CNa. This result shows that CNa has the most optimal conformation to catalyse pNPP and CNA needs urea to reach its optimal conformation.The scond part is about the effect of regulatory factors on CNA in its equilibrium denaturation process. Results show that CaM, Ca2+ and Mn2+ have different effects on CNA. Besides, they could influence each other. Ca2+ could affect CaM and CaM could affect Mn2+.The third part is comparison of how Mn2+ affects equilibrium denaturation processes of CNa, CNA and BA. Results show that Mn2+ could activate all their phosphatase activities and it could be added by that of urea. So Mn2+ must have a different way to affect enzyme activity from that of urea. Mn2+ might bind to catalytic core and form new conformation. The fourth part is about refolding condition of CNa. Results show that activity couldn’t be recovered if denatured in 8M urea for more than 10min. Ca2+ and Mn2+ are benefit to CNa’s refolding.