Osborn Kenneth D, Zaidi Asma, Urbauer Ramona J Bieber, Michaelis Mary L, Johnson Carey K
Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045, USA.
Biochemistry. 2005 Aug 23;44(33):11074-81. doi: 10.1021/bi050488m.
We used single-molecule fluorescence spectroscopy to probe the conformation of calmodulin (CaM) bound to oxidatively modified plasma-membrane Ca(2+)-ATPase (PMCAox). We found that oxidative modification altered the coupling between the ATP binding domain and the autoinhibitory domain. Oxidative modification of PMCA is known to result in a loss of activity for the enzyme. Conformations of PMCAox-CaM complexes were probed by single-molecule polarization modulation spectroscopy, which measured the orientational mobility of fluorescently labeled CaM bound to PMCAox. We detected an enhanced population of PMCAox-CaM complexes with a low orientational mobility in the presence of ATP, whereas nonoxidized PMCA-CaM complexes existed almost exclusively in a high-mobility state in the presence of ATP. We have previously attributed such high-mobility states to PMCA-CaM complexes with a dissociated autoinhibitory/CaM binding domain, whereas the lower-mobility state was attributed to autoinhibited PMCA-CaM complexes with a nondissociated autoinhibitory domain [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. In the absence of ATP, the orientational mobility distributions are similar for CaM complexed with oxidized PMCA or nonoxidized PMCA. These results suggest that oxidative modification of PMCA reduced the propensity of the autoinhibitory domain to dissociate from binding sites near the catalytic core of the enzyme with bound nucleotide upon CaM stimulation in the presence of Ca(2+). This interpretation was further supported by chymotrypsin proteolysis, which probes the tightness of binding of the autoinhibitory domain to sites near the catalytic core of the enzyme. Enhanced proteolysis was observed for PMCA upon binding CaM or ATP. In contrast, proteolysis was partially blocked for oxidatively modified PMCA, even in the presence of ATP.
我们使用单分子荧光光谱法来探究与氧化修饰的质膜Ca(2+) -ATP酶（PMCAox）结合的钙调蛋白（CaM）的构象。我们发现氧化修饰改变了ATP结合结构域与自身抑制结构域之间的偶联。已知PMCA的氧化修饰会导致该酶活性丧失。通过单分子偏振调制光谱法探测PMCAox -CaM复合物的构象，该方法测量了与PMCAox结合的荧光标记CaM的取向迁移率。我们检测到在ATP存在下，具有低取向迁移率的PMCAox -CaM复合物群体增加，而未氧化的PMCA -CaM复合物在ATP存在下几乎完全处于高迁移率状态。我们之前将这种高迁移率状态归因于自身抑制/CaM结合结构域解离的PMCA -CaM复合物，而较低迁移率状态则归因于自身抑制结构域未解离的自身抑制性PMCA -CaM复合物[奥斯本，K.D.等人（2004年）《生物物理杂志》87卷，1892 - 1899页]。在没有ATP的情况下，与氧化型PMCA或未氧化型PMCA复合的CaM的取向迁移率分布相似。这些结果表明，在Ca(2+)存在下，PMCA的氧化修饰降低了自身抑制结构域在CaM刺激下与结合核苷酸的酶催化核心附近结合位点解离的倾向。胰凝乳蛋白酶蛋白水解实验进一步支持了这一解释，该实验探测了自身抑制结构域与酶催化核心附近位点结合的紧密程度。观察到PMCA在结合CaM或ATP后蛋白水解增强。相比之下，即使在ATP存在下，氧化修饰的PMCA的蛋白水解也部分受到抑制。
