Chen Baowei, Bigelow Diana J
Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
Biochemistry. 2002 Nov 26;41(47):13965-72. doi: 10.1021/bi0266030.
We have measured conformational changes of phospholamban (PLB) induced both by its interaction with the SR Ca-ATPase and by phosphorylation of Ser-16 by cAMP-dependent protein kinase (PKA) using an engineered PLB having a single cysteine (Cys-24) derivatized with the fluorophore 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (ANSmal). This modified mutant PLB is fully functional when co-reconstituted with the affinity-purified Ca-ATPase in liposomes. ANSmal emission properties and its solvent accessibility indicate that Cys-24 is in an aqueous environment outside the membrane. Fluorescence quenching and time-resolved anisotropy measurements of ANSmal-PLB demonstrate distinct structures for PLB in the free and Ca-ATPase-bound state. Both solvent exposure and probe motions of ANSmal are enhanced upon interaction of PLB with the Ca-ATPase. This conformational transition entails conversion of free PLB in a conformation which is insensitive to one which is sensitive to the phosphorylation state of PLB. Upon phosphorylation of Ca-ATPase-bound PLB, a decreased level of solvent exposure of ANSmal is observed, suggesting that the amino acid sequence of PLB near the lipid-water interface acts as a conformational switch in response to the phosphorylation of PLB. A longer correlation time, resolved by anisotropy measurements, corresponding to polypeptide chain fluctuations, is substantially restricted by interaction of PLB with the Ca-ATPase. This restriction is not reversed by phosphorylation of PLB, indicating that the region around Cys-24 near the lipid-water interface does not undergo dissociation from the Ca-ATPase. These results suggest that the phosphorylation by PKA induces a redistribution of PLB-Ca-ATPase protein contacts to relieve the inhibitory effect of PLB for the activation of calcium transport.
我们使用一种经过工程改造的、带有单个半胱氨酸（Cys-24）且用荧光团2-(4'-马来酰亚胺基苯胺基)萘-6-磺酸（ANSmal）衍生化的磷蛋白（PLB），测量了PLB与肌浆网Ca-ATP酶相互作用以及被环磷酸腺苷依赖性蛋白激酶（PKA）磷酸化丝氨酸-16所诱导的构象变化。当这种修饰的突变型PLB与亲和纯化的Ca-ATP酶共重组成脂质体时，其功能完全正常。ANSmal的发射特性及其溶剂可及性表明Cys-24处于膜外的水环境中。对ANSmal-PLB的荧光猝灭和时间分辨各向异性测量表明，PLB在游离态和与Ca-ATP酶结合态具有不同的结构。PLB与Ca-ATP酶相互作用时，ANSmal的溶剂暴露和探针运动均增强。这种构象转变导致游离PLB从对PLB磷酸化状态不敏感的构象转变为敏感的构象。当Ca-ATP酶结合的PLB被磷酸化时，观察到ANSmal的溶剂暴露水平降低，这表明脂质-水界面附近的PLB氨基酸序列作为响应PLB磷酸化的构象开关。通过各向异性测量解析出的对应于多肽链波动的较长相关时间，在PLB与Ca-ATP酶相互作用时受到显著限制。这种限制不会因PLB的磷酸化而逆转，表明脂质-水界面附近Cys-24周围区域不会与Ca-ATP酶解离。这些结果表明，PKA介导的磷酸化诱导PLB-Ca-ATP酶蛋白接触的重新分布，以减轻PLB对钙转运激活的抑制作用。
