The inhibitory action of phospholamban involves stabilization of alpha-helices within the Ca-ATPase.
The inhibitory action of phospholamban involves stabilization of alpha-helices within the Ca-ATPase.
复制标题
受磷蛋白的抑制作用涉及稳定 Ca-ATP 酶内的 α 螺旋。
DOI：
10.1021/bi011148d
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Squier,ThomasC
中科院分区：
文献类型：
--
作者：
Tatulian,SurenA;Chen,Baowei;Li,Jinhui;Negash,Sewite;Middaugh,CRussell;Bigelow,DianaJ;Squier,ThomasC
我们使用衰减全反射傅立叶变换红外（ATR-FTIR）和圆二色性（CD）光谱来识别由于磷蛋白（PLB）对运输活性的功能性抑制而导致的ca - atp酶内的二级和动态结构变化。从大肠杆菌中表达和纯化了同位素标记的[13C]PLB，并用未标记的Ca-ATPase进行了功能重构，使Ca-ATPase的酰胺I和II吸收带与[13C]PLB的吸收带分离开来。在ca - atp酶与PLB共重构后，在CD光谱和与ca - atp酶相关的酰胺I和酰胺II波段都观察到光谱位移，这表明α-螺旋稳定性增加。与PLB相关的H/D交换动力学发生相应的变化，表明ca - atp酶中通常进行酰胺H/D快速交换的100±20个残基发生交换抗性。PLB的二级结构没有相应的大变化。PLB与Ca-ATPase之间结构相互作用的亲和性与功能抑制的亲和性几乎相同（Kd= 140±30 μM），证实PLB对Ca-ATPase的抑制调节涉及到Ca-ATPase内部α-螺旋的稳定。
We have used attenuated total reflection Fourier transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopies to identify secondary and dynamic structural changes within the Ca-ATPase that result from the functional inhibition of transport activity by phospholamban (PLB). Isotopically labeled [13C]PLB was expressed and purified fromEscherichia coliand was functionally reconstituted with unlabeled Ca-ATPase, permitting the resolution of the amide I and II absorbance bands of the Ca-ATPase from those of [13C]PLB. Upon co-reconstitution of the Ca-ATPase with PLB, spectral shifts are observed in both the CD spectra and the amide I and II bands associated with the Ca-ATPase, which are indicative of increased α-helical stability. Corresponding changes in the kinetics of H/D exchange occur upon association with PLB, indicating that 100 ± 20 residues in the Ca-ATPase that normally undergo rapid amide H/D exchange become exchange resistant. There are no corresponding large changes in the secondary structure of PLB. The affinity of the structural interaction between PLB and the Ca-ATPase is virtually identical to that associated with functional inhibition (Kd= 140 ± 30 μM), confirming that the inhibitory regulation of the Ca-ATPase by PLB involves the stabilization of α-helices within the Ca-ATPase.