Tatulian Suren A, Chen Baowei, Li Jinhui, Negash Sewite, Middaugh C Russell, Bigelow Diana J, Squier Thomas C
Biochemistry and Biophysics Section, Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045-2106, USA.
Biochemistry. 2002 Jan 22;41(3):741-51. doi: 10.1021/bi011148d.
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 [(13)C]PLB was expressed and purified from Escherichia coli and 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 [(13)C]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 alpha-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 (K(d) = 140 +/- 30 microM), confirming that the inhibitory regulation of the Ca-ATPase by PLB involves the stabilization of alpha-helices within the Ca-ATPase.
我们利用衰减全反射傅里叶变换红外光谱（ATR-FTIR）和圆二色光谱（CD）来鉴定钙ATP酶内的二级结构和动态结构变化，这些变化是由受磷蛋白（PLB）功能抑制的转运活性所导致的。从大肠杆菌中表达并纯化出同位素标记的[(13)C]PLB，并将其与未标记的钙ATP酶进行功能重组，从而能够区分钙ATP酶与[(13)C]PLB的酰胺I和酰胺II吸收带。在将钙ATP酶与PLB共同重组后，在CD光谱以及与钙ATP酶相关的酰胺I和酰胺II谱带中均观察到光谱位移，这表明α-螺旋稳定性增加。与PLB结合后，氢/氘交换动力学发生相应变化，表明钙ATP酶中通常经历快速酰胺氢/氘交换的100±20个残基变得抗交换。PLB的二级结构没有相应的大变化。PLB与钙ATP酶之间结构相互作用的亲和力与功能抑制相关的亲和力几乎相同（K(d)=140±30 microM），这证实了PLB对钙ATP酶的抑制调节涉及钙ATP酶内α-螺旋的稳定。
