Calcium-dependent stabilization of the central sequence between Met(76) and Ser(81) in vertebrate calmodulin.
Calcium-dependent stabilization of the central sequence between Met(76) and Ser(81) in vertebrate calmodulin.
复制标题
脊椎动物钙调蛋白中 Met(76) 和 Ser(81) 之间中心序列的钙依赖性稳定性。
DOI：
10.1016/s0006-3495(01)75931-0
复制
发表时间：
2001
影响因子：
3.4
通讯作者：
Squier,TC
中科院分区：
文献类型：
--
作者：
Qin,Z;Squier,TC
Spin-label electron paramagnetic resonance (EPR) provides optimal resolution of dynamic and conformational heterogeneity on the nanosecond time-scale and was used to assess the structure of the sequence between Met76and Ser81in vertebrate calmodulin (CaM). Previous fluorescence resonance energy transfer and anisotropy measurements indicate that the opposing domains of CaM are structurally coupled and the interconnecting central sequence adopts conformationally distinct structures in the apo-form and following calcium activation. In contrast, NMR data suggest that the opposing domains of CaM undergo independent rotational dynamics and that the sequence between Met76and Ser81in the central sequence functions as a flexible linker that connects two structurally independent domains. However, these latter measurements also resolve weak internuclear interactions that suggest the formation of transient helical structures that are stable on the nanosecond time-scale within the sequence between Met76and Asp80in apo-CaM (H. Kuboniwa, N. Tjandra, S. Grzekiek, H. Ren, C. B. Klee, and A. Bax, 1995,Nat. Struct. Biol.2:768–776). This reported conformational heterogeneity was resolved using site-directed mutagenesis and spin-label EPR, which detects two component spectra for 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin labels (MTSSL) bound to CaM mutants T79C and S81C that include a motionally restricted component. In comparison to MTSSL bound within stable helical regions, the fractional contribution of the immobilized component at these positions is enhanced upon the addition of small amounts of the helicogenic solvent trifluoroethanol (TFE). These results suggest that the immobilized component reflects the formation of stable secondary structures. Similar spectral changes are observed upon calcium activation, suggesting a calcium-dependent stabilization of the secondary structure. No corresponding changes are observed in either the solvent accessibility to molecular oxygen or the maximal hyperfine splitting. In contrast, more complex spectral changes in the line-shape and maximal hyperfine splitting are observed for spin labels bound to sites that undergo tertiary contact interactions. These results suggest that spin labels at solvent-exposed positions within the central sequence are primarily sensitive to backbone fluctuations and that either TFE or calcium binding stabilizes the secondary structure of the sequence between Met76and Ser81and modulates the structural coupling between the opposing domains of CaM.
