EFFECTS OF ION-BINDING ON THE BACKBONE DYNAMICS OF CALBINDIN-D9K DETERMINED BY N-15 NMR RELAXATION
EFFECTS OF ION-BINDING ON THE BACKBONE DYNAMICS OF CALBINDIN-D9K DETERMINED BY N-15 NMR RELAXATION
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DOI：
10.1021/bi00088a039
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发表时间：
1993-09-21
期刊：
影响因子：
2.9
通讯作者：
CHAZIN, WJ
中科院分区：
文献类型：
--
作者：
AKKE, M;SKELTON, NJ;CHAZIN, WJ
用N-15核磁共振波谱对载子-和（Cd2+）1-钙结合蛋白D9k的主链动力学进行了表征。利用富含n- 15的样品，通过二维质子检测的异核磁共振实验，在11.74 T磁场强度下测量了自旋-晶格和自旋-自旋弛豫速率常数和稳态{H-1}-N-15核Overhauser效应。利用无模型的形式，用广义阶参数和有效相关时间表征了N-H键向量的动力学特性。将apo和（Cd2+）1状态的数据与（Ca2+）2状态的数据进行比较[Kordel， J., Skelton, n.j., Akke， M., Palmer, a.g ., & Chazin， W. J.（1992）生物化学31,48 56-4866]以确定离子连接对钙结合蛋白D9k主干动力学的影响。两个结合环对金属离子连接的响应不同：n端离子结合环的高频(10（9)-10(12)s-1）波动不受离子结合的影响，而c端离子结合环中的残基G57、D58、G59和E60在载子状态下的阶参数明显低于金属结合状态。4个螺旋对离子结合的动态响应远小于c端结合环，其中对位于连接环和结合位点II之间的螺旋III的影响最大。在载子和（Cd2+）]态的未占据n端离子结合环中也检测到较慢时间尺度上的显著波动；（Cd2+）1态的表观速率更大。这些关于钙结合蛋白D9k中离子结合的动力学响应的结果提供了对结合过程的分子细节的见解，以及离子首先在c端结合的途径中钙结合协同现象的熵贡献的定性证据。
The backbone dynamics of apo- and (Cd2+)1-calbindin D9k have been characterized by N-15 nuclear magnetic resonance spectroscopy. Spin-lattice and spin-spin relaxation rate constants and steady-state {H-1}-N-15 nuclear Overhauser effects were measured at a magnetic field strength of 11.74 T by two-dimensional, proton-detected heteronuclear NMR experiments using N-15-enriched samples. There laxation parameters were analyzed using a model-free formalism that characterizes the dynamics of the N-H bond vectors in terms of generalized order parameters and effective correlation times. The data for the apo and (Cd2+)1 states were compared to those for the (Ca2+)2 state [Kordel, J., Skelton, N. J., Akke, M., Palmer, A. G., & Chazin, W. J. (1 992) Biochemistry 31, 48 56-4866] to ascertain the effects of ion ligation on the backbone dynamics of calbindin D9k. The two binding loops respond differently to ligation by metal ions: high-frequency (10(9)-10(12)s-1) fluctuations of the N-terminal ion-binding loop are not affected by ion binding, whereas residues G57, D58, G59, and E60 in the C-terminal ion-binding loop have significantly lower order parameters in the apo state than in the metal-bound states. The dynamical responses of the four helices to binding of ions are much smaller than that for the C-terminal binding loop, with the strongest effect on helix III, which is located between the linker loop and binding site II. Significant fluctuations on slower time scales also were detected in the unoccupied N-terminal ion-binding loop of the apo and (Cd2+)] states; the apparent rates were greater for the (Cd2+)1 state. These results on the dynamical response to ion binding in calbindin D9k provide insights into the molecular details of the binding process and qualitative evidence for entropic contributions to the cooperative phenomenon of calcium binding for the pathway in which the ion binds first in the C-terminal site.