Resolution of structural changes associated with calcium activation of calmodulin using frequency domain fluorescence spectroscopy.
Resolution of structural changes associated with calcium activation of calmodulin using frequency domain fluorescence spectroscopy.
复制标题
使用频域荧光光谱解析与钙调蛋白的钙激活相关的结构变化。
DOI：
10.1021/bi00191a007
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Squier,TC
中科院分区：
文献类型：
--
作者：
Yao,Y;Schöneich,C;Squier,TC
Revised Manuscript Received April 11, 1994® abstract: Structural changes associated with the calcium-dependent activation of wheat germ calmodulin (CaM) were assessed through measurements of steady-state and time-resolved changes in the fluorescence associated with (1) the unique tyrosine (Tyri39) located in calcium binding loop IV or (2) A-(l-pyrenyl)-maleimide (PM) or 4-(iodoacetamido) salicylic acid (IASA) covalently attached to Cys27 present in calcium binding loop I. These fluorophorespermit themeasurement of calcium-dependent changes in (i) the solvent accessibility and rotational dynamics associated with calcium binding loops I and IV and (ii) the hydrodynamic properties of the entire protein. Specific nitration of the unique tyrosine (Tyri39) in calcium binding loop IV permits the use of fluorescence resonance energy transfer to measure both theaverage spatial separation and distance heterogeneity between Cys27 and Tyri39, providing a direct measurement of the conformational flexibility of the central helix. Upon calcium binding,(i) the solvent accessibility and rotational dynamics of both PM and IASA (covalently boundto Cys27) and Tyri39 increase,(ii) overall protein rotational motion decreases,(iii) the average separation between the chromophores at Cys27 and nitrotyrosine 139 decreases, and (iv) the conformational flexibility associated with the central helix decreases. Therefore, upon calcium binding, the central helix becomes more extended and rigid, while the globular domains adopt a more open tertiary conformation that brings Cys27 and Tyr [39 into closer proximity. This calcium-dependent structural change functions to expose the hydrophobicbinding sites located within the globular domains, and to enhance the probability of binding target sequences through a reduction in conformational heterogeneity.Calmodulin (CaM) 1 is a ubiquitous eukaryotic Ca2+-binding protein that regulates numerous cellular processes, including muscle contraction, neurotransmission, neuronal plasticity, cytoskeletal assembly, and a host of reactions involved in the energy and biosynthetic metabolism of the cell [reviewed by Wylie and Vanaman (1988)]. The crystal structure of the calcium-liganded form of CaM has recently been refined to 1.7 A (Chattopadhyaya et al., 1992) and shows two structurally homologous, globular domains connected by an eight-turn central a-helix (Babu et al., 1985, 1988). Both globular domains consist of two Ca2+-binding sites, each of which shows a helix-loop-helix motif known as an EF-hand (Kretsinger & Nockolds, 1973). Binding of Ca2+ is cooperative with
