Probing the Ca(2+) switch of the neuronal Ca(2+) sensor GCAP2 by time-resolved fluorescence spectroscopy.
Probing the Ca(2+) switch of the neuronal Ca(2+) sensor GCAP2 by time-resolved fluorescence spectroscopy.
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通过时间分辨荧光光谱探测神经元 Ca(2 ) 传感器 GCAP2 的 Ca(2 ) 开关
DOI：
10.1021/cb3000748
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发表时间：
2012
影响因子：
4
通讯作者：
Lienau
中科院分区：
文献类型：
--
作者：
Kollmann;Becker;Shirdel;Scholten;Ostendorp;Lienau
我们报告的荧光寿命和旋转各向异性测量的荧光染料Alexa 647连接到鸟苷酸环化酶激活蛋白2（GCAP 2），细胞内豆蔻酰化的钙传感器蛋白在感光细胞。通过将染料连接到对监测钙诱导的构象变化至关重要的不同蛋白质区域，我们可以测量荧光寿命和旋转相关时间作为豆蔻酰化、钙和所附染料的位置的函数，而GCAP 2仍然能够以Ca 2+敏感的方式调节鸟苷酸环化酶。当外部改变蛋白质构象时，我们观察到荧光动力学中不同的位点特异性变化。荧光寿命的明显减少表明，在无钙状态下，氨基酸位置131处的染料标记物比位置111处感测到更疏水的蛋白质环境。用钙饱和GCAP 2增加荧光寿命，因此导致位置111更多地暴露于溶剂，同时导致位置131移动到疏水蛋白质裂缝中。此外，我们发现不同的，双指数各向异性衰减反映的荧光团偶极和染料/蛋白质复合物的重定向运动，分别。我们的实验数据很好地描述了一个“摇摆锥”模型，并揭示了在位置111的GCAP 2蛋白的染料标记物，钙和豆蔻酰化的结果在一个显着增加的荧光团的取向灵活性。我们的研究结果提供了证据表明，位于位置111和131之间的α-螺旋的上下运动是蛋白质-染料复合物动力学的关键特征。这种活塞样运动的操作是由细胞内信使钙触发的。
We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca2+-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a “wobbling-in-a-cone” model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein–dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.
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