Department of Chemistry, University of Minnesota Duluth, Duluth, Minnesota, USA.
Biophys J. 2012 Jul 18;103(2):238-46. doi: 10.1016/j.bpj.2012.05.051. Epub 2012 Jul 17.
The C2A domain is one of two calcium ion (Ca(2+))- and membrane-binding domains within synaptotagmin I (Syt I), the identified Ca(2+) sensor for regulated exocytosis of neurotransmitter. We propose that the mechanistic basis for C2A's response to Ca(2+) and cellular function stems from marginal stability and ligand-induced redistributions of protein conformers. To test this hypothesis, we used a combination of calorimetric and fluorescence techniques. We measured free energies of stability by globally fitting differential scanning calorimetry and fluorescence lifetime spectroscopy denaturation data, and found that C2A is weakly stable. Additionally, using partition functions in a fluorescence resonance energy transfer approach, we found that the Ca(2+)- and membrane-binding sites of C2A exhibit weak cooperative linkage. Lastly, a dye-release assay revealed that the Ca(2+)- and membrane-bound conformer subset of C2A promote membrane disruption. We discuss how these phenomena may lead to both cooperative and functional responses of Syt I.
C2A 结构域是突触融合蛋白 I（Syt I）中两个钙离子（Ca(2+)）和膜结合结构域之一，是被鉴定的用于神经递质调节胞吐的 Ca(2+)传感器。我们提出，C2A 对 Ca(2+)和细胞功能的响应机制源于其构象的边缘稳定性和配体诱导的再分布。为了验证这一假设，我们结合使用了量热法和荧光技术。我们通过全局拟合差示扫描量热法和荧光寿命光谱变性数据来测量稳定性的自由能，并发现 C2A 结构域的稳定性较弱。此外，我们还使用荧光共振能量转移方法中的配分函数，发现 C2A 的 Ca(2+)和膜结合位点表现出微弱的协同联系。最后，染料释放实验表明，C2A 的 Ca(2+)和膜结合构象亚基可促进膜破裂。我们讨论了这些现象如何导致 Syt I 的协同和功能性响应。
