Harnessing environmental Ca2+ for extracellular protein thermostabilization

Ca2+ is the third-most prevalent metal ion in the environment. EF hands are common Ca2+-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes.  Cytoplasmic EF hand proteins often mediate allosteric control of signal transduction pathway components in response to intracellular Ca2+ concentration fluctuations by coupling Ca2+ binding to changes in protein structure. We show that an extracellular structural Ca2+-binding site mediates protein thermostabilization by such conformational coupling as well. Binding Ca2+ to the EF hand of the extracellular (periplasmic) Escherichia coli glucose-galactose binding protein thermostabilizes this protein by ~17K relative to its Ca2+-free form. Using statistical thermodynamic analysis of a fluorescent conjugate of ecGGBP that reports simultaneously on ligand binding and multiple conformational states, we found that its Ca2+-mediated stabilization is determined by conformational coupling mechanisms in t...钙离子（Ca²+）是环境中第三丰富的金属离子。EF手结构（EF hand）是一类常见的Ca²+结合基序，广泛存在于真核生物与原核生物的胞外及胞内蛋白中。胞质EF手蛋白通常通过将Ca²+结合与蛋白质结构变化相偶联，介导响应胞内Ca²+浓度波动的信号转导通路组分的变构调控。本研究发现，一类胞外结构性Ca²+结合位点同样可通过此类构象偶联机制介导蛋白质的热稳定化。将Ca²+结合至胞外（周质）大肠杆菌葡萄糖-半乳糖结合蛋白（ecGGBP）的EF手结构后，该蛋白的热稳定性相较于无Ca²+状态提升了约17开尔文（K）。我们通过对可同时报告配体结合与多种构象状态的ecGGBP荧光共轭物开展统计热力学分析，发现其Ca²+介导的稳定作用由构象偶联机制所决定……