REQUIREMENT OF THE C-TERMINAL PROLINE RESIDUE FOR STABILITY OF THE CA2+-ACTIVATED PHOTOPROTEIN AEQUORIN
REQUIREMENT OF THE C-TERMINAL PROLINE RESIDUE FOR STABILITY OF THE CA2+-ACTIVATED PHOTOPROTEIN AEQUORIN
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DOI：
10.1042/bj2930181
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发表时间：
1993-07-01
影响因子：
4.1
通讯作者：
CAMPBELL, AK
中科院分区：
文献类型：
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作者：
WATKINS, NJ;CAMPBELL, AK
编码来自维多利亚水母的 Ca2+ 激活光蛋白水母发光蛋白的 cDNA 已被设计用于研究 C 端脯氨酸残基在生物发光中的作用。通过 PCR 合成重组水母蛋白，然后进行体外转录/翻译，并通过比活性、稳定性和对腔肠素的亲和力来表征。水母发光蛋白的 C 端脯氨酸残基对于结合的腔肠素的长期稳定性至关重要。 2小时后，减去脯氨酸的水母发光蛋白的比活性仅为野生型的1%，并且在18小时后几乎失去活性。通过用腔肠素类似物（ε-腔肠素）重新激活进一步证明了该变体的不稳定性，其中15分钟内达到最大重新激活，并且发光活性在3小时内几乎完全消除。用组氨酸或谷氨酸替换 C 端脯氨酸残基，比活性分别降低至野生型的 10% 和 19%。然而，这些变体也不稳定，t1/2 值分别为 2.4 小时和 2.3 小时。当脯氨酸被组氨酸取代时，不依赖于 Ca2+ 的光发射增强，证实了 C 端脯氨酸的稳定作用。没有检测到 C 末端脯氨酸的去除对腔肠素的亲和力有显着影响。
cDNA coding for the Ca2+-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca2+-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.