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Requirement of the C-terminal proline residue for stability of the Ca(2+)-activated photoprotein aequorin

Watkins, N. J. and Campbell, Anthony Keith 1993. Requirement of the C-terminal proline residue for stability of the Ca(2+)-activated photoprotein aequorin. Biochemical Journal 293 (1) , pp. 181-185.

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Abstract

cDNA coding for the Ca(2+)-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 2 h, 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 Ca(2+)-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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Pharmacy
Subjects: Q Science > QD Chemistry
Q Science > QR Microbiology
R Medicine > RM Therapeutics. Pharmacology
Publisher: Portland Press
ISSN: 0264-6021
Last Modified: 05 Jun 2017 02:58
URI: http://orca-mwe.cf.ac.uk/id/eprint/22486

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