Blue fluorescent protein from the calcium-sensitive photoprotein aequorin is a heat resistant enzyme, catalyzing the oxidation of coelenterazine

Abstract 

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin (BFP-aq) was prepared and determined to be a heat resistant enzyme, catalyzing the luminescent oxidation of coelenterazine (luciferin) with molecular oxygen as a general luciferase. After treatment with excess ethylenediaminetetraacetic acid to remove Ca²⁺ from BFP-aq, the blue fluorescence shifted to a greenish fluorescence. This greenish fluorescent protein (gFP-aq) was identified as a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1. By incubation with coelenterazine in the absence of reducing reagents, gFP-aq was converted to aequorin at 25 °C. BFP-aq and gFP-aq possessing both fluorescence and luminescence activities may work as novel reporter proteins.

Abbreviations:  BFP-aq, blue fluorescent protein from aequorin, gFP-aq, greenish fluorescent protein from aequorin, CTZ, coelenterazine, CTM, coelenteramide, CD, circular dichroism, EDTA, ethylenediaminetetraacetic acid, DTT, dithiothreitol, MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, rlu, relative light units, TLC, thin-layer chromatography, SDS–PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis

Keywords:  Bioluminescence, Quantum yield, Luciferin, Enzyme–substrate complex, Refolding