Requirement of the C-terminal proline residue for stability of the Ca(2+)-activated photoprotein aequorin.
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.
Watkins
N J
NJ
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, U.K.
Campbell
A K
AK
D000331:Aequorin / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D004247:DNA / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D006639:Histidine / Q000737:chemistry; D007093:Imidazoles; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011392:Proline / Q000737:chemistry*; D014176:Protein Biosynthesis; D011719:Pyrazines; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D037401:Scyphozoa / Q000378:metabolism
D005971:Glutamates; D007093:Imidazoles; D011719:Pyrazines; D011994:Recombinant Proteins; D018698:Glutamic Acid; C017144:coelenterazine; D006639:Histidine; D000331:Aequorin; D004247:DNA; D011392:Proline; D002118:Calcium