Aequorin variants with improved bioluminescence properties

Abstract

The photoprotein aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoaequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic aequorins. Variants of aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

Fig. 1

Molecular structure of native coelenterazine.

Fig. 2

(A) Bioluminescene emission spectra of (□) cystine-free aequorin with native coelenterazine (••••) and aequorin mutant W86F with coelenterazine hcp, and (□ •) aequorin mutant Y82F with coelenterazine i. (B) Chemical structures of coelenterazine i (left), and coelenterazine hcp.

Fig. 3

Bioluminescence λ_max emission and decay half-lives. • Aequorin mutant F113W paired with coelenterazine hcp. ▪ Cysteine-free aequorin paired with native coelenterazine. ▴ Aequorin mutant W86F paired with coelenterazine i. ♦ Aequorin mutant Y92F paired with coelenterazine i.

Fig. 4

Stability of the apoaequorin mutants at 4°C. At predetermined intervals, an aliquot of the mutant proteins were fully charged with coelenterazine overnight at 4°C and the activity was measured. Data are the average of three points ± the standard deviation. Some error bars are obstructed by the symbols of the points.