Bioluminescence and secondary structure properties of aequorin mutants produced for site-specific conjugation and immobilization

Aequorin is one of several photoproteins that emits visible light upon bind ing to calcium ions. It has been widely used as a Ca2+-indicator and as an alternative highly sensitive bioluminescent label in binding assays. The ap oprotein of aequorin binds an imidazopyrazine compound (coelenterazine) and molecular oxygen to form a stable photoprotein complex. Upon addition of c alcium, the photoprotein undergoes a conformational change leading to the o xidation of the chromophore with the release of CO2 and blue light. To gain more information of structure-function relationships within the photoprote in that will aid in the design of mutants suitable for site-specific conjug ation and immobilization, polymerase chain reaction (PCR)-based site-direct ed mutagenesis was employed to produce five different aequorin mutants. The five mutants included a cysteine-free mutant and four other mutants with s ingle cysteine residues at selected positions within the protein. The aequo rin mutants exhibited different bioluminescence emission characteristics wi th two mutants showing a decrease in relative light production in compariso n to the cysteine-free mutant. Additionally, circular dichroism (CD) spectr a revealed that the single amino acid substitutions made for two of the aeq uorin mutants did alter their secondary structures.