title: Bioluminescence and secondary structure properties of aequorin mutants produced for site-specific conjugation and immobilization.
abstract: Aequorin is one of several photoproteins that emits visible light upon binding to calcium ions. It has been widely used as a Ca(2+)-indicator and as an alternative highly sensitive bioluminescent label in binding assays. The apoprotein of aequorin binds an imidazopyrazine compound (coelenterazine) and molecular oxygen to form a stable photoprotein complex. Upon addition of calcium, the photoprotein undergoes a conformational change leading to the oxidation of the chromophore with the release of CO(2) and blue light. To gain more information of structure-function relationships within the photoprotein that will aid in the design of mutants suitable for site-specific conjugation and immobilization, polymerase chain reaction (PCR)-based site-directed mutagenesis was employed to produce five different aequorin mutants. The five mutants included a cysteine-free mutant and four other mutants with single cysteine residues at selected positions within the protein. The aequorin mutants exhibited different bioluminescence emission characteristics with two mutants showing a decrease in relative light production in comparison to the cysteine-free mutant. Additionally, circular dichroism (CD) spectra revealed that the single amino acid substitutions made for two of the aequorin mutants did alter their secondary structures.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Lewis
authors.forename: J J
authors.initials: JJ
authors.lastname: López-Moya
authors.forename: S
authors.initials: S
authors.lastname: Daunert
mesh_terms: D000331:Aequorin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D005470:Fluorometry; D008163:Luminescent Measurements; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012694:Serine / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D012694:Serine; D000331:Aequorin; D003545:Cysteine