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

被引:22
作者
Lewis, JC [1 ]
López-Moya, JJ [1 ]
Daunert, S [1 ]
机构
[1] Univ Kentucky, Dept Chem, Lexington, KY 40506 USA
关键词
D O I
10.1021/bc9900800
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Aequorin is one of several photoproteins that emits visible light upon binding to calcium ions. It has been widely used as a Ca2+-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 CO2 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.
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页码:65 / 70
页数:6
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