Exploring Bioluminescence Function of the Ca²⁺‐regulated Photoproteins with Site‐directed Mutagenesis

Abstract

Site‐directed mutagenesis is a powerful tool to investigate the structure–function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site‐directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+‐regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.

Luminescence Activity Decreases When v‐coelenterazine Replaces Coelenterazine in Calcium‐Regulated Photoprotein—A Theoretical and Experimental Study

“…is difficult to identify the protonation forms of three histidines (His16, His58 and His169 in aequorin; His22, His64 and His175 in obelin) involved in three key triads surrounding the substrate in cavity. Many site-directed mutagenesis experimental studies found that only the H169Q mutant of aequorin, in which His169 was replaced by glutamine, whose side chain has two protons, retained BL activity (23% of the wild-type protein BL) (36). This implies that His169 is protonated in aequorin.…”

“…Additionally, three histidines (His16, His58 and His169) of three key triads located in the CTZ-binding cavity of aequorin are strictly conserved (36). The other well-known calcium-regulated photoprotein, obelin, also has three histidines, that is, Scheme 1.…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…Cloning of cDNAs encoding several photoproteins and their expression in bacterial cells, as well as effective activation of the recombinant apophotoproteins with a synthetic coelenterazine (under calcium-free conditions in the presence of O 2 ) opened the way to obtaining almost unlimited amounts of the recombinant proteins as well as to producing different mutated and chimeric photoproteins. Many photoprotein mutants with altered characteristics such as higher thermostability and bioluminescence activity, different emission color, faster or slower bioluminescence kinetics, and modified calcium affinity [ 3 , 4 , 24 , 25 ], as well as chimeric photoproteins genetically fused with different polypeptides, were produced. The diversity of photoprotein variants and synthetic coelenterazine analogues allow the development of novel bioluminescent assays or to improve characteristics of the already existing ones.…”

“…Numerous studies on photoprotein EF-hand motif have shown that calcium affinity of the protein can be changed. Different approaches have been used to perform modifications: specific point mutations of the Ca 2+ -binding sites, random mutagenesis and functional screening, replacement of the consensus sequence of the first Ca 2+ -binding site with a loop sequence belonging to other EF-hand Ca 2+ -binding proteins [ 4 , 25 ], or/and the usage of coelenterazine synthetic analogues [ 31 ]. The aequorin variant with a reduced Ca 2+ affinity was produced and proved to be eminently suitable for measuring Ca 2+ within cell compartments with high calcium concentration (endoplasmic reticulum, mitochondria, etc.)…”

“…This suggests a fundamentally linear relationship between the protein amount and the light emitted. Being the in situ stable enzyme–substrate complexes, photoproteins have become of great academic interest—many proteins of the wild type (WT) and their mutant variants with altered bioluminescent properties were obtained, tertiary structures were determined, and the reaction mechanism was studied (e.g., [ 4 ]).…”