Bioluminescent and spectroscopic properties of His—Trp—Tyr triad mutants of obelin and aequorin

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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.

Section: Tyr Residuesmentioning

confidence: 96%

“…). According to various studies, this triad is proposed to participate both in 2‐hydroperoxycoelenterazine stabilization and the formation of active photoprotein from apoprotein, coelenterazine and oxygen . The third triad is formed by His58, Trp108 and Tyr132 (Fig.…”

Section: His−trp−tyr Triadsmentioning

confidence: 99%

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

Eremeeva

Vysotski

2018

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“…The internal cavity of hydromedusan Ca 2+ ‐regulated photoproteins which also use coelenterazine as a substrate of bioluminescence reaction contains many aromatic residues . These residues are involved in stabilizing the 2‐hydroperoxy group of coelenterazine (Trp, His and Tyr) , emitter formation (His, Tyr and Trp) and coelenterazine activation by oxygen (Tyr) . In copepod luciferases, conservative motifs within the repeats contain two Tyr residues one of which is conservative (positions are marked with frames in Fig.…”

Section: Improvement Of Reporter Properties By Mutagenesismentioning

confidence: 99%

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Markova

Larionova

Vysotski

2019

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Copepod luciferases—a family of small secretory proteins of 18.4–24.3 kDa, including a signal peptide—are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine‐dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high‐throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.

“…Three His residues (H16, H58 and H169) are important in stabilizing coelenterazine (Fig. ) and their subsequent conservative or semi‐conservative mutagenesis affected drastically the luminescence activity . Similarly, the C‐terminal proline of aequorin proved to be essential for the stability of the bound coelenterazine and any substitution/deletion of this residue resulted in loss of aequorin activity .…”

Section: Engineering Fp–photoprotein Sensors For Calcium Based On Bretmentioning

confidence: 99%

“…Although aequorin structure has been the most extensively studied by mutagenesis, several reports by Vysotski and collaborators have also shown striking structure–function effects in obelin . In 2008, Rowe et al .…”

Section: Engineering Fp–photoprotein Sensors For Calcium Based On Bretmentioning

confidence: 99%

Fluorescent Protein–photoprotein Fusions and Their Applications in Calcium Imaging

Bakayan

Domingo

Vaquero

et al. 2017

Calcium‐activated photoproteins, such as aequorin, have been used as luminescent Ca2+ indicators since 1967. After the cloning of aequorin in 1985, microinjection was substituted by its heterologous expression, which opened the way for a widespread use. Molecular fusion of green fluorescent protein (GFP) to aequorin recapitulated the nonradiative energy transfer process that occurs in the jellyfish Aequorea victoria, from which these two proteins were obtained, resulting in an increase of light emission and a shift to longer wavelength. The abundance and location of the chimera are seen by fluorescence, whereas its luminescence reports Ca2+ levels. GFP‐aequorin is broadly used in an increasing number of studies, from organelles and cells to intact organisms. By fusing other fluorescent proteins to aequorin, the available luminescence color palette has been expanded for multiplexing assays and for in vivo measurements. In this report, we will attempt to review the various photoproteins available, their reported fusions with fluorescent proteins and their biological applications to image Ca2+ dynamics in organelles, cells, tissue explants and in live organisms.