Aequorin mutants with increased thermostability

Abstract

Bioluminescent labels can be especially useful for in vivo and live animal studies due to the negligible bioluminescence background in cells and most animals, and the non-toxicity of bioluminescent reporter systems. Significant thermal stability of bioluminescent labels is essential, however, due to the longitudinal nature and physiological temperature conditions of many bioluminescent based studies. To improve the thermostability of the bioluminescent protein aequorin we employed random and rational mutagenesis strategies to create two thermostable double mutants, S32T/E156V and M36I/E146K, and a particularly thermostable quadruple mutant, S32T/E156V/Q168R/L170I. The double aequorin mutants, S32T/E156V and M36I/E146K, retained 4 and 2.75 times more of their initial bioluminescence activity than wild type aequorin during thermostability studies at 37 °C. Moreover, the quadruple aequorin mutant, S32T/E156V/Q168R/L170I, exhibited more thermostability at a variety of temperatures than either double mutant alone, producing the most thermostable aequorin mutant identified thus far.

Improving the luminescence properties of aequorin by conjugating to CdSe/ZnS quantum dot nanoparticles: Red shift and slowing decay rate

“…It is noticeable that the increase in spectral diversity of photoprotein will provide an increase in its employment as a reporter in multi-analyte detection and imaging. Researchers have succeeded in creating aequorin with various features such as Ca 2+ sensitivities, varying regeneration times, altered emission kinetics by using different methods including site-specific and random mutagenesis [11,30,31], pairing different coelenterazine analogues with aequorin [30,32] and incorporating non-natural amino acids [33,34] and last but not least is bioluminescence resonance energy transfer (BRET) reactions [35,36].…”

“…There are many approaches to produce a shift in photoprotein light emission such as site-specific mutagenesis [11,30,31], construction of semisynthetic photoproteins which uses different coelenterazine analogues [30,32], incorporating non-natural amino acids [33,34] and bioluminescence resonance energy transfer system (BRET) [35,36].…”

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.)…”

Dissection of Protein Kinase Pathways in Live Cells Using Photoluminescent Probes: Surveillance or Interrogation?

“…As noted above, because bioluminescence does not require photoexcitation, it offers significant advantage for imaging techniques due to reduced autofluorescence of biological specimen and absence of photobleaching. Furthermore, bioluminescent signal can last for a prolonged period of time, as long as the enzyme is constantly supplied with its substrate [88]; this, however, might be problematic in the case of intracellular applications, as the cell membrane-penetrating forms of luciferins need to be used. Even more importantly, luciferases originating from several organisms (e.g., sea pansy, firefly or click beetle) possess relatively high molecular weight (in the range of 36-62 kDa [89]; see Figure 2), making those poorly suitable for application as genetically encoded tags.…”