Laura Rowe

Aequorin variants with improved bioluminescence properties

The photoprotein aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoaequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic aequorins. Variants of aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

Spectral tuning of photoproteins by partnering site-directed mutagenesis strategies with the incorporation of chromophore analogs.

Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2-hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.

Modulating the Bioluminescence Emission of Photoproteins by in Vivo Site-Directed Incorporation of Non-Natural Amino Acids

The in vivo incorporation of non-natural amino acids into specific sites within proteins has become an extremely powerful tool for bio- and protein chemists in recent years. One avenue that has yet to be explored, however, is whether or not the incorporation of non-natural amino acids can tune the color of light emitted by bioluminescent proteins, whose light emission mechanisms are more complex and less well understood than their fluorescent counterparts. Bioluminescent proteins are becoming increasingly important in a variety of research fields, such as in situ imaging and the study of protein-protein interactions in vivo, and an increased spectral variety of bioluminescent reporters is needed for further progress. Thus, herein we report the first successful spectral shifting (44 nm) of a bioluminescent protein, aequorin, via the site-specific incorporation of several non-natural amino acids into an integral amino acid position within the aequorin structure in vivo.

Genetically modified semisynthetic bioluminescent photoprotein variants: simultaneous dual-analyte assay in a single well employing time resolution of decay kinetics.

Progress in the miniaturization and automation of complex analytical processes depends largely on increasing the sensitivity, diversity, and robustness of current labels. Because of their ubiquity and ease of use, fluorescent, enzymatic, and bioluminescent labels are often employed in such miniaturized and multiplexed formats, with each type of label having its own unique advantages and drawbacks. The ultrasensitive detection limits of bioluminescent reporters are especially advantageous when dealing with very small sample volumes and biological fluids. However, bioluminescent reporters currently do not have the multiplexing capability that fluorescent labels do. In an effort to address this limitation, we have developed a method of discriminating two semisynthetic aequorin variants from one another using time resolution. In this work we paired two aequorin conjugates with different coelenterazine analogues and then resolved the two signals from one another using the difference in decay kinetics and half-life times. Utilizing this time-resolution, we then developed a simultaneous, dual-analyte, single well assay for 6-keto-prostaglandin-FI-alpha and angiotensin II, two important cardiovascular molecules.

EF-hand Ca2+-binding bioluminescent proteins: effects of mutations and alternative divalent cations

Bioluminescent photoproteins, such as aequorin and obelin, are proteins that emit light upon binding calcium. Aequorin and obelin contain four EF-hand domains arranged into a globular structure. The loop region of these EF-hand domains binds calcium by coordinating it in a pentagonal bipyramidal structure with oxygen atoms. The binding of calcium to these EF-hands causes a slight conformational change in the protein, which leads to the oxidation of the internally sequestered chromophore, coelenterazine, producing coelenteramide and CO2. The excited coelenteramide then relaxes radiatively, emitting bioluminescence at 471 nm in aequorin or 491 nm in obelin. Although calcium is the traditional, and generally the most powerful, triggering ligand in this bioluminescence reaction, alternative di- and trivalent cations can also bind to the EF-hand loops and stimulate luminescence. Species capable of this cross-reactivity include: Cd2+, Ba2+, Mn2+, Sr2+, Mg2+, and several lanthanides. Magnesium is also known to modulate the bioluminescence of wild-type aequorin, increase its stability, and decrease its aggregation tendency. Both wild-type aequorin and wild-type obelin contain several cysteine residues, aequorin has three and obelin has five. It is believed that these cysteine residues play an important, but as of yet unknown, role in the bioluminescence of these proteins, since mutating most of these residues causes significant loss in bioluminescent activity. In order to explore whether or not these cysteine residues contributed to the specificity of the EF-hand domains for cations we generated four aequorin and obelin mutants and observed their luminescent intensity and decay kinetics by stimulation with calcium, barium, and magnesium. It was found that the cysteine mutations do appear to alter the effects that alternative divalent cations have on the bioluminescence of both aequorin and obelin.

BIOLUMINESCENCE CHARACTERISTICS OF AN OBELIN MUTANT IN VARYING SOLVENT CONDITIONS

INTRODUCTION Obelin is a globular, 22 kDa bioluminescent photoprotein composed of four EFhand domains, and a hydrophobic core cavity in which coelenterazine is located. Upon binding calcium, obelin undergoes a conformational change which leads to the oxidation of coelenterazine to an excited coelenteramide. This excited coelenteramide subsequently relaxes, and releases the characteristic 485 nm bioluminescent light of native obelin.’ Obelin was isolated from the bioluminescent hydroid Obelia longissima, and is structurally analogous to aequorin, but offers certain advantages over aequorin in terms of calcium sensing within a cell. These advantages include the fact that obelin’s bioluminescence is less sensitive to competing intracellular divalent cations, such as Mg”, and that the calcium induced bioluminescence kinetics are faster in obelin.’ This allows for less cross-reactivity and an ability to monitor ultra-fast calcium fluctuations in vivo. Native obelin contains five cysteine residues, at positions 5 1, 67, 75, 15 1, and 158. Obelin has a high ratio of cysteine residues, and these residues are thought to be important for bioluminescent activity.’ However, recent X-ray crystal structures have been resolved for both apoobelin and obelin, and no cysteine residues are found to be close enough to the active site of the photoprotein to be directly involved in the bioluminescence reaction, nor are any of the five cysteines involved in intramolecular disulfide bonds.’ All cysteine residues are, however, located in or very near the a-helices of the EF-hand domains.’ To the best of our knowledge no mutational studies have been undertaken concerning the cysteine residues in obelin, with the exception of mutations of C158 to serine and alanine.’ Therefore, in order to more filly understand the role of cysteine in the bioluminescence of obelin we undertook a mutational study on all five cysteine residues in obelin. The results of this study are currently still under analysis, however, we selected one mutant, in which the cysteine residues at positions 67 and 75 were both mutated to serines, and analyzed the change in bioluminescence properties when the photoprotein was exposed to a range of conditions that may be encountered in a cellular environment. Different areas and organelles within cells exhibit specific microenvironments whose physical properties may differ than the cell in general. Such pH and solvent changes may affect the bioluminescent properties of obelin in general, and our C6576 mutant specifically. Therefore, herein we describe the analysis of the relative activity, decay half-life time, and emission maximum of obelin C67-75 in buffers varying in pH and buffer composition.

Genetically engineered luminescent proteins in biosensing

Luminescent proteins originally isolated from marine or terrestrial organisms have played a key role in the development of several biosensing systems. These proteins have been used in a variety of applications including, immunoassays, binding assays, cell-based sensing, high throughput screening, optical imaging, etc. Among the luminescent proteins isolated, the bioluminescent protein aequorin has been one of the proteins at the forefront in terms of its use in a vast number of biosensing systems. In our laboratory, we have employed aequorin as a label in the development of highly sensitive assays through chemical and genetic modifications from single step analysis of physiologically important molecules in biological fluids. An important aspect of optimizing these assays for clinical use involves understanding the stability of the various aequorin variants that are available. To this end we have designed several stability studies involving three important aequorin mutants, Mutant S, Mutant 5, and Mutant 53. The cysteine free aequorin, Mutant S, has been the most ubiquitously used aequorin variant in our laboratory because of its increased stability and activity as compared to native aequorin. Mutant 5 and Mutant 53 contain a single cyteine residue at position 5 and 53 in the protein, respectively. Because of the presence of a single cysteine residue, Mutant 5 and Mutant 53 both can be site-specifically conjugated. This site specific conjugation capability gives Mutant 5 and Mutant 53 an advantage over native aequorin when developing assays. Additional studies optimizing the expression, purification, and charging of aequorin Mutant S were also performed. A thorough understanding of the efficient expression, purification, and storage of these aequorin mutants will allow for the more practical utilization of these mutants in the development of future biosensing systems.