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Bioluminescence, the production and emission of visible light from a chemical reaction in living organisms, is found in almost every major phylum in the marine ecosystem, but only a few bioluminescent mechanisms are well understood today. The luminous brittlestar Ophiopsila californica produces intense green luminescence from its long arms when mechanically stimulated. Distinctively, the light producing cells (photocytes) of O. californica appear to be of neural origin. Understanding the mechanism of light production in this species could identify novel chemistry and cellular biology for biological light production, which could have additional applied uses in a wide range of fields including neuroscience, other biomedical research, and commercial usages. O. californica bioluminescence is produced by a photoprotein that requires calcium ions as a cofactor. However, previous studies in the Deheyn lab showed that bioluminescence can also be triggered in the absence of calcium in the extracellular environment, thus indicating the possibility that intracellular calcium is somehow involved in the light production mechanism. The goal of my thesis was to investigate whether ryanodine receptors (a type of calcium channel) are involved in the bioluminescence mechanism of O. californica photocytes. I applied pharmacological agents known to activate or inhibit ryanodine receptors from other species on isolated cell suspensions enriched in photocytes and assessed their effect on bioluminescence. My results suggested that ryanodine receptors are not involved in the process. Further research is needed to unveil the cellular mechanisms underlying bioluminescence production by O. californica.

Bioluminescence, the production and emission of visible light from a chemical reaction in living organisms, is found in almost every major phylum in the marine ecosystem, but only a few bioluminescent mechanisms are well understood today. The luminous brittlestar Ophiopsila californica produces intense green luminescence from its long arms when mechanically stimulated. Distinctively, the light producing cells (photocytes) of O. californica appear to be of neural origin. Understanding the mechanism of light production in this species could identify novel chemistry and cellular biology for biological light production, which could have additional applied uses in a wide range of fields including neuroscience, other biomedical research, and commercial usages. O. californica bioluminescence is produced by a photoprotein that requires calcium ions as a cofactor. However, previous studies in the Deheyn lab showed that bioluminescence can also be triggered in the absence of calcium in the extracellular environment, thus indicating the possibility that intracellular calcium is somehow involved in the light production mechanism. The goal of my thesis was to investigate whether ryanodine receptors (a type of calcium channel) are involved in the bioluminescence mechanism of O. californica photocytes. I applied pharmacological agents known to activate or inhibit ryanodine receptors from other species on isolated cell suspensions enriched in photocytes and assessed their effect on bioluminescence. My results suggested that ryanodine receptors are not involved in the process. Further research is needed to unveil the cellular mechanisms underlying bioluminescence production by O. californica.

Bioluminescence in marine systems is dominated by the use of coelenterazine for light production. The bioluminescent reaction of coelenterazine is an enzyme catalyzed oxidative decarboxylation: coelenterazine reacts with molecular oxygen to form carbon dioxide, coelenteramide, and light. One such class is the Ca2+‐regulated photoproteins. These proteins bind coelenterazine and oxygen, and trap 2‐hydroperoxycoelenterazine, an intermediate along the reaction pathway. The reaction is halted until Ca2+ binding triggers the completion of the reaction. There are currently no reported experimental, atomistic descriptions of this ternary Michaelis complex. This study utilized computational techniques to develop an atomistic model of the Michaelis complex. Extensive molecular dynamics simulations were carried out to study the interactions between four tautomeric/protonation states of coelenterazine and wide‐type and mutant obelin. Only minor differences in binding modes were observed across all systems. Interestingly, no basic residues were identified in the vicinity of the N7‐nitrogen of coelenterazine. This observation was surprising considering that deprotonation at this position is a key mechanistic step in the proposed bioluminescent reaction. This work suggests that coelenterazine binds either as the O10H tautomer, or in the deprotonated form. Implicit ligand sampling simulations were used to identify potential O2 binding and migration pathways within obelin. A key oxygen binding site was identified close to the coelenterazine imidazopyrazinone core. The O2 binding free energy was observed to be dependent on the protonation state of coelenterazine. Taken together, the description of the obelin-coelenterazine‐O2 complexes established in this study provides the basis for future computational studies of the bioluminescent mechanism.

Bioluminescence in marine systems is dominated by the use of coelenterazine for light production. The bioluminescent reaction of coelenterazine is an enzyme catalyzed oxidative decarboxylation: coelenterazine reacts with molecular oxygen to form carbon dioxide, coelenteramide, and light. One such class is the Ca2+‐regulated photoproteins. These proteins bind coelenterazine and oxygen, and trap 2‐hydroperoxycoelenterazine, an intermediate along the reaction pathway. The reaction is halted until Ca2+ binding triggers the completion of the reaction. There are currently no reported experimental, atomistic descriptions of this ternary Michaelis complex. This study utilized computational techniques to develop an atomistic model of the Michaelis complex. Extensive molecular dynamics simulations were carried out to study the interactions between four tautomeric/protonation states of coelenterazine and wide‐type and mutant obelin. Only minor differences in binding modes were observed across all systems. Interestingly, no basic residues were identified in the vicinity of the N7‐nitrogen of coelenterazine. This observation was surprising considering that deprotonation at this position is a key mechanistic step in the proposed bioluminescent reaction. This work suggests that coelenterazine binds either as the O10H tautomer, or in the deprotonated form. Implicit ligand sampling simulations were used to identify potential O2 binding and migration pathways within obelin. A key oxygen binding site was identified close to the coelenterazine imidazopyrazinone core. The O2 binding free energy was observed to be dependent on the protonation state of coelenterazine. Taken together, the description of the obelin-coelenterazine‐O2 complexes established in this study provides the basis for future computational studies of the bioluminescent mechanism.

Light producing proteins (photoproteins) are attractive molecules to study because of their potential applications in biotechnology and biomedicine. Photoproteins have been identified in several different phyla, however a photoprotein from the phylum Echinodermata has yet to be characterized. The goal in this work is to extract the calcium-dependent photoprotein from brittlestar Ophiopsila californica for purification and identification. The photoprotein was successfully extracted while maintaining activity. Three chromatographic techniques were used to separate the functional photoprotein from other constituents in the brittlestar extracts. The light producing fractions from each of these chromatography methods were analyzed using gel electrophoresis in attempt to identify a band that correlates with the amount of light production when comparing multiple fractions. A ~45 kDa band showed this positive correlation throughout each purification step. This band was characterized by ESI-QTOF-MS but did not result in the identification of a new photoprotein. Similar methods were attempted on isolated photocytes (light producing cells). However nearly all light producing activity was lost when attempting to extract the photoprotein from the cell. This indicated that the photoprotein relies on an intact membrane in order to function. This leads to the idea that the activation of this photoprotein could be different than the one previously proposed.

Light producing proteins (photoproteins) are attractive molecules to study because of their potential applications in biotechnology and biomedicine. Photoproteins have been identified in several different phyla, however a photoprotein from the phylum Echinodermata has yet to be characterized. The goal in this work is to extract the calcium-dependent photoprotein from brittlestar Ophiopsila californica for purification and identification. The photoprotein was successfully extracted while maintaining activity. Three chromatographic techniques were used to separate the functional photoprotein from other constituents in the brittlestar extracts. The light producing fractions from each of these chromatography methods were analyzed using gel electrophoresis in attempt to identify a band that correlates with the amount of light production when comparing multiple fractions. A ~45 kDa band showed this positive correlation throughout each purification step. This band was characterized by ESI-QTOF-MS but did not result in the identification of a new photoprotein. Similar methods were attempted on isolated photocytes (light producing cells). However nearly all light producing activity was lost when attempting to extract the photoprotein from the cell. This indicated that the photoprotein relies on an intact membrane in order to function. This leads to the idea that the activation of this photoprotein could be different than the one previously proposed.

Background: Bioluminescence is a process in which light is emitted by a living organism. Most creatures that emit light are sea creatures, but some insects, plants, fungi etc, also emit light. The biotechnological application of bioluminescence has become routine and is considered essential for many medical and general technological advances. Identification of bioluminescent proteins is more challenging due to their poor similarity in sequence. So far, no specific method has been reported to identify bioluminescent proteins from primary sequence.Results: In this paper, we propose a novel predictive method that uses a Support Vector Machine (SVM) and physicochemical properties to predict bioluminescent proteins. BLProt was trained using a dataset consisting of 300 bioluminescent proteins and 300 non-bioluminescent proteins, and evaluated by an independent set of 141 bioluminescent proteins and 18202 non-bioluminescent proteins. To identify the most prominent features, we carried out feature selection with three different filter approaches, ReliefF, infogain, and mRMR. We selected five different feature subsets by decreasing the number of features, and the performance of each feature subset was evaluated.Conclusion: BLProt achieves 80% accuracy from training (5 fold cross-validations) and 80.06% accuracy from testing. The performance of BLProt was compared with BLAST and HMM. High prediction accuracy and successful prediction of hypothetical proteins suggests that BLProt can be a useful approach to identify bioluminescent proteins from sequence information, irrespective of their sequence similarity. 2011 Kandaswamy et al; licensee BioMed Central Ltd.

Aequorin belongs to a unique class of photoproteins that emit light upon the binding of certain metals, calcium being the principal intracellular activator. This 'reporting function' of the metal-binding is instantaneous and is very easy to quantitate experimentally. The project objective was to develop a variety of recombinant forms of aequorin so they can be employed as metal biosensors. Three calcium-binding sites of aequorin were modified to examine their roles in the calcium-dependent luminescence as well as potentially binding other metal ions. Aequorins having Site 2 substitutions unexpectedly produce more light than wild-type aequorin.... Calcium, Bioluminescence, Photoprotein, Mutagenesis

[No abstract available]