Studying Light Production in the Sea at the Molecular Level
by Patrick H. Donnan
Dinoflagellates are marine and freshwater microorganisms responsible for the bioluminescent phenomenon dubbed the “phosphorescence of the sea,” which creates dazzling displays of blue light in regions such as Puerto Rico’s bioluminescent bays. Like all known bioluminescent systems, dinoflagellates use an enzyme, called luciferase, in order to catalyze the chemistry necessary for generating light. In addition, all known bioluminescence systems use molecular oxygen and another molecule called luciferin. Luciferin, however, is not the same molecule between systems, as each has evolved its own preferred molecule for making light. However, in contrast to other bioluminescent systems, dinoflagellates are able to cut out many of the other chemicals nature often needs to generate light. For example, fireflies use adenosine triphosphate (ATP) to provide the energy needed, while bacteria utilize another energy source, a molecule known as flavin mononucleotide (FMN). However, dinoflagellates have figured out how to conduct ‘simple’ bioluminescence and only need oxygen and dinoflagellate luciferin, making them unique (and possibly cleverer ) than other bioluminescent organisms.
Dinoflagellate bioluminescence is triggered upon physical agitation of the organism, and dinoflagellates utilize special organelles which house both luciferase and luciferin to make this happen. These organelles, called scintillons, are biochemically configured such that shaking the organisms causes the acidity in the scintillon to increase. The increase in acidity, which is the same as decreasing the pH, turns luciferase active. In order to study the process by which this happens, we have employed a computational technique called molecular dynamics (MD). MD allows us to simulate biomolecules, such as enzymes, and watch how their behavior changes over time. MD simulations use structures determined by methods such as X-ray crystallography as starting points for computer-based investigations. While for many biomolecules, numerous structures have been obtained by X-ray crystallography, only one exists for dinoflagellate luciferase. MD provides a tool for filling in the gaps and determining biomolecular structures, with the added benefit of being able to observe the structure in motion.
In particular, we have been able to study the effect of adding and removing protons on specific places on luciferase and observe the resulting changes in the dynamics of the enzyme. This has allowed us to understand the process of how increasing the acidity in the scintillon, in other words, creating an environment with more protons available to hop onto the enzyme, directly causes movement in the enzyme structure. One challenge faced when modeling luciferase with MD was the size of the enzyme, which makes studying structural changes over the longer timescales computationally taxing (though by our standards, just one millisecond is a long time). Even using graphics processing units (GPUs) that were available to us to accelerate calculations, conducting a simulation of one millisecond could take over one hundred years to conduct for the luciferase enzyme. To overcome this, we employed a methodology to accelerate our calculations by selectively tampering with the system’s potential energy, allowing us to carry out calculations over a time frame of weeks instead of years.
Ultimately, we were able to produce the active form of the enzyme , which is, to date, still inaccessible via X-ray crystallography. Using our results, we have made several testable, experimental predictions, spurring ongoing research undertaken by other students in the laboratory. For our part, the active enzyme structure has opened new avenues of computational investigation. We are currently working to examine interactions between luciferase and luciferin to better understand how bio-luminescence is catalyzed. By using MD to simulate different versions of luciferin, and its chemical derivatives that are potential intermediates in the bio-luminescent reaction, we are studying which atoms in both luciferase and luciferin are important for generating light. Further, we are adding molecular oxygen to our simulations and studying how all three necessary components for bio-luminescence come together in dinoflagellates: enzyme, substrate, and oxygen.
We hope to one day see our work applied in the development of an imaging agent based on the dinoflagellate bio-luminescent system. Specifically, such an agent could report on the acidity of its location, which can be important in medical contexts where even slight changes in pH can have drastic physiological effects. As a final note, dinoflagellates are also responsible for the harmful algal blooms known as red tides. Understanding their unique chemistry lays the groundwork for a truly Auburn goal: fighting crimson tides.
Acknowledgement: This work is supported by the National Sciences Foundation and Alabama EPSCoR and was done in the lab of Prof. Steve Mansoorabadi in the department of Chemistry and Biochemistry, Auburn University.