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This description is mostly intended for people with some scientific background. For a less technical description of my research, click here. When an animal is wounded, it is important to seal the wound in order to stem the loss of internal fluids as well as to keep out harmful pathogens. To accomplish this, clotting processes have evolved independently along different animal lineages. Although the molecular mechanisms that underlie the clotting process vary greatly among the different types of animals, they all result in insoluble plugs which stop the loss of blood (or hemolymph - the invertebrate equivalent of blood), and reduce the risk of infection. The vertebrate clotting system is by far the best characterized of all clotting systems. The lab in which I work, Russell Doolittle's lab at UC San Diego, has been studying vertebrate clotting processes for many years. In particular, Prof. Doolittle has focused on the protein fibrinogen. Fibrinogen is present at high concentration in the blood, and upon injury the proteolytic activation of a series of proteins ends in the conversion of fibrinogen to fibrin, which polymerizes to form the gel-like clot. In the past decade the lab has been learning more about fibrinogen and fibrin by solving x-ray structures of proteolytic fragments of these molecules, alone and in complex with peptides that mimic the interactions in the fibrin polymer. Eventually, this work culminated in the structure of a complete, native chicken fibrinogen. I was lucky enough to be involved with the chicken fibrinogen project, which was my first experience with crystallography. I enjoyed it so much I decided to switch from the molecular evolution projects I had been working on to doing my Ph.D. work in structural biology.
While the focus of the lab is still on vertebrate fibrin, my research involves the crustacean clotting system. Crustaceans have developed a way of making clots completely distinct from vertebrates (and from chelicerates, insects, and echinoderms, for that matter!). More than thirty years ago Prof. Doolittle and one of his first graduate students, Gerry Fuller, worked out the basic mechanisms of clot formation in the California spiny lobster, Panulirus interruptus. The lobster was a convenient choice of test subject because it is easy to get large quantities of hemolymph from them, and they are fairly easy to find here off the coast of Southern California (not to mention, they taste really good - for a good recipe for Panulirus interruptus tails click here). The primary component of the crustacean clotting system is the clottable protein (CP), a 420kDa homodimer found at relatively high concentrations in hemolymph. Upon injury, circulating hemocytes release a transglutaminase often referred to as the clotting enzyme. Transglutaminases catalyze the formation of a peptide bond between lysine and gluatmine sidechains. And so the clotting enzyme introduces covalent crosslinks between CP dimers, which form long branching chains. The polymers form a gel, which serves to staunch the flow of hemolymph out of the animal. Clotting is also an immune response, and can be induced by components of bacterial cell walls.
My goal the last several years has been to solve the crystal structure of the lobster CP. I have purified the protein from hemolymph, obtained two different crystal forms of CP, and collect data to a maximum resolution of about 3Å . Unfortunately, attempts to phase the crystal data by soaking atoms with heavy metals have so far not been fruitful. Recently, however, I have been working with some great people at the Automated Molecular Imaging Group at TSRI to solve the structure of CP by single particle reconstruction from cryoelectron micrographs. In this technique, electron micrograph images of individual molecules in many different projections are re-assembled to give a low-resolution (on the order of 15-20Å for a protein like mine, probably) structure. While a low-resolution structure will be able to tell us some things about CP, the ultimate goal is to use this cryo-EM model to obtain phases for the crystal data, and work my way to a high-resolution structure.
What are we likely to learn from the structure of CP? My primary interest is the evolution of this protein. CP is a homolog of vitellogenins, a class of proteins which transport lipids from the liver or hepatopancreas to the ovaries, where they are deposited in yolk to provide nourishment for growing embryos. Like vitellogenins, CP binds lipids, at least some part of which is the carotenoid astaxanthin. It seems obvious that evolution co-opted a protein already present in the hemolymph for a new job forming clots. A vitellogenin structure is already known, and comparison with this structure will help us to understand the structural changes which accompanied the development of crustacean clotting. Another benefit of a more complete understanding of the clotting process in crustaceans, which is part of their immune response, is that it may aid us in keeping them healthier. Since crustaceans are a major agricultural product, especially the shrimp farms of Southeast Asia, efforts at maintaining their health are of great importance. |
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