42166-B4
Change in Phospholipase D Solvent Accessibility Due to Ligand Binding: A Study by Hydrogen-Deuterium Exchange and Mass Spectrometry
Previous work in our laboratory has characterized two commercially available PLD enzymes from different strains of the bacterium Streptomyces, a calcium dependent form (PLD+Ca) and a calcium independent form (PLD-Ca). While these two enzymes both carry out the same two reactions, their physical characteristics appear to be quite different suggesting they may utilize different catalytic mechanisms. This project will allow us to test for ligand interactions on the surface of each enzyme, to test suppositions found in the literature, and provide physical support for a catalytic mechanism. It will be interesting to discover if these two enzymes utilize different catalytic mechanisms.
Work to date has shown that both enzymes are not sold in sufficient purity to be used directly in the next stage of our work, sequencing of each enzyme. Both samples contain multiple protein impurities, some of which may be due to PLD degradation. During the last year we have been able to develop methods to purify the PLD+Ca using anion exchange FPLC, obtaining near baseline separation. Analysis of the FPLC fractions by silver stained SDS-PAGE demonstrated that the PLD+Ca has been purified to near homogeneity. Work continues on trying to improve the FPLC methods to advance and complete the purification process especially for the PLD-Ca, and efforts also continue to stabilize both enzymes against degradation.
The undergraduate students whom participated in this project have developed a set of hands on skills and critical experience in biochemical research. They had an opportunity to develop a feeling for the limitations of data, for the pitfalls of statistics, and for the creation of artifacts. They have tackled: learning how to run and maintain complex instruments, how to read and understand the biochemical literature, and how to organize and present their work both orally and in writing to the faculty and their peers. Work on the project has developed a sense of confidence and professionalism in their skills, which is evidenced by their postgraduate successes.
Over the course of this grant six undergraduates have been supported, four have graduated with Bachelors of Science degrees in either chemistry or biochemistry, with four students having been from demographic groups underrepresented in chemistry. Two graduates are now working as chemistry technicians, while one is in medical school and the other is teaching high school science. The two remaining students anticipate graduating in 2009 and 2010, with Bachelors of Science degrees in biochemistry. One already has a part time job as a chemistry technician, which he plans to continue full time upon graduation, while the other student is considering attending graduate school in chemistry upon graduation in 2010.
