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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 significantly purify each PLD. Using anion exchange chromatography and step gradients with our newly acquired FPLC we were able to obtain near baseline separations. The higher signal to noise ratio, improved reproducibility, and improved reliability of the FPLC greatly aided our method development. Work continues on trying to improve these methods to advance and complete the purification process.
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 critical for their future success.
Two students involved in this work during the summer of 2006, graduated in 2007 with degrees in biochemistry and chemistry respectively. One student, after finishing his chemistry degree, obtained his teaching credential and is now teaching high school science. The two students who worked with me this last summer anticipate graduating in 2008, with degrees in biochemistry and chemistry, respectively. One plans to attend graduate school in chemical engineering while the other plans to attend graduate school in chemistry.
