SUMMER RESEARCH TRAINING IN MEMBRANE SCIENCE AND TECHNOLOGY 2008 NSF REU SITE PROGRAM at the UNIVERSITY OF CINCINNATI
The Department of Pharmacology & Cell Biophysics, College of Medicine is pleased to offer this research project as part of the 2008 summer NSF-REU Site Program administered by the Department of Pharmacology & Cell Biophysics.  Students interested in this project are urged to contact Professor Kirley to discover more about the project, learn what your responsibilities will be during the ten-week research training program.
Project #:  08 - 013 Faculty Supervisor/Mentor: Terence L. Kirley, Ph.D., Professor, Pharmacology & Cell Biophysics College of Medicine Email:  terry.kirley@uc.edu
Molecular Dynamics of a Bio-Membrane Protein: Uncoupling Transmembrane Domain from Active Site Movements in an Ecto-Nucleotidase
General background and significance of the project: Bio-membrane embedded receptor proteins for extracellular nucleotides (purinergic and pyrimidinergic receptors) occur in many parts of the body.  These receptors perform many different physiological functions, and respond to different types of nucleotides (e.g., ATP vs. UTP), as well as to different phosphorylation states of each nucleotide (e.g., ATP vs. ADP vs. AMP vs. adenosine). The ecto-nucleotidases are important for the regulation of the many physiological and pathological signaling processes under purinergic control, including pain perception and maintenance of hemostasis via hydrolysis of the platelet activator, ADP. Ecto-nucleotidases have also been implicated to be important for cell adhesion, new blood vessel growth (angiogenesis), and cancer development.  The activity of these integral membrane protein enzymes is modulated by characteristics of their transmembrane domains and by changes in the properties of the cell membrane in which they are embedded.
Brief description of proposed research and activities for the 10-week REU period: The REU student will learn and utilize techniques of site-directed mutagenesis and recombinant protein expression in mammalian cells.  Experiments will be designed to test hypotheses developed in our laboratory as to how the movements known to occur in the transmembrane helices of the ecto-nucleotidases are functionally coupled to the predicted physical movements in extracellular lobes of the active site of the enzyme at the molecular level that are believed to be essential for its activity.  Enzyme assays, Western blots, proteolytic cleavage and chemical cross-linking experiments will be devised and performed to examine the hypothesis that certain amino acid residues located near the interface of the transmembrane domain and the extracellular region are important for enzyme activity and biological function via coupling of the movements in the transmembrane regions to the extracellular active site.
What the REU Student can gain from participating in this project: The REU student will learn and apply basic biochemical and molecular biology techniques to molecules integrated within a bio-membrane.  The REU student will also be expected to be actively engaged in experimental design and critical reading of scientific literature. The REU student will be challenged to independently design additional experiments, and will learn from, and interact with, other members of the Kirley laboratory working on related projects. The REU student will be expected to participate in laboratory group meetings and presentations, and interact with Dr. Kirley and other laboratory personnel daily. The REU student will be guided in scientific presentation and writing skills and will have the opportunity through significant research contributions to earn co-authorship when the studies are published.