An award is made to St. Mary's University in San Antonio TX to purchase a fluorometer for the use of at least eight faculty members in the School of Science, Engineering, and Technology. Fluorometry, the measurement of light emission from fluorescent chemicals, is versatile and very sensitive, so it can be used in multiple fields. The eight faculty members will use the instrument in research that varies from chemosensor design, to detection of genetic transcription control elements, to the design of quantum dots for use in LEDs, to water-quality studies in natural aquifers. It will also be used to describe protein-protein interactions and protein dynamics in studies of enzymes.
The faculty members using the fluorometer in their research engage exclusively with undergraduate students in their projects, and since St. Mary's University is a Hispanic Serving Institution, the instrument will be used by undergraduate students from an underserved minority. The exposure of students to this high-quality instrument will expand their experimental, verbal and analytical skills, and enable them to perform meaningful research. Students will make discoveries that will be disseminated through publications in reviewed journals, and they will attend research conferences to discuss their findings. Since the projects to be performed are so varied, the instrument will impact students in various majors, from biophysics to organic chemistry to cell biology. The water quality studies have the potential to benefit users of the Edwards Aquifer, which serves San Antonio and surrounding communities. The instrument will be shared with students at neighboring universities.
Report for the Public on NSF MRI #7353674 Report from Dr. S. C. Daubner, PI During the summer of 2014 experiments were initiated on the binding strength of tyrosine hydroxylase (TyrH), the enzyme that controls levels of dopamine and epinephrine, to a putative activator protein named 14-3-3. 14-3-3 proteins bind to many phosphorylated target proteins and either activates them or inhibits them. Binding of 14-3-3 to TyrH is complex since TyrH is phosphorylated not once but at three positions, and is a tetramer, so actually is phosphorylated up to 12 times. Further there are seven different isoforms (identified by Greek letters) and it is not known whether one or more of them is specific for TyrH. To study the binding between the two proteins we optimized a protocol for tagging the 14-3-3 with a fluorescent molecule, DANSYL-Cl. The Horiba Fluorolog Fluorometer was used to measure the relative size of the tagged 14-3-3 protein in the presence and absence of TyrH. Polarization anistotropy is the technique we employed; this technique follows whether the protein "tumbles" more or less in the presence of the binding partner. Preliminary data suggest that the 14-3-3 protein is hindered in tumbling when TyrH phosphorylated at two positions (eight in the tetramer) is added to the mixture. Anisotropy measurements for 1 µM dansylated 14-3-3 protein zeta is about 0.2, and additions of TyrH phosphorylated at serine19 and serine40 (1, 2, 4, and 8 µM) cause that value to increase to 0.24. The Horiba fluorometer provides very sensitive and reproducible data. These early experiments show promise that actual equilibrium binding constants will be obtained for the interactions of the two proteins. In the summer of 2014 one undergraduate in particular was occupied with these experiments. Since the academic year began that has expanded to three students. One of them is an international student from Honduras and the other two are Hispanic Americans, fulfilling our goal of introducing minority students to research. The two Americans are both funded by scholarships which require them to carry out research projects. The Honduran, Eduardo Cruz, has already presented his summer work at local research symposia, once via oral presentation and once with a poster. He and Kevin De La Cruz will take their findings to the Enzyme Mechanisms Meeting in Galveston in January 2015. Report from Dr. S. P. Oxley, Co-PI Fluorescence chemosensors are based on the change in the fluorescence properties of a probe molecule upon the selective binding of a target molecule or ion. The design challenge for the development of fluorescence chemosensors is the generation of active sites that have both a high affinity and high selectivity for the target species. Dye 1 was synthesized and its structure confirmed by FTIR and NMR spectroscopy. Its fluorescent properties were studied under a different solvents, mixtures of organic solvents and water, and in the presence of various metal ions. 1 contains ethylamine groups that are known to bind metal ions. Ca2+ was added to a solution of 80/20 acetone/water containing 1. At low 1 concentration (< 1 mM), a linear increase in fluorescence intensity was observed with increasing [Ca2+]. At high 1 concentration, precipitation was observed with increasing [Ca2+]. Addition of Cu2+, Hg2+, and Fe2+ resulted in a decrease in fluorescence intensity in a 50/50 methanol/water mixture, while Zn2+, Na2+, and K+ showed minimal change in fluorescent intensity. While addition of Cu2+, Hg2+ and Fe2+ reduced fluorescence, a color change from yellow to orange was observed, confirmed by the appearance of an absorbance band at 500 nm. Continuing work includes concentration studies with the above metal ions using both fluorescence and absorbance spectroscopy. Further, two related compounds were synthesized in which the ethylamine group was replaced by a methyl group or a crown ether group. Their fluorescence properties were examined in solvent mixtures as a comparison to 1. The Horiba Scientific FluoroLog Spectrophotometer purchased with funding from NSF MRI 1337392 was used on an almost daily basis, approximately 6 hours a day, for a 10-week summer research session. It continues to be used on a weekly basis during the fall 2014 semester. 1 has a quantum yield of 0.0008.1 The high sensitivity of the FluoroLog Spectrophotometer allowed for both high wavelength resolution and fluorescence intensity for this weakly fluorescent compound. The experiments described above were performed by Jenny Diaz, a rising senior B.S. Chemistry major at St. Mary’s University. She presented her work as a poster at the St. Mary’s University Summer Research Symposium and as an oral presentation at the St. Mary’s University Cimadevilla Lecture Series. She will be presenting a poster at the American Chemical Society Southwest Regional Meeting in October 2014. The project is continuing with 3 students, a senior B.S. Chemistry major, a senior B.S. Biology major, and a freshman Biochemistry major.
