A critical number of investigators at the Kimmel Cancer Center of Thomas Jefferson University have a number of pending projects that require a quantitative method for measuring the quaternary structure of biological samples and their association constants. To this end, we propose to acquire the Beckman Optima XL-I analytical ultracentrifuge. This instrument is well suited for these projects and significantly extends the capabilities of our molecular interaction core facility. Specific areas that we will immediately address using this instrumentation are: 1) Thermodynamic characterization of multimeric dynein and dynactin complexes in relation to the regulation of dynein activity and cargo binding; 2) To study the oligomerization state and hydrodynamic properties of peptide segments derived from viral glycoproteins involved in membrane fusion; 3) tRNA-protein complexes and the mechanism of decoding genetic information; 4) Characterization of Protein-Protein Interaction Antagonists; 5) Characterize conformational changes induced by activation of arrestins, a family of proteins that regulate G protein-coupled receptor signaling; 6) Characterization of the equilibrium binding constants and the stochiometry of the ectodomains of the TCR, the pMHC and the CD8. In addition, the instrument will enhance two additional core facilities (X-ray and small molecule screening facilities) as well as graduate education (PR613, a graduate course on structural biology, is structured such that students apply each technique taught to an actual sample). The analytical ultracentrifuge of choice is the Beckman Optima XL-I. This instrument permits detection using either interference optics or UV/visible optics. The interference mode is sensitive to the refractive index, and thus, does not require the analyte to have a natural chromaphor. Thus, this will permit measurement of samples with low molecular extinction coefficients, small molecule antagonist and membrane bound proteins. The interference optics also provides rapid data collection useful for producing high quality data for sedmentation velocity analysis. On the other hand, the ability to measure absorbance over a large range (190 to 800 nM) allows simultaneous measurement of individual components in a multicomponent system. ? ? ?
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