With this award from the Chemistry Major Research Instrumentation Program, Professor Ernest Petersson from the University of Pennsylvania and colleague Tobias Baumgart will acquire an autosampling stopped-flow spectrometer for users at U Penn, Drexel University, Temple University, Thomas Jefferson University and Swarthmore College. The proposal is aimed at enhancing research and education at all levels, especially in areas such as (a) development of thioamide quenching probes; (b) study of protein/membrane interactions; (c) investigation of assembly kinetics of synergistic protein-nanoparticle assemblies; (d) small molecules to probe and control RNA tertiary structure; (e) determining the dynamics of programmed frame-shifting on the ribosome; (f) study of the folding mechanism of intrinsically disordered proteins; (g) enzymological studies of activation-induced deaminases; (h) studies to provide insight into class II tRNA nucleotidyltransferase (CCA)-addition; (i) study of the mechanism of orotate phosphoribosyltransferase; and (j) mechanistic studies of cellulase enzymes.
The stop-flow system allows rapid mixing of samples allowing the acquisition of real time UV/visible absorbance and fluorescence data with millisecond time resolution for the study of chemical kinetics of molecules binding to proteins and nucleic acids or enzyme activities and simultaneously studies the changes in the spectra. In general, in a stopped flow spectrometer the enzymatic reaction is initiated by opening of valves followed by injection of the enzyme and substrate. The quantities mixed are controlled by the action of the enzyme, substrate, and stop syringe plungers. The mixed solution is irradiated at one wavelength through an excitation monochromator, and absorbance (one wavelength) and fluorescence (two wavelengths) outputs are simultaneously detected using separate filter channels. The data are then processed by a computer and fit to kinetic models specific to the reaction. The kinetic characterization of biomolecules enabled by stopped flow instrumentation is fundamental to understanding their function. Because most cellular processes take place far from equilibrium they are thus governed by the relative rates of enzymes. The instrumentation will be located in the Biological Chemistry Facility and will be used by a large number of researchers and students in various institutions.