MRI: Acquisition of Autosampling Stopped-Flow Spectrometer for in vitro Kinetic Characterization of Biomolecule Binding and Enzymatic Activity

凭借化学专业研究仪器计划的这一奖项，宾夕法尼亚大学的Ernest Petersson教授和同事Tobias Baumgart将为宾夕法尼亚大学，德雷克塞尔大学，坦普尔大学，托马斯杰斐逊大学和斯沃斯莫尔学院的用户购买自动采样停流光谱仪。该提案旨在加强各级的研究和教育，特别是在以下领域：（a）硫代酰胺淬灭探针的开发;（B）蛋白质/膜相互作用的研究;（c）协同蛋白质-纳米颗粒组装体组装动力学的研究;（d）探测和控制RNA三级结构的小分子;（e）确定核糖体上程序化移码的动力学;（f）核蛋白质-纳米颗粒组装体的研究;（g）核蛋白质-纳米颗粒组装体的研究（f）研究内在无序蛋白质的折叠机制;（g）对活化诱导的脱氨酶进行酶学研究;（h）深入了解第二类tRNA核苷酸转移酶（CCA）加成的研究;（i）乳清酸磷酸核糖基转移酶的机制研究;和（j）纤维素酶的机理研究。停流系统允许样品的快速混合，从而允许获得真实的时间UV/UV。具有毫秒级时间分辨率的可见光吸收和荧光数据，用于研究分子与蛋白质和核酸结合或酶活性的化学动力学，同时研究光谱的变化。一般而言，在停流光谱仪中，通过打开阀门然后注入酶和底物来启动酶促反应。通过酶、底物和停止注射器柱塞的作用控制混合量。通过激发单色仪以一个波长照射混合溶液，并使用单独的滤波器通道同时检测吸光度（一个波长）和荧光（两个波长）输出。然后，数据由计算机处理，并拟合到特定于反应的动力学模型。通过停流仪器实现的生物分子的动力学表征是理解其功能的基础。由于大多数细胞过程发生在远离平衡的地方，因此它们受酶的相对速率的支配。仪器将设在生物化学设施内，供各机构的大量研究人员和学生使用。

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.