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

宾夕法尼亚大学的Ernest Petersson教授和他的同事Tobias Baumgart将获得化学主要研究仪器项目的这一奖项，为宾夕法尼亚大学、德雷塞尔大学、天普大学、托马斯杰斐逊大学和斯沃斯莫尔学院的用户购买一台自动采样止流光谱仪。该提案旨在加强各级的研究和教育，特别是在以下领域：(a)开发硫酰胺淬火探针；(b)蛋白质/膜相互作用的研究；(c)协同蛋白质-纳米颗粒组装动力学的研究；(d)小分子，探测和控制RNA三级结构；(e)确定核糖体的程序化帧移动力学；(f)内在无序蛋白折叠机制的研究；(g)活化诱导脱氨酶的酶学研究；(h)研究II类tRNA核苷酸转移酶（CCA）加成；(1)羊角酸磷酸核糖基转移酶的机理研究；(j)纤维素酶的机理研究。停止流动系统允许样品的快速混合，允许以毫秒时间分辨率获取实时紫外/可见吸光度和荧光数据，用于研究分子与蛋白质和核酸或酶活性结合的化学动力学，同时研究光谱的变化。一般来说，在止流光谱仪中，酶促反应是通过打开阀门，然后注射酶和底物来开始的。混合量由酶、底物和停止注射器柱塞的作用控制。混合溶液通过激发单色仪在一个波长照射，吸光度（一个波长）和荧光（两个波长）输出使用单独的滤光器通道同时检测。然后用计算机处理这些数据，使其符合特定于反应的动力学模型。通过停止流动仪器对生物分子进行动力学表征是了解其功能的基础。因为大多数细胞过程发生在远离平衡状态的地方，所以它们是由酶的相对速率控制的。该仪器将位于生物化学设施，并将由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.