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

宾夕法尼亚大学的欧内斯特·彼得森教授和他的同事Tobias Baumgart获得了化学专业研究仪器项目颁发的这一奖项，他们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一台自动采样停流光谱仪。该建议旨在加强各级的研究和教育，特别是在下列领域：(A)硫代酰胺猝灭探针的开发；(B)蛋白质/膜相互作用的研究；(C)协同蛋白质-纳米颗粒组装的组装动力学的研究；(D)用于探测和控制RNA三级结构的小分子；(E)确定核糖体上程序性框架移动的动力学；(F)研究固有无序蛋白质的折叠机制；(G)激活诱导脱氨酶的酶学研究；(H)深入了解第二类tRNA核苷酸转移酶(CCA)加成的研究；(I)轮转磷酸核糖转移酶机理的研究；和(J)纤维素酶的机理研究。停流系统允许快速混合样品，允许获取毫秒时间分辨率的实时UV/可见光吸光度和荧光数据，用于研究与蛋白质和核酸或酶活性结合的分子的化学动力学，并同时研究光谱的变化。一般来说，在停流光谱仪中，酶反应是通过打开阀门，然后注入酶和底物来启动的。混合量由酶、底物和止动注射器柱塞的作用控制。混合溶液通过激发单色仪以一个波长照射，并使用单独的滤光片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，并符合特定于反应的动力学模型。由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。因为大多数细胞过程远离平衡，所以它们受酶的相对速率的控制。该仪器将设在生物化学设施中，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.