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

MRI：获取自动采样停流光谱仪，用于生物分子结合和酶活性的体外动力学表征

• 批准号: 1337449
• 负责人: Ernest Petersson
• 金额: $ 11.03万
• 依托单位: University of Pennsylvania
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2016-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1337449&HistoricalAwards=false
• 关键词: MRI; Acquisition; Autosampling; Stopped; Flow

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.

通过化学主要研究仪器计划的奖项，宾夕法尼亚大学的欧内斯特·彼得森教授和同事托比亚斯·鲍姆加特（Tobias Baumgart）将为U Penn，DREXEL大学，Temple University，Temple University，Thomas Jefferson University，Thomas Jefferson University和Swarthmore College的用户购买自动采样的停止流量光谱仪。该提案旨在增强各个层次的研究和教育，尤其是在（a）硫酰胺淬火探针的开发等领域； （b）研究蛋白质/膜相互作用； （c）研究协同蛋白 - 纳米颗粒组件的组装动力学； （d）小分子以探测和控制RNA三级结构； （e）确定核糖体上编程的框架移动的动力学； （f）研究本质上无序蛋白的折叠机制； （g）激活诱导的脱氨酶的酶学研究； （h）研究以深入了解II类TRNA核苷酸转移酶（CCA）的研究； （i）研究口磷酸磷酸糖基转移酶的机理； 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.通常，在停止的流量光谱仪中，酶促反​​应是通过打开瓣膜开始的，然后注射酶和底物。混合的数量由酶，底物和停止注射器柱的作用控制。通过激发单色器在一个波长下辐照混合溶液，并使用单独的滤波器通道同时检测到吸光度（一个波长）和荧光（两个波长）输出。然后通过计算机处理数据，并适合于反应特定的动力学模型。通过停止流量仪器启用生物分子的动力学表征对于理解其功能至关重要。由于大多数细胞过程都远离平衡发生，因此它们受酶的相对速率控制。该仪器将位于生物化学设施中，并将被各种机构的大量研究人员和学生使用。

项目成果

Improving the fluorescent probe acridonylalanine through a combination of theory and experiment

理论与实验相结合改进荧光探针吖啶丙氨酸

• DOI: 10.1002/poc.3813
• 发表时间: 2018
• 期刊: Journal of Physical Organic Chemistry
• 影响因子: 1.8
• 作者: Sungwienwong, Itthipol;Ferrie, John J.;Jun, Joomyung V.;Liu, Chunxiao;Barrett, Taylor M.;Hostetler, Zachary M.;Ieda, Naoya;Hendricks, Amara;Muthusamy, Anand K.;Kohli, Rahul M.
• 通讯作者: Kohli, Rahul M.

Fluorescent Probes for Studying Thioamide Positional Effects on Proteolysis Reveal Insight into Resistance to Cysteine Proteases

• DOI: 10.1002/cbic.201900115
• 发表时间: 2019-08-16
• 期刊: CHEMBIOCHEM
• 影响因子: 3.2
• 作者: Liu, Chunxiao;Barrett, Taylor M.;Petersson, E. James
• 通讯作者: Petersson, E. James