Steady-State and Stopped-flow Spectropolarimeter at NCSU

描述(由申请人提供)：三名NCSU科学家在蛋白质-蛋白质或蛋白质-核酸相互作用是主要成分的领域工作，建议购买能够进行稳态和停流圆二色(CD)研究的光谱偏振仪。该仪器由应用光物理学公司制造，旨在替换和升级1989年购买的现有和过时的Jasco CD。现有的乐器仍然是NCSU校园内唯一的CD乐器，校园内的几个教员大量使用它。然而，为了获得足够的数据，将仪器保持在工作状态变得越来越困难。现有的仪器在200 nm以下无法提供可靠的数据，温度控制差，软件和计算机陈旧，没有停流或滴定能力。在过去的三年里，该仪器需要数千美元的维修费用才能继续运行。拟议的仪器将使研究蛋白质和核酸构象成为可能，并提供有关蛋白质和/或核酸稳定性以及蛋白质折叠机制的信息。结合谱的测量将允许确定结合常数和化学计量，将获得对圆二向色性参数的热依赖关系，并将测量动力学反应。该仪器将为研究人员目前的实验方法提供便利，包括核磁共振光谱、荧光光谱、X射线结晶学、差示扫描和等温滴定热法、圆二色谱、质谱仪等生化和生物物理技术。稳态和停流光谱旋光仪将补充美国国立卫生研究院资助的结构和功能研究，重点是了解半胱氨酸天冬氨酸酶的折叠和组装、参与细菌反应和保护的过渡状态调节蛋白，以及修饰的核苷对RNA和RNA：肽和RNA：金属相互作用的影响。

DESCRIPTION (provided by applicant): Three NCSU scientists working in fields in which protein-protein or protein-nucleic acids interactions are a primary component, propose to purchase a spectropolarimeter capable of steady-state and stopped-flow circular dichroism (CD) studies. The instrument, manufactured by Applied Photophysics, is intended as a replacement and upgrade for an existing and outdated Jasco CD purchased in 1989. The existing instrument remains the only CD instrument on the NCSU campus and is used heavily by several faculty across the campus. However, it becomes increasingly difficult to maintain the instrument in working condition in order to acquire adequate data. The existing instrument will not provide reliable data below 200 nm, the temperature control is poor, the software and computer are antiquated, and it does not have stopped-flow or titration capabilities. In the past three years, the instrument has required several thousand dollars in repair costs to remain functional. The proposed instrument will enable the study of protein and nucleic acid conformations and provide information on protein and/or nucleic acid stability as well as protein folding mechanisms. Measurements of binding profiles will allow the determination of binding constants and stoichiometries, thermal dependence to circular dichroism parameters will be obtained, and kinetic reactions will be measured. The instrument will facilitate the current experimental approaches of the investigators which include NMR spectroscopy, fluorescence spectroscopy, X-ray crystallography, differential scanning and isothermal titration calorimetry, circular dichroism, mass spectrometry, and other biochemical and biophysical techniques. The steady-state and stopped-flow spectropolarimeter will complement NIH funded structural and functional studies focused on understanding the folding and assembly of caspases, transition state regulator proteins involved in bacterial response and protection, and characterization of modified nucleoside contributions to RNA and to RNA:peptide and RNA:metal interactions.