Fluorescence and Thermodynamics Studies with Proteins

蛋白质的荧光和热力学研究

• 批准号: 9106377
• 负责人: Maurice Eftink
• 金额: $ 33.99万
• 依托单位: University of Mississippi
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1995-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9106377&HistoricalAwards=false
• 关键词: Fluorescence; Thermodynamics; Studies; Proteins

The proposed research program deals with fundamental questions regarding unfolding transitions in proteins and the interaction of specific ligands with proteins. The work will focus on two proteins, nuclease A and trp aporepressor, and certain site- directed mutants of these proteins. Dr. Eftink will study the cold (as well as high temperature) unfolding and pressure unfolding of relatively unstable mutants of nuclease. The goal is to evaluate and correlate various thermodynamic parameters for the unfolding of several mutants of nuclease in order to better understand the forces responsible for stabilizing proteins. The "structure" and dynamic characteristics of the various unfolding states (i.e., produced by low and high temperature, pressure, urea, low pH) will be studied by a combination of several techniques, including i) time-resolved fluorescence anisotropy to measure local motion (of the single trp residue), ii) resonance energy transfer between sites to obtain intramolecular distance information, iii) size exclusion chromatography to measure the overall hydrodynamic radius of the molecule, and iv) CD to observe any differences ins secondary structure. In addition to comparing the various unfolded states, the effect of the destabilizing mutations on the folded state of the proteins will be studied by some of the above methods and by v) room temperature phosphorescence to compare the rigidity of the environment of the trp residue, and vi) titration microcalorimetry to measure thermodynamic parameters for the binding of specific ligands to the protein.

拟议的研究计划涉及基本问题 关于蛋白质中的解折叠转变和 与蛋白质的特异性配体。 工作将集中在两个方面 蛋白质，核酸酶A和trp辅基阻遏物，以及某些位点- 这些蛋白质的定向突变体。 Eftink博士将研究 冷（以及高温）展开和压力 相对不稳定的核酸酶突变体的解折叠。 目标 是评估和关联各种热力学参数 用于核酸酶的几种突变体的解折叠， 更好地理解稳定的力量 proteins. 的“结构”和动态特性， 各种展开状态（即，生产的低和高 温度、压力、尿素、低pH值）将通过 几种技术的组合，包括i）时间分辨 荧光各向异性，以测量局部运动（单个 色氨酸残基），ii）位点之间的共振能量转移， 获得分子内距离信息，iii）尺寸排阻 色谱法来测量总的流体动力学半径 iv）CD以观察分子中的任何差异， 二级结构。 除了比较各种 在未折叠状态下，不稳定突变对 蛋白质的折叠状态将由一些 和通过v）室温磷光， 比较色氨酸残基环境的刚性，和 vi）滴定微量热法以测量热力学 特定配体与蛋白质结合的参数。