Fluorescence and Thermodynamics Studies with Proteins

拟议的研究计划涉及有关蛋白质中展开转变和特定配体与蛋白质相互作用的基本问题。这项工作将集中在两种蛋白质，核酸酶A和trp载前蛋白，以及这些蛋白质的某些位点定向突变体。埃夫丁克博士将研究相对不稳定的核酸酶突变体的冷（以及高温）展开和压力展开。目的是为了更好地理解稳定蛋白质的力量，评估和关联几种核酸酶突变体展开的各种热力学参数。各种展开状态（即由低温和高温、高压、尿素、低pH值产生）的“结构”和动态特性将通过几种技术的组合来研究，包括i)时间分辨荧光各向异性来测量局部运动（单个色氨酸残基）；ii)位点之间的共振能量转移，以获得分子内距离信息，iii)尺寸排斥色谱，以测量分子的整体水动力半径，iv) CD观察二级结构的差异。除了比较各种未展开的状态外，不稳定突变对蛋白质折叠状态的影响将通过上述一些方法进行研究，并通过室温磷光来比较trp残基环境的刚性。6)滴定微热法测量特定配体与蛋白质结合的热力学参数。

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.