Fluorescence and Thermodynamics Studies with Proteins

拟议的研究计划涉及与蛋白质的展开转变和特定配体与蛋白质的相互作用有关的基本问题。这项工作将集中在两种蛋白质，核酸酶A和色氨酸抑制物，以及这些蛋白质的某些定点突变。Eftink博士将研究相对不稳定的核酸酶突变体的低温(以及高温)展开和压力展开。目的是评估和关联几个核酸酶突变体的展开的各种热力学参数，以便更好地了解负责稳定蛋白质的力。将结合几种技术来研究各种展开状态(即，由低温和高温、压力、尿素、低pH产生)的“结构”和动态特性，包括：i)时间分辨荧光各向异性以测量(单个Trp残基)的局部运动，ii)位置之间的共振能量转移以获得分子内距离信息，Iii)尺寸排除层析测量分子的整体流体动力学半径，以及iv)CD观察二级结构中的任何差异。除了比较不同的未折叠状态外，还将用上述方法中的一些方法和v)室温磷光比较Trp残基的环境刚性，以及vi)滴定微量热法测量特定配体与蛋白质结合的热力学参数来研究不稳定突变对蛋白质折叠状态的影响。

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.