INTRINSIC FLUORESCENCE OF PROTEINS

固有蛋白质吸光度和荧光的限制是 不能区分特定的蛋白质或多肽与 光谱相似分子的背景。我们已经证明了一个 大肠杆菌色氨酸营养缺陷体能利用5-羟色氨酸(5-OHTrp) 在体内取代色氨酸，产生一种光谱增强的蛋白质 (9月)。这种代谢工程蛋白，噬菌体lambda Ci 抑制子，在功能上与野生型蛋白没有区别 在蛋白质-蛋白质相互作用和协同结合方面 操作者DNA。用这种SEP研究DNA对自组装的影响 我们已经证明了高阶低聚单位是一个 能够与操作者DNA结合的八聚体。因此，目前- 公认的细胞发育控制机制假说 这种重要的DNA结合蛋白对外界信号的反应 需要重新评估。 这项提案的第一个目的是调查 Lambda功能中的蛋白质-蛋白质和蛋白质-DNA相互作用 CI抑制者。我们将使用野生型和突变型抑制子，包含 色氨酸或色氨酸类似物，如5-OHTrp，以产生SEP。 将使用N-末端研究特定的DNA相互作用 已知其X射线晶体结构的阻遏因子的结构域。 合作互动将使用完整长度的 抑制者。RNA聚合酶-lambda Ci抑制子的相互作用也将 接受检查。 将使用多学科方法。同聚体和异构体 蛋白质-DNA组装将用荧光共振能量进行研究 转移、荧光各向异性和超速离心法。足迹 分析将被用来检查DNA结合的热力学 全长抑制物，而荧光法将评估结合 通过N-端结构域构建。数据解释与分析 特定的N-末端结构域突变，包括SEP，将得到协助 通过分子建模。 这项建议的第二个目的是继续研究 芳香族氨基酸的光物理，并将这一知识应用于 特定蛋白质系统的特征，如lambda Ci 抑制者。我们将研究分子的荧光强度衰变。 芳香族氨基酸和色氨酸类似物用于SEP 模型化合物、小肽和较大多肽的上下文 已知的三级结构，包括N端结构域 抑制者。假设，包括侧链旋转体模型，将是 根据数据、结构和光物理进行评估。核磁共振 实验将提供有关侧链的独立信息 构象和种群。这些研究将使我们能够充分 解释在抑制物及其SEP类似物上获得的数据(目的 1)。

A limitation of intrinsic protein absorbance and fluorescence is the inability to distinguish a specific protein or polypeptide against a background of spectrally similar molecules. We have shown that a tryptophan auxotroph of E coli can utilize 5-hydroxytryptophan (5-OHTrp) to replace tryptophan in vivo, generating a spectrally enhanced protein (SEP). This metabolically engineered protein, bacteriophage lambda cI repressor, is functionally indistinguishable from wild-type protein both in terms of protein-protein interactions and cooperative binding to operator DNA. Using this SEP to study the effects of DNA on self-assembly of repressor, we have shown that the high order oligomeric unit is an octamer that is competent to bind operator DNA. Thus, the currently - accepted hypothesis for the mechanism of control of cellular development by this important DNA-binding protein in response to external signals needs to be re-evaluated. The first aim of this proposal is to investigate the role of protein-protein and protein-DNA interactions in the function of lambda cI repressor. We will use wild-type and mutant repressor, containing tryptophan or tryptophan analogues, such as 5-OHTrp, to generate SEPS. Specific DNA interactions will be investigated using the N-terminal domain of the repressor for which an x-ray crystal structure is known. Cooperative interactions will be investigated using full-length repressor. RNA polymerase - lambda cI repressor interactions will also be examined. A multi-disciplinary approach will be used. Homomeric and heteromeric protein-DNA assemblies will be studied by fluorescence resonance energy transfer, fluorescence anisotropy, and ultracentrifugation. Footprint analysis will be used to examine the thermodynamics of DNA binding by full-length repressors, while fluorescence methods will assess binding by N-terminal domain constructs. Data interpretation and analysis of specific N-terminal domain mutations, including SEPs, will be assisted by molecular modeling. The second aim of this proposal is to continue our studies on the photophysics of the aromatic amino acids and to apply this knowledge to the characterization of specific protein systems, such as lambda cI repressor. We will study the fluorescence intensity decay of the aromatic amino acids and the tryptophan analogues used for SEPs in the context of model compounds, small peptides, and larger polypeptides of known tertiary structure including the N-terminal domain of the repressor. Hypotheses, including the side chain rotamer model, will be evaluated with respect to the data, structure, and photophysics. NMR experiments will provide independent information about side chain conformations and populations. These studies will permit us to fully interpret the data obtained on the repressor and its SEP analogues (aim 1).