Partial Protein Unfolding as a Novel Signaling Mechanism

部分蛋白质折叠作为一种新型信号机制

• 批准号: 6607120
• 负责人: WOUTER D HOFF
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6607120
• 关键词: acidity /alkalinity; active sites; bacterial proteins; biological signal transduction; calorimetry; chemical kinetics; chromophore; circular dichroism; conformation; crystallization; electrospray ionization mass spectrometry; flash photolysis; infrared spectrometry; interferometry; nonvisual photoreceptor; nuclear magnetic resonance spectroscopy; phase change; photoactivation; protein folding; protein structure function; protonation; radiotracer; site directed mutagenesis; solvents; structural biology; thermodynamics

Receptor activation is at the basis of all biological signal transduction events. Malfunctioning of signaling pathways can cause cancer. We study the mechanism of receptor activation using photoactive yellow protein (PYP), a PAS domain photoreceptor, as a powerful model system. PYP exhibits rhodopsin-like photochemistry based on its p-coumaric acid (pCA) chromophore, and has been investigated by X-ray crystallography. We have recently shown that the presence of a protein crystal lattice greatly reduces the structural changes that occur during the formation of the pB photocycle intermediate, the presumed signaling state of PYP. In addition, our preliminary results strongly indicate that the pB intermediate is partially unfolded. In this proposal we will examine the role of partial protein unfolding upon receptor activation as a novel signal transduction mechanism. (i) We will establish the extent of partial protein unfolding during pB formation by NMR spectroscopy, and by two novel methods: light-induced H/D exchange and light-induced differential scanning calorimetry. In addition, we will examine the hypothesis that the pB intermediate is a molten globule state by studying changes in ANS fluorescence, changes in CD spectrum, and changes in the radius of gyration upon pB formation. (ii) We will examine the effects of the presence of a crystal lattice on the kinetics and thermodynamics of the photocycle using both wt- PYP and the E46Q mutant in both P6 3 and P6 5 crystals by flash photolysis at a range of temperatures. (iii) We will test the hypothesis that the Glu46-pCA couple constitutes the built-in default for the light-triggered protein quake in PYP. These experiments will reveal changes in functional dynamics imposed by a crystal lattice, and will determine the role of transient partial protein unfolding as a novel signal transduction mechanism that is anticipated to play a role not only in PYP but also in other signal transduction systems.

受体激活是所有生物信号转导事件的基础。 信号通路故障可能会导致癌症。 我们使用光活性黄蛋白（PYP）（PAS结构域光感受器）作为强大的模型系统研究受体激活的机制。 PYP基于其p-美乳酸（PCA）发色团表现出类似的光蛋白样光化学，并已通过X射线晶体学研究。我们最近表明，蛋白质晶格的存在大大降低了在Pb光循环中间体形成期间发生的结构变化，即PYP的假定信号传导状态。 此外，我们的初步结果强烈表明PB中间体是部分展开的。在此提案中，我们将研究部分蛋白在受体激活中的作用作为一种新型的信号转导机制。 （i）我们将通过NMR光谱法建立PB形成过程中部分蛋白的程度，并通过两种新颖的方法：光诱导的H/D交换和光诱导的差异扫描量热法。 此外，我们将通过研究ANS荧光的变化，CD频谱的变化以及PB形成后回旋半径的变化来研究以下假设。 （ii）我们将使用WT-PYP和E46Q突变体在P6 3和P6 5晶体中通过闪光照射在一系列温度下使用WT-PYP和E46Q突变体的光循环的动力学研究对光循环的动力学的影响。 （iii）我们将检验以下假设：GLU46-PCA夫妇构成了PYP中光触发蛋白质地震的内置默认值。 这些实验将揭示由晶体晶格施加的功能动力学变化，并将确定瞬态部分蛋白展开的作用，作为一种新型信号转导机制，预计不仅在PYP中，而且在其他信号传输系统中都起着作用。