Conformational Properties of Cytochrome c in Apoptosis

描述（由申请人提供）：细胞凋亡机制的缺陷导致癌症、神经变性、心血管和自身免疫性疾病。血红素蛋白质细胞色素c（cyt c）从线粒体释放到胞质溶胶中是启动细胞凋亡的关键事件。Cyt c是一种天然状态下的缓慢过氧化物酶，当与心磷脂（CL）结合时，它催化心磷脂过氧化，这反过来又有助于外膜渗透和Cyt c释放。细胞色素c-CL相互作用的调节可能是许多人类疾病的有价值的药物靶点，但这种策略的合理设计首先取决于对CL结合的细胞色素c的构象特性的理解。我们假设，CL结合的细胞色素c是一个动态的不同构象组成的状态。因此，细胞色素c过氧化物酶活性在某些构象中可能更稳健。使用一系列位点特异性的生物物理探针，拟议的研究将阐明CL结合的细胞色素c的构象特性，细胞色素c构象和过氧化物酶活性的生理调节剂的影响，并开发用于评估细胞色素c在体内的构象动力学的方法。在多个荧光团标记的cyt c衍生物中的荧光共振能量转移（FRET）动力学的测量将通过估计荧光供体（D）和受体（A）之间的距离分布来产生结构信息。锌取代的细胞色素c的三重态的寿命分析将探测血红素基团的可及性。再加上计算，这些实验将提供一个模型的异质CL结合的细胞色素C状态。我们将探讨生理相关的离子强度，ATP结合和CL过氧化的影响，对细胞色素C构象合奏的组合物和相关的人口变化与细胞色素C过氧化物酶活性的增加。与一些细胞色素c突变体，我们将测试和区分蛋白质的稳定性和强度的铁-蛋氨酸80键CL结合的细胞色素c构象合奏和相关的过氧化物酶活性的性质的影响。这些研究将确定细胞色素c的哪些结构变化对其CL诱导的过氧化物酶功能特别重要。用荧光标记的cyt c在活的C中进行FRET实验。elegans将在体内评估我们的结构和机制模型，并说明构象动力学如何影响细胞色素c凋亡释放。不仅将拟议的研究确定的构象组成的难以捉摸的CL结合的细胞色素C状态，但也揭示了细胞色素C释放的机制，并提供了一个基础的设计调节细胞凋亡。

DESCRIPTION (provided by applicant): Defects in apoptotic mechanisms contribute to cancer, neurodegeneration, cardiovascular and autoimmune diseases. The release of the heme protein cytochrome c (cyt c) from mitochondria to the cytosol is a key event in initiation of apoptosis. A sluggish peroxidase in its native state, when bound to cardiolipin (CL), cyt c catalyzes cardiolipin peroxidation, which, in turn, contributes to the outer membrane permeation and cyt c release. Modulation of the cyt c-CL interactions could be a valuable drug target for many human diseases but the rational design of such strategies depends first on understanding of the conformational properties of CL-bound cyt c. We hypothesize that CL-bound cyt c is a dynamic state consisting of diverse conformations. As such, the cyt c peroxidase activity may be more robust in certain conformations. Using a collection of site-specific photophysical probes, the proposed studies will elucidate conformational properties of the CL-bound cyt c, effects of physiological modulators of cyt c conformations and peroxidase activity, and also develop methods for assessing cyt c conformational dynamics in vivo. Measurements of fluorescence resonance energy transfer (FRET) kinetics in multiple fluorophore-labeled cyt c derivatives will yield structural information through estimates of the distributions of distances between a fluorescent donor (D) and acceptor (A). Analysis of the lifetime of the triplet state of Zn-substituted cyt c will probe the accessibility of the heme group. Coupled with computations, these experiments will provide a model of the heterogeneous CL-bound cyt c state. We will probe the effects of physiologically-relevant increase in ionic strength, ATP binding and CL peroxidation on the composition of the cyt c conformational ensemble and correlate populations changes with cyt c peroxidase activity. With a number of cyt c mutants, we will test and differentiate effects of protein stability and the strength of the Fe-Met80 bond on the nature of CL-bound cyt c conformational ensemble and associated peroxidase activity. These studies will determine which structural changes in cyt c are particularly important for its CL-induced peroxidase function. FRET experiments with fluorescently-labeled cyt c in live C. elegans will evaluate our structural and mechanistic model in vivo and illustrate how conformational dynamics influence cyt c apoptotic release. Not only will the proposed studies determine the conformational composition of the elusive CL-bound cyt c state but also shed light on the mechanism of the cyt c release and provide a foundation for the design of regulators of apoptosis.