Conformational Properties of Cytochrome c in Apoptosis

描述（由申请人提供）：细胞凋亡机制缺陷导致癌症、神经退行性疾病、心血管疾病和自身免疫性疾病。血红素蛋白细胞色素c （cyt c）从线粒体释放到细胞质是细胞凋亡起始的关键事件。cyt c是一种天然状态下的迟缓过氧化物酶，当与心磷脂（CL）结合时，cyt c催化心磷脂过氧化，这反过来有助于外膜渗透和cyt c释放。对于许多人类疾病来说，调节cyt - cl相互作用可能是一个有价值的药物靶点，但这种策略的合理设计首先取决于对cl结合的cyt - c的构象特性的理解。我们假设cl结合的cyt - c是一个由多种构象组成的动态状态。因此，在某些构象中，cyt - c过氧化物酶活性可能更强。利用一系列位点特异性光物理探针，提出的研究将阐明cl结合的cyt - c的构象性质，cyt - c构象和过氧化物酶活性的生理调节剂的影响，并开发评估体内cyt - c构象动力学的方法。测量荧光共振能量转移（FRET）动力学在多个荧光团标记的cyt - c衍生物将产生结构信息，通过估计的距离分布之间的荧光供体(D)和受体(a)。分析zn -取代cyt - c的三重态寿命将探讨血红素基团的可及性。结合计算，这些实验将提供一个异质cl结合cyt - c状态的模型。我们将探讨生理相关的离子强度增加、ATP结合和CL过氧化对cyt - c构象集合组成的影响，以及与cyt - c过氧化物酶活性相关的种群变化。通过大量的cyt - c突变体，我们将测试和区分蛋白质稳定性和Fe-Met80键强度对cl结合的cyt - c构象集合性质和相关过氧化物酶活性的影响。这些研究将确定cyt 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。在线虫活体中荧光标记cyt - c的FRET实验将评估我们在体内的结构和机制模型，并说明构象动力学如何影响cyt - c凋亡释放。这些研究不仅可以确定难以捉摸的cl结合的cyt - c状态的构象组成，还可以揭示cyt - c释放的机制，并为细胞凋亡调节剂的设计提供基础。

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.