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、心血管疾病和自身免疫性疾病。血红素蛋白细胞色素c(Cytc)从线粒体释放到胞浆是启动细胞凋亡的关键事件。细胞色素c在天然状态下是一种缓慢的过氧化物酶，当与心磷脂(CL)结合时，它催化心磷脂过氧化，进而促进外膜通透和细胞色素c的释放。细胞色素c-CL相互作用的调控可能是许多人类疾病的重要药物靶点，但这些策略的合理设计首先取决于对CL结合的细胞色素c构象性质的了解。我们假设，CL结合的细胞色素c是由不同构象组成的动态状态。因此，在某些构象中，细胞色素C过氧化物酶的活性可能更强。利用一系列特定部位的光物理探针，这些研究将阐明CL结合的细胞色素c的构象特性、生理调节剂对细胞色素c构象和过氧化物酶活性的影响，并建立体内细胞色素c构象动力学的评估方法。多个荧光团标记的Cytc衍生物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)之间的距离分布来获得结构信息。对锌取代的Cytc三重态寿命的分析将探索血红素基团的可及性。结合计算，这些实验将提供一个非均相CL束缚的Cytc态的模型。我们将探索与生理相关的离子强度增加、ATP结合和CL过氧化对细胞色素C构象系综组成的影响，以及与细胞色素C过氧化物酶活性相关的群体变化。通过一些Cyt c突变体，我们将测试和区分蛋白质稳定性和Fe-Met80键的强度对CL结合的Cyt c构象集合的性质和相关的过氧化物酶活性的影响。这些研究将确定Cytc的哪些结构变化对其CL诱导的过氧化物酶功能特别重要。在活体线虫中用荧光标记的细胞色素c进行的FRET实验将评估我们在体内的结构和机制模型，并阐明构象动力学如何影响细胞色素c的凋亡释放。所提出的研究不仅将确定难以捉摸的CL结合的Cyt c状态的构象组成，而且还将有助于阐明Cyt c的释放机制，并为设计凋亡调节因子提供基础。

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.