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.

Public Health Relevance

Defects in apoptotic mechanisms contribute to cancer, neurodegeneration, cardiovascular and autoimmune diseases. A critical no-return step in the execution of apoptosis is release of cytochrome c from mitochondria, which depends on cytochrome c peroxidase activity and its dissociation from the cardiolipin-rich mitochondrial membrane. This study will uncover structural properties of cardiolipin- bound cytochrome c and factors that govern its peroxidase activity and apoptotic release thus providing a foundation for the design of apoptotic regulators.

National Institute of Health (NIH)
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Macromolecular Structure and Function A Study Section (MSFA)
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Anderson, Vernon
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Dartmouth College
Schools of Arts and Sciences
United States
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Leung, Chris W T; Hong, Yuning; Hanske, Jonas et al. (2014) Superior fluorescent probe for detection of cardiolipin. Anal Chem 86:1263-8
Muenzner, Julia; Pletneva, Ekaterina V (2014) Structural transformations of cytochrome c upon interaction with cardiolipin. Chem Phys Lipids 179:57-63
Muenzner, Julia; Toffey, Jason R; Hong, Yuning et al. (2013) Becoming a peroxidase: cardiolipin-induced unfolding of cytochrome c. J Phys Chem B 117:12878-86
Snider, Erik J; Muenzner, Julia; Toffey, Jason R et al. (2013) Multifaceted effects of ATP on cardiolipin-bound cytochrome c. Biochemistry 52:993-5
Vincelli, Amber J; Pottinger, Danielle S; Zhong, Fangfang et al. (2013) Recombinant expression, biophysical characterization, and cardiolipin-induced changes of two Caenorhabditis elegans cytochrome c proteins. Biochemistry 52:653-66
Hanske, Jonas; Toffey, Jason R; Morenz, Anna M et al. (2012) Conformational properties of cardiolipin-bound cytochrome c. Proc Natl Acad Sci U S A 109:125-30