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Multifaceted effects of ATP on cardiolipin-bound cytochrome c.

ABSTRACT: Using a collection of dye-labeled cytochrome c (cyt c) variants, we identify transformations of the heterogeneous cardiolipin (CL)-bound cyt c ensemble with added ATP. Distributions of dye-to-heme distances P(r) from time-resolved fluorescence resonance energy transfer show that ATP decreases the population of largely unfolded cyt c conformers, but its effects are distinct from those of a simple salt. The high peroxidase activity of CL-bound cyt c with added ATP suggests binding interactions that favor protein structures with the open heme pocket. Although ATP weakens cyt c-CL binding interactions, it also boosts the apoptosis-relevant peroxidase activity of CL-bound cyt c.

SUBMITTER: Snider EJ

PROVIDER: S-EPMC3658621 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Multifaceted effects of ATP on cardiolipin-bound cytochrome c.

Snider Erik J EJ Muenzner Julia J Toffey Jason R JR Hong Yuning Y Pletneva Ekaterina V EV

Biochemistry 20130130 6

Using a collection of dye-labeled cytochrome c (cyt c) variants, we identify transformations of the heterogeneous cardiolipin (CL)-bound cyt c ensemble with added ATP. Distributions of dye-to-heme distances P(r) from time-resolved fluorescence resonance energy transfer show that ATP decreases the population of largely unfolded cyt c conformers, but its effects are distinct from those of a simple salt. The high peroxidase activity of CL-bound cyt c with added ATP suggests binding interactions tha ...[more]

PMID: 23331169

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Project description:The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly (13)C,(15)N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.

Dataset Information

Multifaceted effects of ATP on cardiolipin-bound cytochrome c.

Publications

Multifaceted effects of ATP on cardiolipin-bound cytochrome c.

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