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Methods to Probe Conformational Activation and Mitochondrial Activity of Proapoptotic BAK

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BCL-2 Family Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1877))

Abstract

Mitochondrial outer membrane permeabilization (MOMP) is a crucial initiating event in apoptosis that activates the caspase cascade to execute cell demise. The effector B-cell lymphoma 2 (BCL-2) antagonist killer (BAK) forms mitochondrial apoptotic pores to mediate MOMP. In healthy cells, BAK resides at the outer mitochondrial membrane as a dormant monomer. Upon direct interactions with the BCL-2 homology 3 (BH3)-only proapoptotic proteins during apoptosis, BAK undergoes conformational changes to form the active species associated with apoptotic pores. We describe methods to purify mitochondria for MOMP assays and to detect conformational changes in native BAK associated with MOMP by using limited proteolysis and cross-linking analyses.

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Authors and Affiliations

  1. Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Geetika Singh & Tudor Moldoveanu

  2. Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Geetika Singh & Tudor Moldoveanu

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  1. Geetika Singh
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  2. Tudor Moldoveanu
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Editors and Affiliations

  1. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Evripidis Gavathiotis

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Singh, G., Moldoveanu, T. (2019). Methods to Probe Conformational Activation and Mitochondrial Activity of Proapoptotic BAK. In: Gavathiotis, E. (eds) BCL-2 Family Proteins. Methods in Molecular Biology, vol 1877. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8861-7_13

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  • DOI: https://doi.org/10.1007/978-1-4939-8861-7_13

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