title: Conformational rearrangements in the pro-apoptotic protein, Bax, as it inserts into mitochondria: a cellular death switch.
abstract: The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the activation of apoptosis through the mitochondria pathway. Pro- and anti-apoptotic members of this family keep each other in check until the correct time to commit to apoptosis. The point of no return for this commitment is the permeabilization of the outer mitochondrial membrane. Translocation of the pro-apoptotic member, Bax, from the cytosol to the mitochondria is the molecular signature of this event. We employed a novel method to reliably detect Förster resonance energy transfer (FRET) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in Bax as this translocation occurs in live cells. In the cytosol, our FRET measurement indicated that the C-terminal helix is exposed instead of tucked away in the core of the protein. In addition fluorescence correlation spectroscopy (FCS) showed that cytosolic Bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. Cross-linking the C-terminal helix (α9) to helix α4 reduced the potential of those interactions to occur. After translocation, our FRET measurements showed that Bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the BH3 domain (helix α2) and the C-terminal helix. These findings have implications for possible contacts with other Bcl-2 proteins necessary for the regulation of apoptosis.
authors.affiliation: From the Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Gahl
authors.affiliation: From the Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Yi
authors.initials: Y
authors.lastname: He
authors.affiliation: From the Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Shiqin
authors.initials: S
authors.lastname: Yu
authors.affiliation: From the Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 tjandran@nhlbi.nih.gov.
authors.forename: Nico
authors.initials: N
authors.lastname: Tjandra
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011806:Quinolinium Compounds / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019311:Staurosporine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: C481052:Alexa fluor 546; D011806:Quinolinium Compounds; D051028:bcl-2-Associated X Protein; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D019311:Staurosporine
keywords: Apoptosis; B-cell Lymphoma 2 (Bcl-2) Family; Bax; Conformational Change; Fluorescence Correlation Spectroscopy (FCS); Fluorescence Resonance Energy Transfer (FRET)