Apoptosis, the process of programmed cell death, must be carefully regulated in multi-cellular organisms to ensure proper tissue homeostasis, embryonic development and immune system activity.   The Bcl-2 family of proteins regulates the activation of apoptosis through the mitochondria pathway.  Dynamic interactions between pro- and anti-apoptotic members of this family keep each other in check until the proper time to commit to apoptosis.   The point of no return for this commitment is the permeabilization of the outer-mitochondrial membrane (OMM).  Translocation of the pro apoptotic member, Bax, from the cytosol to the mitochondria is the molecular signature of this event.  Molecular interactions and conformational changes associated with this event have been difficult to obtain due to challenges associated with taking subtle measurements in the complex environment of live cells. To circumvent these challenges, we developed 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 measurements indicated that the C-terminal helix is exposed instead of tucked away in the core of the protein. This coincided with measurements using fluorescence correlation spectroscopy (FCS) that showed that cytosolic Bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. We propose that this exposed helix allows for this contact to occur.  Cross-linking the C-terminal helix (a9) to helix a4 reduced the instances of these interactions while at the same time yielded FRET measurements that are consistent with the a9 helix tucked into the core of the protein.  After translocation, our FRET measurements showed that Bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the BH3 domain (helix a2) and the C-terminal helix.  We extended our study by looking at conformational changes of another Bcl-2 member, Bid, as it associates to the OMM. These findings provide insight into the molecular architecture that involve contacts among Bcl-2 proteins to permeabilize the OMM, which is a key necessity for the regulation of apoptosis.

Probing the conformational rearrangements in Bcl-2 proteins, Bax and Bid at the initiation of apoptosis

254th National Meeting (2017)

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