Characterization of Bax Pore Formation Using Fluorescence Techniques

Abstract

<p> Bax is a pro-apoptotic protein believed to permeabilize mitochondria during apoptosis. The mechanism Bax uses is not well understood. In this work, we use fluorescence techniques to shed light on how tBid activates Bax and we examine the topology of the pore-forming domain of Bax. </p> <p> The manner in which tBid promotes apoptosis via Bax activation is not known. Study of tBid and Bax interaction using a new FRET pair showed that the proteins only interacted in the presence ofmembranes. The Bax pore was shown to have a variable size distribution. A fluorescence technique of simultaneously measuring pore formation, Bax insertion and FRET showed that tBid interaction with Bax occurred before all the Bax inserted or formed pores in the liposomes. A chronological order is proposed for Bax pore formation. tBid first binds to liposomes. tBid proceeds to interact with Bax, and Bax inserts into the membrane. After insertion, Bax oligomerizes and forms small pores. More Bax is recruited and the pores become larger. </p> <p> The two central hairpin helices of Bax, helices 5 and 6, are known as the pore-forming domain. We used cysteine scanning with the environment sensitive fluoroprobe NBD to gain insight into the topology of these helices. Fluorescence intensity changes and emission blue shifts showed that residues in these helices undergo conformational reorganization during pore formation. In the activated oligomeric conformation, fluorescence lifetimes showed that helix 5 was more inaccessible to water than helix 6. Cobalt, a cationic NBD quencher, effectively quenched residues in the pore-forming domain, consistent with a pore that is lined with anionic lipid head groups. Quenching with nitroxide groups at various lipid depths showed that residues on helix 6 were most quenched by a shallow quencher, while residues on helix 5 were quenched by deeper quenchers. Compared to beta sheet pore-forming proteins, the data obtained suggests that Bax and possibly other alpha helical pore-forming proteins form a lipidic pore in a dynamic environment. Combined together, the data suggest a model for Bax in which helix 5 spans the bilayer, and helix 6 is buried just below the lipid headgroups of a toroidal pore. </p>