NMR studies of the anti-apoptotic protein Bcl-x(L) in micelles

The Bcl-2 family of proteins play a pivotal role in the regulation of progr ammed cell death. One of the postulated mechanisms for the function of thes e proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporat ed into small detergent micelles. This form of Bcl-x(L) lacks the loop (res idues 49-88) between helix 1 and helix 2 and the putative C-terminal transm embrane helix (residues 214-237). Below the critical micelle concentration (CMC). Bcl-x(L) binds detergents in the hydrophobic groove that binds to pr o-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary s tructure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous so lution, the structure of the protein when dissolved in dodecylphosphocholin e (DPC) micelles consists of several alpha-helices separated by loops. Howe ver, the length and position of the individual helices of Bcl-x(L) in micel les differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and li mited proteolysis. In addition, the mobility of the micelle-bound form of B cl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and locatio n of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dy namic structure in micelles, with helices 1 and 6 and possibly helix 5 part ially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.