SOLUTION TO MEMBRANE CONFORMATIONAL CHANGE OF BCL-XLΔTM
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The central question that this thesis project addresses is the pH-dependent solution-to-membrane conformational change of Bcl-XLΔTM in vitro. Evidence exists for both solution and membrane-inserted conformations playing important roles in mediating the pro-survival activity of Bcl-XL, a Bcl-2 family protein. Our hypotheses were that acidic pH conditions mediate the solution-to-membrane conformational change (i) by destabilizing the soluble conformation and form a “molten-globule” intermediate that favorably inserts into membranes and/or (ii) by favoring the oligomerization of the solution conformation resulting in the insertion of Bcl-XL into the membrane and/or (iii) by altering the electrostatic surface of the profile and enhancing an electrostatic attraction between the protein and the surface of the membrane. Biophysical characterization under neutral and acidic pH conditions in solution indicated that there were no significant changes in the secondary, tertiary or quarternary structure of the protein suggesting that there is no formation of an “acid-induced moltenglobule” or an “acid-induced oligomeric” state. However, we observed interesting changes in the dynamics of the C-terminal end of the protein suggesting a role for protein dynamics in mediating this conformational change. The requirement for anionic lipids in the membrane suggested that pH-modulated electrostatic interactions between the protein and membrane mediated the conformational change and was confirmed by a saltdependence study. Though there were no significant positively charged surfaces on the protein that could interact electrostatically with the membrane surface, we identified a surface on the protein capable of an electrostatic interaction with the membrane. Relative iii to wild-type, a mutant, E153Q/D156N, showed altered pH-dependence of binding to lipid vesicles, altered membrane insertion properties and an enhanced ability to inhibit Baxinduced release of dextran from lipid vesicles indicating that the membrane-inserted form might play a critical role in mediating the pro-survival activity of Bcl-XL. The protonation of histidines and the presence of Ca2+ were shown not to play a significant role in the conformational change. These findings have led to the development of a simple thermodynamic model coupling protonation of ionizable groups to a partitioning into the membrane that explains this pH dependence.