Acid destabilization of the solution conformation of Bcl‐X_L does not drive its pH‐dependent insertion into membranes

Abstract

Regulation of programmed cell death by Bcl-x L is dependent on both its solution and integral membrane conformations. A conformational change from solution to membrane is also important in this regulation. This conformational change shows a pH-dependence similar to the translocation domain of diphtheria toxin, where an acid-induced molten globule conformation in the absence of lipid vesicles mediates the change from solution to membrane conformations. By contrast, Bcl-x L DTM in the absence of lipid vesicles exhibits no gross conformational changes upon acidification as observed by near-and far-UV circular dichroism spectropolarimetry. Additionally, no significant local conformational changes upon acidification were observed by heteronuclear NMR spectroscopy of Bcl-x L DTM. Under conditions that favor the solution conformation (pH 7.4), the free energy of folding for Bcl-x L DTM (DG°) was determined to be 15.8 kcalÁmol. Surprisingly, under conditions that favor a membrane conformation (pH 4.9), DG°was 14.6 kcal Á mol -1. These results differ from those obtained with many other membraneinsertable proteins where acid-induced destabilization is important. Therefore, other contributions must be necessary to destabilize the solution conformation Bcl-x L and favor the membrane conformation at pH 4.9. Such contributions might include the presence of a negatively charged membrane or an electrostatic potential across the membrane. Thus, for proteins that adopt both solution and membrane conformations, an obligatory molten globule intermediate may not be necessary. The absence of a molten globule intermediate might have evolved to protect Bcl-x L from intracellular proteases as it undergoes this conformational change essential for its activity.Keywords: Bcl-2 family; Bcl-x L ; solution to membrane conformational change; diphtheria toxin; pHdependence; pore-forming toxins; protein folding Supplemental material: see www.proteinscience.orgThe Bcl-2 proteins regulate programmed cell death by acting in the cytosol and organellar membranes (Adams and Cory 1998;Chao and Korsmeyer 1998;Green and Reed 1998;Ng and Shore 1998;Harris and Thompson 2000;Hengartner 2000). Some Bcl-2 proteins act by adopting at least two different structural conformations: a solution conformation and an integral membrane conformation. For example, pro-apoptotic Bax is a monomeric, helical bundle protein localized in the cytosol until an apoptotic signal causes translocation to the mitochondrial outer membrane (Suzuki et al. 2000). At the mitochondrial outer membrane, Bax inserts and folds into a large, multimeric integral membrane protein that is thought to regulate the release of cytochrome c Reprint requests to: R. Blake Hill, Department of Biology, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA; e-mail: hill@jhu.edu; fax: (702) 441-2490.Abbreviations: TM, transmembrane anchor; NMR, nuclear magnetic resonance; CD, circular dichroism; GdnHCl, guanidine hydrochloride; HSQC, heteronuclear single quantum coherence.Ar...

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