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

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA

(RECEIVED August 24, 2005; FINAL REVISION October 22, 2005; ACCEPTED October 22, 2005)

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_LTM 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_LTM. Under conditions that favor the solution conformation (pH 7.4), the free energy of folding for Bcl-x_LTM (G°) was determined to be 15.8 kcal·mol^–1. Surprisingly, under conditions that favor a membrane conformation (pH 4.9), G° was 14.6 kcal·mol^–1. These results differ from those obtained with many other membrane-insertable 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; pH-dependence; pore-forming toxins; protein folding

Abbreviations: TM, transmembrane anchor • NMR, nuclear magnetic resonance • CD, circular dichroism • GdnHCl, guanidine hydrochloride • HSQC, heteronuclear single quantum coherence

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051807706.

Reprint requests to: R. Blake Hill, Department of Biology, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA; e-mail: hill{at}jhu.edu; fax: (702) 441-2490.

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