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Biochemistry. 2013 Nov 12;52(45):7901-9. doi: 10.1021/bi400926k. Epub 2013 Nov 1.

Comparison of membrane insertion pathways of the apoptotic regulator Bcl-xL and the diphtheria toxin translocation domain.

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Department of Biochemistry and Molecular Biology, University of Kansas Medical Center , Kansas City, Kansas 66160, United States.


The diphtheria toxin translocation domain (T-domain) and the apoptotic repressor Bcl-xL are membrane proteins that adopt their final topology by switching folds from a water-soluble to a membrane-inserted state. While the exact molecular mechanisms of this transition are not clearly understood in either case, the similarity in the structures of soluble states of the T-domain and Bcl-xL led to the suggestion that their membrane insertion pathways will be similar, as well. Previously, we have applied an array of spectroscopic methods to characterize the pH-triggered refolding and membrane insertion of the diphtheria toxin T-domain. Here, we use the same set of methods to describe the membrane insertion pathway of Bcl-xL, which allows us to make a direct comparison between both systems with respect to the thermodynamic stability in solution, pH-dependent membrane association, and transmembrane insertion. Thermal denaturation measured by circular dichroism indicates that, unlike the T-domain, Bcl-xL does not undergo a pH-dependent destabilization of the structure. Förster resonance energy transfer measurements demonstrate that Bcl-xL undergoes reversible membrane association modulated by the presence of anionic lipids, suggesting that formation of the membrane-competent form occurs close to the membrane interface. Membrane insertion of the main hydrophobic helical hairpin of Bcl-xL, α5-α6, was studied by site-selective attachment of environment-sensitive dye NBD. In contrast to the insertion of the corresponding TH8-TH9 hairpin into the T-domain, insertion of α5-α6 was found not to depend strongly on the presence of anionic lipids. Taken together, our results indicate that while Bcl-xL and the T-domain share structural similarities, their modes of conformational switching and membrane insertion pathways are distinctly different.

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