Modulation of the Membrane Orientation and Secondary Structure of the C-Terminal Domains of Bak and Bcl-2 by Lipids

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Abstract
Infrared spectroscopy was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domains of the pro-apoptotic protein Bak (Bak-C) and the anti-apoptotic protein Bcl-2 (Bcl-2-C) when incorporated into different lipid vesicles. Whereas β-pleated sheet was the predominant type of secondary structure of Bak-C in the absence of membranes, the same peptide adopted different structures depending on lipid composition when incorporated into membranes, with the predominance of the α-helical structure in the case of DMPC and other phospholipids, such as POPC and POPG. However, β-pleated sheet was the predominant structure in other membranes containing phospholipids with longer fatty acyl chains and cholesterol, as well as in a mixture which imitates the composition of the outer mitochondrial membrane (OMM). Similarly, Bcl-2-C adopted a structure with a predominance of intermolecularly bound pleated β-sheet in the absence of membranes, with α-helix as the main component in the presence of DMPC and POPG, but intermolecular β-sheet in the presence of EYPC and cholesterol. Using ATR-IR, it was found that the orientation of the α-helical components of both domains was nearly perpendicular to the plane of the membrane in the presence of DMPC membranes, but not in EYPC or OMM membranes. 2H NMR spectroscopy of DMPC-d54 confirmed the transmembrane disposition of the domains, revealing that they broadened the phase transition temperature, although the order parameter of the C−D bonds was not affected, as might have been expected for intrinsic peptides. When all these results are taken together, it was concluded that the domains only form transmembrane helices in membranes of reduced thickness and that hydrophobic mismatching occurs in thicker membranes, as happens in the membrane imitating the composition of the OMM, where the peptides were partially located outside the membranes.