Pro‐apoptotic bax‐α1 synthesis and evidence for β‐sheet to α‐helix conformational change as triggered by negatively charged lipid membranes

Abstract

Solid phase synthesis of Bax-alpha1, the 25 amino acids domain (14TSSEQIMKTGALLLQGFIQDRAGRM38) of the pro-apoptotic Bax protein has been accomplished using Fmoc chemistry. A new fast and harmless protocol is described for complete TFA removal from the purified peptide powder leading to a final purity greater than 98% as controlled by 19F-NMR, UV and MALDI-TOF mass spectrometry. Secondary structure was determined in various solution and membrane media using UV Circular Dichroism. In water solution, Bax-alpha1 is present as a mixture of beta-sheet and unstructured (random coil) conformations. A marked change from beta-sheet to alpha-helix secondary structures is observed upon interaction with negatively charged phospholipids vesicles whereas neutral lipid membranes have no significant effect on the aqueous peptide conformation. Results are discussed in terms of Bax binding to mitochondrial membranes.

Alpha-Helical Destabilization of the Bcl-2-BH4-Domain Peptide Abolishes Its Ability to Inhibit the IP3 Receptor

“…A membrane- or detergent-enriched solution resembles the most the physiological or experimental conditions in which the IP 3 R/Bcl-2 interaction presumably occurs, since they are both membrane proteins. Therefore, the BH4 region of the small Bcl-2 protein would be located in the proteinaceous binding pocket on the IP 3 R, in the vicinity of the membrane environment This is compatible with the relatively high doses of BH4 peptide necessary in the electrophysiological assays (Figure 2C-D) and in the 45 Ca 2+ flux assays (IC 50 ≈ 30 µM, Figure 3B), which is likely due to the mixed α-helix/unstructured/β-sheet conformations present in the largely aqueous medium [22,49,52]. Accordingly, Bcl-2’s BH4 peptide, which is amphiphilic and has a net positive charge at physiological pH, would still need to accumulate onto the acidic membrane environment of the ER and to assume a more α-helical conformation for gaining its ability to inhibit the IP 3 Rs.…”

The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

“…It is known the importance of electrostatic interactions for extrinsic protein-lipid interaction (Hernández-Caselles et al, 1993) and that many proteins and peptides change their secondary structure upon interaction with negatively charged membranes, for example, Notch ligand Jagged-1 (Popovic et al, 2007), apocytochrome c (Rankin et al, 1998), the Alzheimer beta-amyloid peptide (25-35) (Del Mar Martínez-Senac et al, 1999), the amyloid beta (1-40) peptide (Maltseva et al, 2005) or even Bax-alpha1, where the interaction with anionic phospholipids induces a transition from b-pleated sheet to a-helix (Sani et al, 2007). The effect of high salt concentrations on the binding of Bax-C to POPG vesicles, when added from the outside, indicate that hydrophobic interactions are decisive in this case, although a limited contribution is also given by electrostatic forces.…”

“…Two of these helices coincide with C-and Nterminal domains and they seem to be fundamental for the protein mobility and activity (Cartron et al, 2003;Gao and Dou, 2000;Goping et al, 1998;Wolter et al, 1997). However it has recently been seen that the N-terminal peptide structure, encompassing residues 14-38 of Bax, shows a predominant presence of b-sheet and random coil and a lower contribution of ahelix and b-turn structures in the absence of lipids, but the peptide conformation is mainly a-helical in the presence of liposomes, particularly when the lipid membrane mixtures have a negative charge character (Sani et al, 2007).…”

Maculatin 1.1 Disrupts Staphylococcus aureus Lipid Membranes via a Pore Mechanism

“…Purified Mac1 (2,144.4 Da, observed; 2,144.23 Da, calculated) was analyzed by using an Esquire-HCT electrospray ionization-mass spectrometry system (Bruker Daltonics). Purified peptide (Ͼ99% purity) was lyophilized and residual TFA was removed by 3 rounds of lyophilization in 5 mM HCl (13) and then stored at Ϫ20°C.…”