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Pro-apoptotic bax-α1 synthesis and evidence for β-sheet to α-helix conformational change as triggered by negatively charged lipid mβembranes
Umeå University, Faculty of Science and Technology, Chemistry.
UMR 5144 MOBIOS, CNRS-Université Bordeaux, France.
Umeå University, Faculty of Science and Technology, Chemistry.
UMR 5144 MOBIOS, CNRS-Université Bordeaux, France.
2007 (English)In: Journal of Peptide Science, ISSN 1075-2617, Vol. 13, no 2, 100-106 p.Article in journal (Refereed) Published
Abstract [en]

Solid phase synthesis of Bax-α1, 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-α1 is present as a mixture of β-sheet and unstructured (random coil) conformations. A marked change from β-sheet to α-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.

Place, publisher, year, edition, pages
2007. Vol. 13, no 2, 100-106 p.
Keyword [en]
solid phase synthesis, TFA removal, 19F NMR, UV circular dichroism, electrostatic interaction, apoptotic peptides
URN: urn:nbn:se:umu:diva-12371DOI: doi:10.1002/psc.803OAI: diva2:152042
Available from: 2007-04-03 Created: 2007-04-03Bibliographically approved
In thesis
1. Apoptosis Regulation via the Mitochondrial Pathway: Membrane Response upon Apoptotic Stimuli
Open this publication in new window or tab >>Apoptosis Regulation via the Mitochondrial Pathway: Membrane Response upon Apoptotic Stimuli
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems.

The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-α1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-α1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-α1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface.

Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.

Place, publisher, year, edition, pages
Umeå: Kemi, 2008. 58 p.
Apoptosis, BH4 peptide, Bax-α1 peptide, model membrane, cardiolipin, solid phase peptide synthesis, circular dichroism, solid-state nuclear magnetic resonance spectroscopy.
National Category
urn:nbn:se:umu:diva-1883 (URN)978-91-7264-676-6 (ISBN)
Public defence
2008-11-07, BiA201, Biologihuset, Umeå, 13:00 (English)
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2015-11-13Bibliographically approved

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