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Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH4
Umeå University, Faculty of Science and Technology, Chemistry.
Umeå University, Faculty of Science and Technology, Chemistry.
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2006 (English)In: Journal of Peptide Science, ISSN 1075-2617, Vol. 12, 58-64 p.Article in journal (Refereed) Published
Abstract [en]

Solid phase synthesis of BH4, the 26 amino-acid domain (6RTGYDNREIVMKYIHYKLSQRGYEWD31) of the anti-apoptotic Bcl-2 protein has been accomplished using Fmoc chemistry. The use of peculiar cleavage conditions provided high yields after purification such that tens to hundreds of mg could be obtained. A 15N-labelled version of the peptide could also be synthesized for NMR studies in membranes. The peptide purity was not lower than 98% as controlled by UV and MALDI-TOF mass spectrometry. The secondary structure was determined in water, trifluoroethanol (TFE) and in lipid membrane using UV circular dichroism. The peptide shows dominant -sheeted structures in water that convert progressively into -helical features upon addition of TFE or membrane. The amphipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

Place, publisher, year, edition, pages
2006. Vol. 12, 58-64 p.
Keyword [en]
solid phase synthesis, reverse-phase HPLC, MALDI-TOF mass spectrometry, circular dichroism, non-protected tryptophan, apoptotic peptides, 15N-labelled amino acids
URN: urn:nbn:se:umu:diva-12545DOI: doi:10.1002/psc.686OAI: diva2:152216
Available from: 2007-06-12 Created: 2007-06-12Bibliographically 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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