ORCID Profile
0000-0002-8221-9326
Current Organisation
CNRS-Universite de Bordeaux
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Publisher: Wiley
Date: 09-06-2005
DOI: 10.1002/PSC.686
Abstract: 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 beta-sheeted structures in water that convert progressively into alpha-helical features upon addition of TFE or membrane. The hipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes.
Publisher: American Chemical Society (ACS)
Date: 30-05-2012
DOI: 10.1021/CG300279Z
Publisher: Wiley
Date: 24-04-2009
DOI: 10.1096/FJ.09-134114
Abstract: Using a noninvasive, solid-state magic-angle spinning nuclear magnetic resonance (MAS NMR) approach, we track ex vivo the behavior of in idual membrane components in isolated, active mitochondria (model system: potato tubers) during physiological processes. The in idual phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) membrane constituents were identified as distinct lines by applying MAS (31)P NMR on extracted lipid membranes. However, the CL NMR signal appeared to be very broad in functional mitochondria, indicating a tight complex formation with membrane protein. Calcium stress induced severe membrane degradation without recovery of a single CL NMR resonance. This suggests that calcium overload destroys the outer mitochondrial membrane and does not modify strongly the CL protein complexes in the inner membrane a conclusion confirmed by respiratory controls. Conversely, mitochondrial membrane disruption on time degradation or mechanical stress generates clearly visible identical CL NMR signals, similar to those observed in rehydrated lipid extracts. Similarly, noninvasive based NMR tracking of lipids in response to erse physiological stimuli can easily be used for other organelles and whole living cells.
Publisher: Wiley
Date: 2007
DOI: 10.1002/PSC.803
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.
Publisher: Bentham Science Publishers Ltd.
Date: 11-2012
DOI: 10.2174/138920312804142138
Abstract: Membrane interacting peptides are reviewed in terms of structure and mode of action on lipid membranes. Helical, β-stranded, peptides containing both helices and strands, cyclic, lipopeptides and short linear peptides are seen to considerably modulate membrane function. Among peptides that lead to membrane alteration or permeation, antimicrobial peptides play an important role and some of them may be foreseen as potential new antibiotics. Alternatively, peptides that do not destroy the membrane are also very important in modulating the structure and dynamics of the lipid bilayer and play important roles in membrane protein functions. Peptide lipid complexes are shown to be very variable in structure and dynamics: "carpet", "barrel stave", toroid and disordered pores, electrostatic wedge and molecular electroporation models are discussed. Their assembly is reviewed in terms of electric, hipathic and dynamic properties of both lipids and peptides.
Publisher: Elsevier BV
Date: 03-2009
DOI: 10.1016/J.BBAMEM.2008.12.014
Abstract: A key event in programmed cell death is the translocation of the apoptotic Bax protein from the cytosol towards mitochondria. The first helix localized at the N-terminus of Bax (Bax-alpha1) can act here as an addressing sequence, which directs activated Bax towards the mitochondrial surface. Solid state NMR (nuclear magnetic resonance), CD (circular dichroism) and ATR (attenuated total reflection) spectroscopy were used to elucidate this recognition process of a mitochondrial membrane system by Bax-alpha1. Two potential target membranes were studied, with the outer mitochondrial membrane (OM) mimicked by neutral phospholipids, while mitochondrial contact sites (CS) contained additional anionic cardiolipin. (1)H and (31)P magic angle spinning (MAS) NMR revealed Bax-alpha1 induced pronounced perturbations in the lipid headgroup region only in presence of cardiolipin. Bax-alpha1 could not insert into CS membranes but at elevated concentrations it inserted into the hydrophobic core of cardiolipin-free OM vesicles, thereby adopting beta-sheet-like features, as confirmed by ATR. CD studies revealed, that the cardiolipin mediated electrostatic locking of Bax-alpha1 at the CS membrane surface promotes conformational changes into an alpha-helical state a process which seems to be necessary to induce further conformational transition events in activated Bax which finally causes irreversible membrane permeabilization during the mitochondrial apoptosis.
Publisher: Wiley
Date: 08-01-2008
DOI: 10.1111/J.1742-4658.2007.06222.X
Abstract: The regulative BH4 domain of human Bcl-2 protein exerts its anti-apoptic activity via the mitochondrion. In the present study, we investigated the molecular interactions of this domain with negatively charged liposomes mimicking the outer mitochondrial membrane. To model the overproduction of Bcl-2 found in cancer processes, we studied the impact of elevated concentrations of its regulative BH4 segment on these mitochondrial membranes from the peptide and lipid perspective. Combined solid state (2)H-NMR and differential scanning calorimetry revealed the coexistence of small sized fluid and rigid membrane domains over a large temperature range, which is confirmed by (31)P-NMR at 30 degrees C. The latter are stabilized, in a cholesterol-like manner, by the presence of a BH4 peptide. In the same time scale, the reduction of the headgroup order is seen in the static (14)N and (31)P-NMR spectra when BH4 inserts into the bilayers. Indeed, attenuated total reflection spectroscopy indicated a dominant aggregated beta-sheet secondary structure of BH4 with a 42 degrees tilt relative to the membrane surface. These results are discussed in terms of membrane stabilization versus apoptotic mechanisms at the outer mitochondrial membrane location.
No related grants have been discovered for Erick J. Dufourc.