Nadège Jamin | CEA - Academia.edu (original) (raw)
Papers by Nadège Jamin
Molecular Endocrinology, 2005
binding. In the present work, we functionally and structurally characterized this CRAC motif usin... more binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria. (Molecular Endocrinology 19: 588-594, 2005)
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2008
Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR)... more Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from 1 H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using 1 H-15 N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.
Journal of Structural Biology, 2005
A structural model of the murine PrP small b-sheet was obtained by synthesizing the RGYMLGSADPNGN... more A structural model of the murine PrP small b-sheet was obtained by synthesizing the RGYMLGSADPNGNQVYYRG peptide comprising the two b-strands 127-133 and 159-164 linked by a four-residue sequence of high turn propensity. The DPNG turn sequence is a ''short circuit'' replacing the original protein sequence between the two strands. This 19-residue peptide spontaneously forms very long single fibrils as observed by electron microscopy. The X-ray diffraction patterns of a partially oriented sample reveals an average arrangement of the hairpin peptides into a structure which can be geometrically approximated by an empty-core cylinder. The hairpins are oriented perpendicular to the cylinder axis and a 130 Å helix period is observed. Based on X-ray diffraction constraints and on more indirect general protein structure considerations, a precise and consistent fibril model was built. The structure consists of two b-sheet ribbons wound around a cylinder and assembled into a single fibril with a hairpin orientation perpendicular to the fibril axis. Subsequent implicit and explicit solvent molecular dynamics simulations provided the final structure at atomic resolution and further insights into the stabilizing interactions. Particularly important are the zipper-like network of polar interactions between the edges of the two ribbons, including the partially buried water molecules. The hydrophobic core is not optimally compact explaining the low density of this region seen by X-ray diffraction. The present findings provide also a simple model for further investigating the sequence-stability relationship using a mutational approach with a quasi-independent consideration of the polar and apolar interactions.
A structural model of the murine PrP small b-sheet was obtained by synthesizing the RGYMLGSADPNGN... more A structural model of the murine PrP small b-sheet was obtained by synthesizing the RGYMLGSADPNGNQVYYRG peptide comprising the two b-strands 127-133 and 159-164 linked by a four-residue sequence of high turn propensity. The DPNG turn sequence is a ''short circuit'' replacing the original protein sequence between the two strands. This 19-residue peptide spontaneously forms very long single fibrils as observed by electron microscopy. The X-ray diffraction patterns of a partially oriented sample reveals an average arrangement of the hairpin peptides into a structure which can be geometrically approximated by an empty-core cylinder. The hairpins are oriented perpendicular to the cylinder axis and a 130 Å helix period is observed. Based on X-ray diffraction constraints and on more indirect general protein structure considerations, a precise and consistent fibril model was built. The structure consists of two b-sheet ribbons wound around a cylinder and assembled into a single fibril with a hairpin orientation perpendicular to the fibril axis. Subsequent implicit and explicit solvent molecular dynamics simulations provided the final structure at atomic resolution and further insights into the stabilizing interactions. Particularly important are the zipper-like network of polar interactions between the edges of the two ribbons, including the partially buried water molecules. The hydrophobic core is not optimally compact explaining the low density of this region seen by X-ray diffraction. The present findings provide also a simple model for further investigating the sequence-stability relationship using a mutational approach with a quasi-independent consideration of the polar and apolar interactions.
FEBS Letters, 2006
Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of ... more Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of two synthetic peptides, D82-R101 and D82-I109, encompassing the caveolin scaffolding domain (D82-R101), in the presence of dodecylphosphocholine (DPC) micelles. Our data show that a stable helical conformation of the caveolin scaffolding domain in a membrane mimicking system is only obtained for the peptide including the L102-I109 hydrophobic stretch, a part of the caveolin intra-membrane domain. Through chemical shift variations, an ensemble of six residues of the D82-L109 peptide, mainly located in the V 94 TKYWFYR 101 motif were found to detect the presence of phosphatidylserine solubilized in DPC micelles. Our results constitute a first step for elucidating at a residue level the conformational properties of the central region of the caveolin-1 protein.
The Journal of biological chemistry, Jan 14, 1997
BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residu... more BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 +/- 1.0 A. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K+ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the three-dimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.
Trends in Biochemical Sciences, 2008
The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological funct... more The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological functions such as exocrine and endocrine secretions, feeding behaviour, metabolism, growth, and neuro-and immuno-modulations. These receptors are activated by endogenous peptide hormones including secretin, glucagon, vasoactive intestinal peptide, corticotropin-releasing factor and parathyroid hormone. We have identified a common structural motif that is encoded in all class B GPCRligand N-terminal sequences. We propose that this local structure, a helix N-capping motif, is formed upon receptor binding and constitutes a key element underlying class B GPCR activation. The folded backbone conformation imposed by the capping structure could serve as a template for a rational design of drugs targeting class B GPCRs in several diseases.
European Journal of Biochemistry, 1993
The DNA-binding domain of the c-Myb oncoprotein contains two repeats, R2 and R3, both of which ha... more The DNA-binding domain of the c-Myb oncoprotein contains two repeats, R2 and R3, both of which have been proposed to be related to the helix-turn-helix(HTH) motif. As a first step towards determination of the three-dimensional structure of this domain and of the mode of interaction with the DNA, we have undertaken multidimensional heteronuclear NMR studies using uniformly "N-labeled and 13C, 15N double-labeled R2R3 and, a selectively I5N-enriched sample on all lysine, histidine and leucine residues of R2R3. We present almost complete assignments of the backbone 'H, "N and 'TC" atoms and determine the secondary structure of R2R3 in solution.
Review of Scientific Instruments, 2002
Combining IR spectroscopy with fluorescence imaging in a single microscope: Biomedical applicatio... more Combining IR spectroscopy with fluorescence imaging in a single microscope: Biomedical applications using a synchrotron infrared source (invited). [Review of Scientific Instruments 73, 1357 (2002)]. Lisa M. Miller, Paul Dumas ...
Protein Science, 1998
DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein f... more DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pK, of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 'HN, I5N, I3Ca, "CO, 'Ha, and I3Cp NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple ( 'H-'3C-'5N) and double resonance ('H-'5N or 'H-I3C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, ? T H~H U and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro15 1, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large I3Ca chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.
Molecular Endocrinology, 2005
binding. In the present work, we functionally and structurally characterized this CRAC motif usin... more binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria. (Molecular Endocrinology 19: 588-594, 2005)
Journal of the American Chemical Society, 2010
Nanofabrication by molecular self-assembly involves the design of molecules and self-assembly str... more Nanofabrication by molecular self-assembly involves the design of molecules and self-assembly strategies so that shape and chemical complementarities drive the units to organize spontaneously into the desired structures. The power of self-assembly makes it the ubiquitous strategy of living organized matter and provides a powerful tool to chemists. However, a challenging issue in the self-assembly of complex supramolecular structures is to understand how kinetically efficient pathways emerge from the multitude of possible transition states and routes. Unfortunately, very few systems provide an intelligible structure and formation mechanism on which new models can be developed. Here, we elucidate the molecular and supramolecular self-assembly mechanism of synthetic octapeptide into nanotubes in equilibrium conditions. Their complex hierarchical self-assembly has recently been described at the mesoscopic level, and we show now that this system uniquely exhibits three assembly stages and three intermediates: (i) a peptide dimer is evidenced by both analytical centrifugation and NMR translational diffusion experiments; (ii) an open ribbon and (iii) an unstable helical ribbon are both visualized by transmission electron microscopy and characterized by small angle X-ray scattering. Interestingly, the structural features of two stable intermediates are related to the final nanotube organization as they set, respectively, the nanotube wall thickness and the final wall curvature radius. We propose that a specific self-assembly pathway is selected by the existence of such preorganized and stable intermediates so that a unique final molecular organization is kinetically favored. Our findings suggests that the rational design of oligopeptides can encode both molecular-and macro-scale morphological characteristics of their higherorder assemblies, thus opening the way to ultrahigh resolution peptide scaffold engineering.
Journal of Structural Biology, 2005
A structural model of the murine PrP small beta-sheet was obtained by synthesizing the RGYMLGSADP... more A structural model of the murine PrP small beta-sheet was obtained by synthesizing the RGYMLGSADPNGNQVYYRG peptide comprising the two beta-strands 127-133 and 159-164 linked by a four-residue sequence of high turn propensity. The DPNG turn sequence is a "short circuit" replacing the original protein sequence between the two strands. This 19-residue peptide spontaneously forms very long single fibrils as observed by electron microscopy. The X-ray diffraction patterns of a partially oriented sample reveals an average arrangement of the hairpin peptides into a structure which can be geometrically approximated by an empty-core cylinder. The hairpins are oriented perpendicular to the cylinder axis and a 130 A helix period is observed. Based on X-ray diffraction constraints and on more indirect general protein structure considerations, a precise and consistent fibril model was built. The structure consists of two beta-sheet ribbons wound around a cylinder and assembled into a single fibril with a hairpin orientation perpendicular to the fibril axis. Subsequent implicit and explicit solvent molecular dynamics simulations provided the final structure at atomic resolution and further insights into the stabilizing interactions. Particularly important are the zipper-like network of polar interactions between the edges of the two ribbons, including the partially buried water molecules. The hydrophobic core is not optimally compact explaining the low density of this region seen by X-ray diffraction. The present findings provide also a simple model for further investigating the sequence-stability relationship using a mutational approach with a quasi-independent consideration of the polar and apolar interactions.
International Journal of Quantum Chemistry, 1996
The interaction between the R2R3 DNA binding domain of the oncoprotein c-Myb and oligodeoxynucleo... more The interaction between the R2R3 DNA binding domain of the oncoprotein c-Myb and oligodeoxynucleotides was investigated by 1 H-NMR spectroscopy and fluorescence anisotropy assays. Titration of 12 and 16 base-pair DNA fragments containing the TAACGGTC sequence with R2R3 ...
FEBS Letters, 2002
The conversion of the cellular prion protein into the L L-sheet-rich scrapie prion protein is tho... more The conversion of the cellular prion protein into the L L-sheet-rich scrapie prion protein is thought to be the key step in the pathogenesis of prion diseases. To gain insight into this structural conversion, we analyzed the intrinsic structural propensity of the amino acid sequence of the murine prion C-terminal domain. For that purpose, this globular domain was dissected into its secondary structural elements and the structural propensity of the protein fragments was determined.
Faraday Discussions, 2004
It has become increasingly clear that synchrotron infrared micro-spectroscopy is an extremely val... more It has become increasingly clear that synchrotron infrared micro-spectroscopy is an extremely valuable analysis tool when determining the chemical composition of biological and biomedical samples, at the diffraction-limited spatial resolution. Highly resolved infrared micro-spectroscopy, together with the high signal-to-noise level of the recorded spectra, is essential in generating chemical and statistical (multivariate) images. This is illustrated in the case of individual cell and hair section studies. Unprecedented chemical images of lipid distribution and secondary structure relative concentration have been achieved using the synchrotron source. A comparison with a Focal plane Array imaging system, on the same hair section, shows that, despite the fast imaging processing and improved quality achieved with the focal plane array detectors, spectral quality is markedly superior in the case of the synchrotron source. It is clear that the two approaches could be very complementary if combined on the same sample area, in a synchrotron facility.
European Biophysics Journal, 2010
Heteronuclear single quantum correlated spectroscopy MALDI/TOF Matrix-assisted laser desorption i... more Heteronuclear single quantum correlated spectroscopy MALDI/TOF Matrix-assisted laser desorption ionization time of flight MEM Maximum entropy method N-MAD N-terminal membrane attachment domain NATA N-acetyltryptophanamide NOESY Nuclear Overhauser effect spectroscopy POPC 1-Palmitoyl-2-oleoyl-sn-glycero-3phosphocholine POPS 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] POPG 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] TOCSY Total correlated spectroscopy Eur Biophys J (2010) 39:307-325 309
Biophysical Journal, 2011
PMP1, a regulatory subunit of the yeast plasma membrane H þ -ATPase, is a single transmembrane he... more PMP1, a regulatory subunit of the yeast plasma membrane H þ -ATPase, is a single transmembrane helix protein. Its cytoplasmic C-terminus possesses several positively charged residues and interacts with phosphatidylserine lipids as shown through both 1 H-and 2 H-NMR experiments. We used all-atom molecular dynamics simulations to obtain atomic-scale data on the effects of membrane interface lipid composition on PMP1 structure and tilt. PMP1 was embedded in two hydrated bilayers, differing in the composition of the interfacial region. The neutral bilayer is composed of POPC (1-palmitoyl-2-oleoyl-3-glycerophosphatidylcholine) lipids and the negatively charged bilayer is composed of POPC and anionic POPS (1-palmitoyl-2-oleoyl-3glycero-phosphatidylserine) lipids. Our results were consistent with NMR data obtained previously, such as a lipid sn-2 chain lying on the W28 aromatic ring and in the groove formed on one side of the PMP1 helix. In pure POPC, the transmembrane helix is two residues longer than the initial structure and the helix tilt remains constant at 6 5 3 . By contrast, in mixed POPC-POPS, the initial helical structure of PMP1 is stable throughout the simulation time even though the C-terminal residues interact strongly with POPS headgroups, leading to a significant increase of the helix tilt within the membrane to 20 5 5 .
Bioorganic & Medicinal Chemistry, 2011
For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become ... more For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become a powerful tool, provided the approach can bridge the gap in sensitivity. Here we propose constructs based on the non-selective grafting of cryptophane precursors on holo-transferrin. This biological system was chosen because there are many receptors on the cell surface, and endocytosis further increases this density. The study of these biosensors with K562 cell suspensions via fluorescence microscopy and (129)Xe NMR indicates a strong interaction, as well as interesting features such as the capacity of xenon to enter the cryptophane even when the biosensor is endocytosed, while keeping a high level of polarization. Despite a lack of specificity for transferrin receptors, undoubtedly due to the hydrophobic character of the cryptophane moiety that attracts the biosensor into the cell membrane, these biosensors allow the first in-cell probing of biological events using hyperpolarized xenon.
Molecular Endocrinology, 2005
binding. In the present work, we functionally and structurally characterized this CRAC motif usin... more binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria. (Molecular Endocrinology 19: 588-594, 2005)
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2008
Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR)... more Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from 1 H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using 1 H-15 N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.
Journal of Structural Biology, 2005
A structural model of the murine PrP small b-sheet was obtained by synthesizing the RGYMLGSADPNGN... more A structural model of the murine PrP small b-sheet was obtained by synthesizing the RGYMLGSADPNGNQVYYRG peptide comprising the two b-strands 127-133 and 159-164 linked by a four-residue sequence of high turn propensity. The DPNG turn sequence is a ''short circuit'' replacing the original protein sequence between the two strands. This 19-residue peptide spontaneously forms very long single fibrils as observed by electron microscopy. The X-ray diffraction patterns of a partially oriented sample reveals an average arrangement of the hairpin peptides into a structure which can be geometrically approximated by an empty-core cylinder. The hairpins are oriented perpendicular to the cylinder axis and a 130 Å helix period is observed. Based on X-ray diffraction constraints and on more indirect general protein structure considerations, a precise and consistent fibril model was built. The structure consists of two b-sheet ribbons wound around a cylinder and assembled into a single fibril with a hairpin orientation perpendicular to the fibril axis. Subsequent implicit and explicit solvent molecular dynamics simulations provided the final structure at atomic resolution and further insights into the stabilizing interactions. Particularly important are the zipper-like network of polar interactions between the edges of the two ribbons, including the partially buried water molecules. The hydrophobic core is not optimally compact explaining the low density of this region seen by X-ray diffraction. The present findings provide also a simple model for further investigating the sequence-stability relationship using a mutational approach with a quasi-independent consideration of the polar and apolar interactions.
A structural model of the murine PrP small b-sheet was obtained by synthesizing the RGYMLGSADPNGN... more A structural model of the murine PrP small b-sheet was obtained by synthesizing the RGYMLGSADPNGNQVYYRG peptide comprising the two b-strands 127-133 and 159-164 linked by a four-residue sequence of high turn propensity. The DPNG turn sequence is a ''short circuit'' replacing the original protein sequence between the two strands. This 19-residue peptide spontaneously forms very long single fibrils as observed by electron microscopy. The X-ray diffraction patterns of a partially oriented sample reveals an average arrangement of the hairpin peptides into a structure which can be geometrically approximated by an empty-core cylinder. The hairpins are oriented perpendicular to the cylinder axis and a 130 Å helix period is observed. Based on X-ray diffraction constraints and on more indirect general protein structure considerations, a precise and consistent fibril model was built. The structure consists of two b-sheet ribbons wound around a cylinder and assembled into a single fibril with a hairpin orientation perpendicular to the fibril axis. Subsequent implicit and explicit solvent molecular dynamics simulations provided the final structure at atomic resolution and further insights into the stabilizing interactions. Particularly important are the zipper-like network of polar interactions between the edges of the two ribbons, including the partially buried water molecules. The hydrophobic core is not optimally compact explaining the low density of this region seen by X-ray diffraction. The present findings provide also a simple model for further investigating the sequence-stability relationship using a mutational approach with a quasi-independent consideration of the polar and apolar interactions.
FEBS Letters, 2006
Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of ... more Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of two synthetic peptides, D82-R101 and D82-I109, encompassing the caveolin scaffolding domain (D82-R101), in the presence of dodecylphosphocholine (DPC) micelles. Our data show that a stable helical conformation of the caveolin scaffolding domain in a membrane mimicking system is only obtained for the peptide including the L102-I109 hydrophobic stretch, a part of the caveolin intra-membrane domain. Through chemical shift variations, an ensemble of six residues of the D82-L109 peptide, mainly located in the V 94 TKYWFYR 101 motif were found to detect the presence of phosphatidylserine solubilized in DPC micelles. Our results constitute a first step for elucidating at a residue level the conformational properties of the central region of the caveolin-1 protein.
The Journal of biological chemistry, Jan 14, 1997
BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residu... more BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 +/- 1.0 A. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K+ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the three-dimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.
Trends in Biochemical Sciences, 2008
The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological funct... more The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological functions such as exocrine and endocrine secretions, feeding behaviour, metabolism, growth, and neuro-and immuno-modulations. These receptors are activated by endogenous peptide hormones including secretin, glucagon, vasoactive intestinal peptide, corticotropin-releasing factor and parathyroid hormone. We have identified a common structural motif that is encoded in all class B GPCRligand N-terminal sequences. We propose that this local structure, a helix N-capping motif, is formed upon receptor binding and constitutes a key element underlying class B GPCR activation. The folded backbone conformation imposed by the capping structure could serve as a template for a rational design of drugs targeting class B GPCRs in several diseases.
European Journal of Biochemistry, 1993
The DNA-binding domain of the c-Myb oncoprotein contains two repeats, R2 and R3, both of which ha... more The DNA-binding domain of the c-Myb oncoprotein contains two repeats, R2 and R3, both of which have been proposed to be related to the helix-turn-helix(HTH) motif. As a first step towards determination of the three-dimensional structure of this domain and of the mode of interaction with the DNA, we have undertaken multidimensional heteronuclear NMR studies using uniformly "N-labeled and 13C, 15N double-labeled R2R3 and, a selectively I5N-enriched sample on all lysine, histidine and leucine residues of R2R3. We present almost complete assignments of the backbone 'H, "N and 'TC" atoms and determine the secondary structure of R2R3 in solution.
Review of Scientific Instruments, 2002
Combining IR spectroscopy with fluorescence imaging in a single microscope: Biomedical applicatio... more Combining IR spectroscopy with fluorescence imaging in a single microscope: Biomedical applications using a synchrotron infrared source (invited). [Review of Scientific Instruments 73, 1357 (2002)]. Lisa M. Miller, Paul Dumas ...
Protein Science, 1998
DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein f... more DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pK, of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 'HN, I5N, I3Ca, "CO, 'Ha, and I3Cp NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple ( 'H-'3C-'5N) and double resonance ('H-'5N or 'H-I3C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, ? T H~H U and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro15 1, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large I3Ca chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.
Molecular Endocrinology, 2005
binding. In the present work, we functionally and structurally characterized this CRAC motif usin... more binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria. (Molecular Endocrinology 19: 588-594, 2005)
Journal of the American Chemical Society, 2010
Nanofabrication by molecular self-assembly involves the design of molecules and self-assembly str... more Nanofabrication by molecular self-assembly involves the design of molecules and self-assembly strategies so that shape and chemical complementarities drive the units to organize spontaneously into the desired structures. The power of self-assembly makes it the ubiquitous strategy of living organized matter and provides a powerful tool to chemists. However, a challenging issue in the self-assembly of complex supramolecular structures is to understand how kinetically efficient pathways emerge from the multitude of possible transition states and routes. Unfortunately, very few systems provide an intelligible structure and formation mechanism on which new models can be developed. Here, we elucidate the molecular and supramolecular self-assembly mechanism of synthetic octapeptide into nanotubes in equilibrium conditions. Their complex hierarchical self-assembly has recently been described at the mesoscopic level, and we show now that this system uniquely exhibits three assembly stages and three intermediates: (i) a peptide dimer is evidenced by both analytical centrifugation and NMR translational diffusion experiments; (ii) an open ribbon and (iii) an unstable helical ribbon are both visualized by transmission electron microscopy and characterized by small angle X-ray scattering. Interestingly, the structural features of two stable intermediates are related to the final nanotube organization as they set, respectively, the nanotube wall thickness and the final wall curvature radius. We propose that a specific self-assembly pathway is selected by the existence of such preorganized and stable intermediates so that a unique final molecular organization is kinetically favored. Our findings suggests that the rational design of oligopeptides can encode both molecular-and macro-scale morphological characteristics of their higherorder assemblies, thus opening the way to ultrahigh resolution peptide scaffold engineering.
Journal of Structural Biology, 2005
A structural model of the murine PrP small beta-sheet was obtained by synthesizing the RGYMLGSADP... more A structural model of the murine PrP small beta-sheet was obtained by synthesizing the RGYMLGSADPNGNQVYYRG peptide comprising the two beta-strands 127-133 and 159-164 linked by a four-residue sequence of high turn propensity. The DPNG turn sequence is a "short circuit" replacing the original protein sequence between the two strands. This 19-residue peptide spontaneously forms very long single fibrils as observed by electron microscopy. The X-ray diffraction patterns of a partially oriented sample reveals an average arrangement of the hairpin peptides into a structure which can be geometrically approximated by an empty-core cylinder. The hairpins are oriented perpendicular to the cylinder axis and a 130 A helix period is observed. Based on X-ray diffraction constraints and on more indirect general protein structure considerations, a precise and consistent fibril model was built. The structure consists of two beta-sheet ribbons wound around a cylinder and assembled into a single fibril with a hairpin orientation perpendicular to the fibril axis. Subsequent implicit and explicit solvent molecular dynamics simulations provided the final structure at atomic resolution and further insights into the stabilizing interactions. Particularly important are the zipper-like network of polar interactions between the edges of the two ribbons, including the partially buried water molecules. The hydrophobic core is not optimally compact explaining the low density of this region seen by X-ray diffraction. The present findings provide also a simple model for further investigating the sequence-stability relationship using a mutational approach with a quasi-independent consideration of the polar and apolar interactions.
International Journal of Quantum Chemistry, 1996
The interaction between the R2R3 DNA binding domain of the oncoprotein c-Myb and oligodeoxynucleo... more The interaction between the R2R3 DNA binding domain of the oncoprotein c-Myb and oligodeoxynucleotides was investigated by 1 H-NMR spectroscopy and fluorescence anisotropy assays. Titration of 12 and 16 base-pair DNA fragments containing the TAACGGTC sequence with R2R3 ...
FEBS Letters, 2002
The conversion of the cellular prion protein into the L L-sheet-rich scrapie prion protein is tho... more The conversion of the cellular prion protein into the L L-sheet-rich scrapie prion protein is thought to be the key step in the pathogenesis of prion diseases. To gain insight into this structural conversion, we analyzed the intrinsic structural propensity of the amino acid sequence of the murine prion C-terminal domain. For that purpose, this globular domain was dissected into its secondary structural elements and the structural propensity of the protein fragments was determined.
Faraday Discussions, 2004
It has become increasingly clear that synchrotron infrared micro-spectroscopy is an extremely val... more It has become increasingly clear that synchrotron infrared micro-spectroscopy is an extremely valuable analysis tool when determining the chemical composition of biological and biomedical samples, at the diffraction-limited spatial resolution. Highly resolved infrared micro-spectroscopy, together with the high signal-to-noise level of the recorded spectra, is essential in generating chemical and statistical (multivariate) images. This is illustrated in the case of individual cell and hair section studies. Unprecedented chemical images of lipid distribution and secondary structure relative concentration have been achieved using the synchrotron source. A comparison with a Focal plane Array imaging system, on the same hair section, shows that, despite the fast imaging processing and improved quality achieved with the focal plane array detectors, spectral quality is markedly superior in the case of the synchrotron source. It is clear that the two approaches could be very complementary if combined on the same sample area, in a synchrotron facility.
European Biophysics Journal, 2010
Heteronuclear single quantum correlated spectroscopy MALDI/TOF Matrix-assisted laser desorption i... more Heteronuclear single quantum correlated spectroscopy MALDI/TOF Matrix-assisted laser desorption ionization time of flight MEM Maximum entropy method N-MAD N-terminal membrane attachment domain NATA N-acetyltryptophanamide NOESY Nuclear Overhauser effect spectroscopy POPC 1-Palmitoyl-2-oleoyl-sn-glycero-3phosphocholine POPS 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] POPG 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] TOCSY Total correlated spectroscopy Eur Biophys J (2010) 39:307-325 309
Biophysical Journal, 2011
PMP1, a regulatory subunit of the yeast plasma membrane H þ -ATPase, is a single transmembrane he... more PMP1, a regulatory subunit of the yeast plasma membrane H þ -ATPase, is a single transmembrane helix protein. Its cytoplasmic C-terminus possesses several positively charged residues and interacts with phosphatidylserine lipids as shown through both 1 H-and 2 H-NMR experiments. We used all-atom molecular dynamics simulations to obtain atomic-scale data on the effects of membrane interface lipid composition on PMP1 structure and tilt. PMP1 was embedded in two hydrated bilayers, differing in the composition of the interfacial region. The neutral bilayer is composed of POPC (1-palmitoyl-2-oleoyl-3-glycerophosphatidylcholine) lipids and the negatively charged bilayer is composed of POPC and anionic POPS (1-palmitoyl-2-oleoyl-3glycero-phosphatidylserine) lipids. Our results were consistent with NMR data obtained previously, such as a lipid sn-2 chain lying on the W28 aromatic ring and in the groove formed on one side of the PMP1 helix. In pure POPC, the transmembrane helix is two residues longer than the initial structure and the helix tilt remains constant at 6 5 3 . By contrast, in mixed POPC-POPS, the initial helical structure of PMP1 is stable throughout the simulation time even though the C-terminal residues interact strongly with POPS headgroups, leading to a significant increase of the helix tilt within the membrane to 20 5 5 .
Bioorganic & Medicinal Chemistry, 2011
For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become ... more For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become a powerful tool, provided the approach can bridge the gap in sensitivity. Here we propose constructs based on the non-selective grafting of cryptophane precursors on holo-transferrin. This biological system was chosen because there are many receptors on the cell surface, and endocytosis further increases this density. The study of these biosensors with K562 cell suspensions via fluorescence microscopy and (129)Xe NMR indicates a strong interaction, as well as interesting features such as the capacity of xenon to enter the cryptophane even when the biosensor is endocytosed, while keeping a high level of polarization. Despite a lack of specificity for transferrin receptors, undoubtedly due to the hydrophobic character of the cryptophane moiety that attracts the biosensor into the cell membrane, these biosensors allow the first in-cell probing of biological events using hyperpolarized xenon.