Martine Moulin

Institut Laue-Langevin, Grenoble, Rhône-Alpes, France

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Publications (42)180.56 Total impact

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    ABSTRACT: Phosphatidylcholine (PC) is a major component of eukaryotic cell membranes and one of the most commonly used phospholipids for reconstitution of membrane proteins into carrier systems such as lipid vesicles, micelles and nanodiscs. Selectively deuterated versions of this lipid have many applications, especially in structural studies using techniques such as NMR, neutron reflectivity and small-angle neutron scattering. Here we present a comprehensive study of selective deuteration of phosphatidylcholine through biosynthesis in a genetically modified strain of Escherichia coli. By carefully tuning the deuteration level in E. coli growth media and varying the deuteration of supplemented carbon sources, we show that it is possible to achieve a controlled deuteration for three distinct parts of the PC lipid molecule, namely the (a) lipid head group, (b) glycerol backbone and (c) fatty acyl tail. This biosynthetic approach paves the way for the synthesis of specifically deuterated, physiologically relevant phospholipid species which remain difficult to obtain through standard chemical synthesis.
    Applied microbiology and biotechnology. 10/2014;
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    ABSTRACT: The Slc26 proteins are a ubiquitous superfamily of anion transporters conserved from bacteria to man, amongst which four have been identified as human disease genes. Our functional knowledge of this protein family has increased but limited structural information is available. These proteins contain a transmembrane (TM) domain and a C-terminal cytoplasmic Sulfate Transporter and Anti-Sigma factor (STAS) domain. In a fundamental step toward understanding the structure/function relationships within the family we have used Small Angle Neutron Scattering (SANS) on two distantly related bacterial homologues to show that there is a common, dimeric, structural architecture amongst Slc26A transporters. Pulsed ELectron-electron DOuble Resonance (PELDOR) spectroscopy supports the dimeric, SANS-derived model. Using chimeric/truncated proteins we have determined the domain organisation, the STAS domains project away from the TM core and are essential for protein stability. We use the SANS generated envelopes to assess a homology model of the TM core.
    The Biochemical journal. 07/2014;
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    ABSTRACT: Structural studies of membrane proteins remain a great experimental challenge. Functional reconstitution into artificial nanoscale bilayer disc carriers that mimic the native bilayer environment allows the handling of membrane proteins in solution. This enables the use of small-angle scattering techniques for fast and reliable structural analysis. The difficulty with this approach is that the carrier discs contribute to the measured scattering intensity in a highly nontrivial fashion, making subsequent data analysis challenging. Here, an elegant solution to circumvent the intrinsic complexity brought about by the presence of the carrier disc is presented. In combination with small-angle neutron scattering (SANS) and the D2O/H2O-based solvent contrast-variation method, it is demonstrated that it is possible to prepare specifically deuterated carriers that become invisible to neutrons in 100% D2O at the length scales relevant to SANS. These `stealth' carrier discs may be used as a general platform for low-resolution structural studies of membrane proteins using well established data-analysis tools originally developed for soluble proteins.
    Acta Crystallographica Section D Biological Crystallography 02/2014; 70(Pt 2):317-328. · 12.67 Impact Factor
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    Dataset: nsmbSI
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    ABSTRACT: The concentration profile of deuterated myoglobin (Mb) adsorbed onto polystyrene substrates displaying poly(ethylene glycol) (PEG) brushes is characterized by neutron reflectometry (NR). The method allows to directly distinguish between primary adsorption at the grafting surface, ternary adsorption within the brush, and secondary adsorption at the brush outer edge. It complements depth-insensitive standard techniques, such as ellipsometry, radioactive labeling and quartz crystal microbalance. The study explores the effect of the PEG polymerization degree, N, and the grafting density, σ, on Mb adsorption. In the studied systems there is no indication of secondary or ternary adsorption but there is evidence of primary adsorption involving a dense inner layer at the polystyrene surface. For sparsely grafted brushes the primary adsorption involves an additional dilute outer protein layer on top of the inner layer. The amount of protein adsorbed in the inner layer is independent of N but varies with σ, while for the outer layer it is correlated to the amount of grafted PEG and thus sensitive to both N and σ. The use of deuterated proteins enhances the sensitivity of NR and enables monitoring exchange between deuterated and hydrogenated species.
    Langmuir 10/2013; · 4.38 Impact Factor
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    Chemical Physics 07/2013; 424:84-8. · 1.96 Impact Factor
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    ABSTRACT: Small angle neutron scattering (SANS) is a powerful technique for investigating association states and conformational changes of biological macromolecules in solution. SANS is of particular interest for the study of the multi-component systems, as membrane protein complexes, for which in vitro characterisation and structure determination are often difficult. This article details the important physical properties of surfactants in view of small angle neutron scattering studies and the interest to deuterate membrane proteins for contrast variation studies. We present strategies for the production of deuterated membrane proteins and methods for quality control. We then review some studies on membrane proteins, and focus on the strategies to overcome the intrinsic difficulty to eliminate homogeneously the detergent or surfactant signal for solubilised membrane proteins, or that of lipids for membrane proteins inserted in liposomes.
    The European Physical Journal E 07/2013; 36(7):9889. · 2.18 Impact Factor
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    ABSTRACT: By specifically labeling leucine/valine methyl groups and lysine side chains "inside" and "outside" dynamics of proteins on the nanosecond timescale are compared using neutron scattering. Surprisingly, both groups display similar dynamics as a function of temperature, and the buried hydrophobic core is sensitive to hydration and undergoes a dynamical transition.
    Angewandte Chemie International Edition 11/2012; · 11.34 Impact Factor
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    ABSTRACT: The structure of adsorbed globular protein layers on hydrophobic surfaces is elucidated in detail by combining the use of a fully deuterated protein, myoglobin, and the neutron reflectivity technique. The hydrophobic surfaces consist of grafted self-assembled monolayer of octadecyltrichlorosilane (OTS) and polystyrene (PS) layer on silicon substrates. Different protein concentrations ranging from 1mg/ml to 0.01mg/ml are used. On the OTS surface and for low protein concentration, the adsorbed protein layer consists of a dense layer of thickness around 13Å indicating that proteins are denaturated when adsorbed on the hydrophobic interface - myoglobin being a globular protein with an average diameter of about 40Å. At high protein concentration, an additional layer is observed on the top of this first denaturated layer. The thickness of this layer corresponds roughly to the dimensions of the myoglobin suggesting that additional proteins in their bulk conformation are adsorbed on the top. In the case of PS, the protein is significantly less flattened at the interface, PS being a less hydrophobic surface.
    Journal of Colloid and Interface Science 09/2012; · 3.55 Impact Factor
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    ABSTRACT: The observation of biological activity in solvent-free protein-polymer surfactant hybrids challenges the view of aqueous and nonaqueous solvents being unique promoters of protein dynamics linked to function. Here, we combine elastic incoherent neutron scattering and specific deuterium labeling to separately study protein and polymer motions in solvent-free hybrids. Myoglobin motions within the hybrid are found to closely resemble those of a hydrated protein, and motions of the polymer surfactant coating are similar to those of the hydration water, leading to the conclusion that the polymer surfactant coating plasticizes protein structures in a way similar to hydration water.
    Journal of the American Chemical Society 08/2012; 134(32):13168-71. · 10.68 Impact Factor
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    ABSTRACT: Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context.
    Biophysical Journal 07/2012; 103(1):129-36. · 3.67 Impact Factor
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    ABSTRACT: The coherent density fluctuations of a perdeuterated dry protein have been studied by Brillouin neutron spectroscopy. Besides a nearly wavevector-independent branch located around 5 meV, a propagating mode with a linear trend at low wavevector Q is revealed. The corresponding speed of 3780 ± 130 m/s is definitely higher than that of hydrated proteins. Above Q = 0.8 Å(-1), this mode becomes overdamped, with lifetimes shorter than 0.1 ps, in fashion similar to glassy materials. The present results indicate that dry proteins sustain coherent density fluctuations in the THz frequency regime. The trend of the longitudinal modulus indicates that in this frequency range dry biomolecules are more rigid than hydrated proteins.
    The Journal of Physical Chemistry B 02/2012; 116(12):3861-5. · 3.61 Impact Factor
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    ABSTRACT: The structure of dense poly(N-isopropylacrylamide) (PNIPAM) brushes and oligo(ethylene glycol) (OEG) monolayers has been probed using neutron reflectometry and ellipsometry. The PNIPAM brush is swollen below the Lower Critical Solution Temperature (LCST) of 32 ∘C and is collapsed at 37 ∘C. Neutron reflectivity shows that below the LCST, the brush is described by a two-layer model: an inner dense layer and a hydrated outer layer. Above the LCST the collapsed brush forms a homogenous layer. With a fully deuterated myoglobin protein to increase the neutron scattering length density contrast, the reflectivity data show no detectable primary adsorption on the grafted OEG surface. A bound on the ternary adsorption onto PNIPAM chains forming dense brushes below and above the LCST is obtained.
    The European Physical Journal Special Topics 01/2012; 213(1):343-353. · 1.80 Impact Factor
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    ABSTRACT: Water dynamics plays a fundamental role for the fulfillment of biological functions in living organisms. Decades of hydrated protein powder studies have revealed the peculiar dynamical properties of hydration water with respect to pure water, due to close coupling interactions with the macromolecule. In such a framework, we have studied coherent collective dynamics in protein and DNA hydration water. State-of-the-art neutron instrumentation has allowed us to observe the propagation of coherent density fluctuations within the hydration shell of the biomolecules. The corresponding dispersion curves resulted to be only slightly affected by the coupling with the macromolecules. Nevertheless, the effects of the interaction appeared as a marked increase of the mode damping factors, which suggested a destructuring of the water hydrogen-bond network. Such results were interpreted as the signature of a "glassy" dynamical character of macromolecule hydration water, in agreement with indications from measurements of the density of vibrational states. Extending the investigations to living organisms at physiological conditions, we present here an in-vivo study of collective dynamics of intracellular water in Escherichia coli cells. The cells and water were fully deuterated to minimise the incoherent neutron scattering background. The water dynamics observed in the living cells is discussed in terms of the dynamics of pure bulk water and that of hydration water measured in powder samples.
    Journal of Physics Conference Series 01/2012; 340(1):012091.
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    ABSTRACT: Freeze-dried perdeuterated sperm whale myoglobin was kept in a water-saturated atmosphere in order to obtain a hydration degree of 335 H(2)O molecules per one myoglobin molecule. Incoherent neutron scattering was performed at the neutron spectrometer TOFTOF at the FRM II in an angular range of q from 0.6 to 1.8 Å(-1) and a temperature range from 4 to 297 K. We used neutrons with a wavelength of λ αE 6 Å and an energy resolution of about 65 μeV corresponding to motions faster than 10 ps. At temperatures above 225 K, broad lines appear in the spectra caused by quasielastic scattering. For an explanation of these lines, we assumed that there are only two types of protons, those that are part of the hydration water (72%) and those that belong to the protein (28%). The protons of the hydration water were analyzed with the diffusion model of Singwi and Sjölander [Phys. Rev. 119, 863 (1960)]. In this model, a water molecule stays for a time τ(0) in a bound state performing oscillatory motions. Thereafter, the molecule performs free diffusion for the time τ(1) in a nonbound state followed again by the oscillatory motions for τ(0) and so forth. We used the general formulation with no simplifications as τ(0)≫τ(1) or τ(1)≫τ(0). At room temperature, we obtained τ(0) αE 104 ps and τ(1) αE 37 ps. For the protein bound hydrogen, the dynamics is described by a Brownian oscillator where the protons perform overdamped motions in limited space.
    Physical Review E 10/2011; 84(4 Pt 1):041930. · 2.31 Impact Factor
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    ABSTRACT: Nuclear hormone receptors (NHRs) control numerous physiological processes through the regulation of gene expression. The present study provides a structural basis for understanding the role of DNA in the spatial organization of NHR heterodimers in complexes with coactivators such as Med1 and SRC-1. We have used SAXS, SANS and FRET to determine the solution structures of three heterodimer NHR complexes (RXR-RAR, PPAR-RXR and RXR-VDR) coupled with the NHR interacting domains of coactivators bound to their cognate direct repeat elements. The structures show an extended asymmetric shape and point to the important role played by the hinge domains in establishing and maintaining the integrity of the structures. The results reveal two additional features: the conserved position of the ligand-binding domains at the 5' ends of the target DNAs and the binding of only one coactivator molecule per heterodimer, to RXR's partner.
    Nature Structural & Molecular Biology 05/2011; 18(5):564-70. · 11.90 Impact Factor
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    ABSTRACT: Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.
    Biophysical Journal 03/2011; 100(5):1252-60. · 3.67 Impact Factor
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    ABSTRACT: INTRODUCTION Membrane proteins correspond to about 30% of the proteome of all organisms. They perform a wide range of essential cellular functions, are involved in a number of genetic diseases and have considerable therapeutic importance: 60 % of drug targets are membrane receptors or ion channels. Membrane proteins are also essential for the virulence of pathogens. The determination of their structures, required for understanding their role and function, designing drugs, etc is often difficult. The structural knowledge concerning membrane proteins remains very limited. The number of available structures increases, but only 250 unique membrane protein structures are known (http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html) to date (June 2010), a small number as compared to that (≈ 50000) for soluble proteins. Small angle neutron scattering (SANS) can be carried out in solution and can be used to provide ab initio low resolution envelops, confirming a 3D structure or addressing conformation changes (Petoukhov & Svergun, 2007). In the case of solubilized membrane proteins, detergent and/or lipid contributions may be masked by specific deuteration of solvent and/or of one of the protein/detergent complex components (Johs et al. 2006, Nogales et al. 2010, Cardoso et al. 2009). Deuteration of the protein part is advantageous for increasing the signal over noise ratio in the scattering curve, studying conformation of a partner within multi-protein complexes. In addition, we are investigating the potentialities of new classes of surfactants (fluorinated or neutral amphiphilic polymer surfactants) designed to improve the stability of solubilized membrane protein assemblies. They are matched in solvent conditions (at a D 2 O percentage) close to that of a hydrogenated protein (Gohon et al. 2006, Gohon et al. 2008, Breyton et al., 2009), which imposes membrane protein deuteration in SANS.
    07/2010;