Sébastien Buchoux

Plant sterols in “rafts”: a better way to regulate membrane thermal shocks

Specialized lipid domains (rafts) that are generally enriched in sterols and sphingolipids, are most likely present in cell membranes of animals, plants and fungi. While cholesterol and ergosterol are predominant in vertebrates and fungi, plants possess complex sterol profiles, dominated by sitosterol and stigmasterol in Arabidopsis thaliana. Fully hydrated model membranes of composition approaching those found in rafts of mammals, fungi and plants were investigated by means of solid‐state 2H‐NMR, using deuterated dipalmitoylphosphatidylcholine (2H62‐DPPC). The dynamics of such membranes was determined through measuring of membrane ordering or disordering properties. The presence of the liquid‐ordered, lo, phase, which may be an indicator of rigid sterol‐sphingolipid domains, was detected in all binary or ternary mixtures of all sterols investigated. Of great interest, the dynamics of ternary mixtures mimicking rafts in plants (phytosterol/glucosylcerebroside/ DPPC), showed a lesser temperature sensitivity to thermal shocks, on comparing to systems mimicking rafts in mammals and fungi. This effect was particularly marked with sitosterol. The presence of an ethyl group branched on the alkyl chain of sitosterol and stigmas‐terol is proposed as reinforcing the membrane cohesion by additional attractive van der Waals interactions with the alkyl chains of sphingolipids and phospholip‐ids. As a side result, the elevated resolution of NMR spectra in the presence of sitosterol also suggests domains of smaller size than with other sterols. Finally, the role of phytosterols in maintaining plant membranes in a state of dynamics less sensitive to temperature shocks is discussed.—Johannes G. Beck, Damien Mathieu, Cecile Loudet, Sebastien Buchoux and Erick J. Dufourc. Plant sterols in “rafts”: a better way to regulate membrane thermal shocks. FASEB J. 21, 1714–1723 (2007)

Surfactin-Triggered Small Vesicle Formation of Negatively Charged Membranes: A Novel Membrane-Lysis Mechanism

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri = 200) and is complete for Ri = 50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.

Key Role of Polyphosphoinositides in Dynamics of Fusogenic Nuclear Membrane Vesicles

The role of phosphoinositides has been thoroughly described in many signalling and membrane trafficking events but their function as modulators of membrane structure and dynamics in membrane fusion has not been investigated. We have reconstructed models that mimic the composition of nuclear envelope precursor membranes with naturally elevated amounts of phosphoinositides. These fusogenic membranes (membrane vesicle 1(MV1) and nuclear envelope remnants (NER) are critical for the assembly of the nuclear envelope. Phospholipids, cholesterol, and polyphosphoinositides, with polyunsaturated fatty acid chains that were identified in the natural nuclear membranes by lipid mass spectrometry, have been used to reconstruct complex model membranes mimicking nuclear envelope precursor membranes. Structural and dynamic events occurring in the membrane core and at the membrane surface were monitored by solid-state deuterium and phosphorus NMR. “MV1-like” (PC∶PI∶PIP∶PIP2, 30∶20∶18∶12, mol%) membranes that exhibited high levels of PtdIns, PtdInsP and PtdInsP2 had an unusually fluid membrane core (up to 20% increase, compared to membranes with low amounts of phosphoinositides to mimic the endoplasmic reticulum). “NER-like” (PC∶CH∶PI∶PIP∶PIP2, 28∶42∶16∶7∶7, mol%) membranes containing high amounts of both cholesterol and phosphoinositides exhibited liquid-ordered phase properties, but with markedly lower rigidity (10–15% decrease). Phosphoinositides are the first lipids reported to counterbalance the ordering effect of cholesterol. At the membrane surface, phosphoinositides control the orientation dynamics of other lipids in the model membranes, while remaining unchanged themselves. This is an important finding as it provides unprecedented mechanistic insight into the role of phosphoinositides in membrane dynamics. Biological implications of our findings and a model describing the roles of fusogenic membrane vesicles are proposed.

Effect of monolayer lipid charges on the structure and orientation of protein VAMP1 at the air-water interface.

SNARE proteins are implicated in membrane fusion during neurotransmission and peptide hormone secretion. Relatively little is known about the molecular interactions of their trans- and juxtamembrane domains with lipid membranes. Here, we report the structure and the assembling behavior of one of the SNARE proteins, VAMP1/synaptobrevin1 incorporated in a lipid monolayer at an air-water interface which mimics the membrane environment. Our results show that the protein is extremely sensitive to surface pressure as well as the lipid composition. Monolayers of proteins alone or in the presence of the neutral phospholipid DMPC underwent structural transition from alpha-helix to beta-sheet upon surface compression. In contrast, the anionic phospholipid DMPG inhibited this transition in a concentration-dependent manner. Moreover, the orientation of the proteins was highly sensitive to the charge density of the lipid layers. Thus, the structure of VAMP1 is clearly controlled by protein-lipid interactions.

How to Easily Extract Physical Properties from MD Simulations of Lipid Membranes with Fatslim

Extracting physical parameters from MD simulations of lipid membranes is crucial when comparing simulations with experimental observations. Unfortunately, the analysis of the trajectories may be problematic because of the size of the generated files and/or because extracting relevant data from atom coordinates stored in the trajectories may not be trivial.In order to overcome these difficulties, FATSLiM (“Fast Analysis Toolbox for Simulations of Lipid Membranes” - https://fatslim.github.io/) was developed. The main goal of this free open source software is to provide a fast and robust analytical tool to extract physical parameters such as bilayer thickness, area per lipid, lateral diffusion or lipid order parameter from molecular dynamics simulations of lipid membranes.FATSLiM is computationally efficient as it scales linearly with the number of lipids and is parallelized. As an illustration, on a 8-core machine, extracting both the thickness and the area per lipid from a 100-frame trajectory of a MD simulation of 12800 coarse-grained lipids takes about 15 seconds (35 seconds for All-Atom lipids) while needing 56 MB of memory (155 MB for All-Atom lipids). APL@voro, one the fastest tools, gives comparable results in 28 seconds (3.5 min for All-Atom lipids) but requires almost 1.6 GB of memory (11 GB for All-Atom lipids) and is not able to work with non-planar bilayers.Indeed, as opposed to other softwares, FATSLiM does not rely on any assumption regarding the bilayer planarity. In contrast, It calculates the local topology so it can then perform analysis on a planar membrane, an ondulating bilayer, or even a vesicle with the same accuracy and reliability. As such, it is perfectly suited to investigate the effect of a protein (on any molecule) on the properties/morphology of a membrane.

CHAPTER 6:Magnetic Liposomes and Bicelles: New Tools for Membrane-Peptide Structural Studies

The structure and topology of membrane peptides and proteins in a natural membrane environment can be approached with solid-state NMR by making use of the magnetic properties of lipids, which in certain conditions lead to magnetically oriented membrane samples. Lipids possess an intrinsic very small magnetic susceptibility anisotropy, Δχ, which leads to interesting annealing properties in very high magnetic fields of ca. 20 Tesla. Saturated chain lipids have a negative Δχ, leading to liposome (multilamellar vesicle, MLV) deformation to prolate or to lipid bicelles (40–100 nm nanodiscs) oriented with the normal to the disc surface perpendicular to the field. Biphenyl chain-containing lipids exhibit a positive Δχ that leads to MLV oblate deformation or bicelle orientation with the normal to the nanodisc parallel to the field. Using different lipids either in the form of liposomes or bicelles to tune the membrane orientation with respect to the field, a collection of NMR experiments can be performed to gain membrane peptide/protein topology and structural information. Representative examples are given of the Pf1 coat membrane protein, the antimicrobial peptide surfactin and the neurotransmitter methionine enkephalin.